The fungal cell wall serves as the interface between the organism and its environment. Complex carbohydrates are a major component of the cell wall, , glucan, mannan and chitin. β-Glucan is a pathogen associated molecular pattern (PAMP) composed of β-(1 → 3,1 → 6)-linked glucopyranosyl repeat units. This PAMP plays a key role in fungal structural integrity and immune recognition. Glycogen is an α-(1 → 4,1 → 6)-linked glucan that is an intracellular energy storage carbohydrate. We observed that glycogen was co-extracted during the isolation of β-glucan from SC5314. We hypothesized that glucan and glycogen may form a macromolecular species that links intracellular glycogen with cell wall β-(1 → 3,1 → 6)-glucan. To test this hypothesis, we examined glucan-glycogen extracts by multi-dimensional NMR to ascertain if glycogen and β-glucan were interconnected. H NMR analyses confirmed the presence of glycogen and β-glucan in the macromolecule. Diffusion Ordered SpectroscopY (DOSY) confirmed that the β-glucan and glycogen co-diffuse, which indicates a linkage between the two polymers. We determined that the linkage is not via peptides and/or small proteins. Our data indicate that glycogen is covalently linked to β-(1 → 3,1 → 6) glucan via the β -(1 → 6)-linked side chain. We also found that the glucan-glycogen complex was present in , and , but was not present in or hyphal glucan. These data demonstrate that glucan and glycogen form a novel macromolecular complex in the cell wall of and other species This new and unique structure expands our understanding of the cell wall in species.

Atherosclerosis is a multifactorial inflammatory disease of the arterial wall and its clinical manifestations, including myocardial infarction and stroke, are the leading causes of death in western societies. Recent data has suggested that disruption of protein homeostasis in a cell's endoplasmic reticulum (ER), a condition known as ER stress, is associated with the progression of atherosclerosis. Furthermore, signaling by the serine/threonine kinase glycogen synthase kinase (GSK)-3α/β mediates pro-atherogenic processes. This thesis examines the role of ER stress and GSK3α/β signaling in atherosclerosis. Initially, three apolipoprotein-E deficient (ApoE-/-) mouse models of accelerated atherosclerosis were established. Relative to ApoE-/- mice fed a chow diet, pro-atherogenic conditions promoted hepatic steatosis, atherosclerosis, ER stress and GSK3β activity. A subset of mice from each group were given the GSK3α/β inhibitor valproate. Valproate supplementation suppressed hepatic steatosis, atherosclerosis and GSK3β activity in each mouse model without altering ER stress levels. This study revealed a role for ER stress and GSK3α/β in multiple murine models of atherosclerosis. Next, we investigated ER stress and GSK3α/β signaling in macrophage foam cell formation. In macrophages, ER stress induced GSK3α/β activity in a protein kinase R-like endoplasmic reticulum kinase (PERK) dependent manor. GSK3α/β inhibition attenuated ER stress induced lipid accumulation and the expression of distal components of the PERK pathway. Overexpression of constitutively active GSK3β induced foam cell formation. In mice, valproate supplementation attenuated PERK signaling in peritoneal macrophages and macrophages within atherosclerotic lesions. Together, these results point to GSK3α/β being a downstream component of the PERK pathway and that PERK-GSK3α/β signaling mediates ER stress induced foam cell formation. Lastly, we investigated the tissue and homolog specific functions of GSK3α and GSK3β in atherosclerosis. In high fat diet (HFD) fed low-density lipoprotein receptor deficient (LDLR-/-) mice, deletion of GSK3α or GSK3β in hepatocytes did not alter liver lipid content or atherosclerosis. Myeloid cell deletion of GSK3α, but not GSK3β, attenuated HFD induced atherosclerosis. Mechanistically, deletion of GSK3α in macrophages promotes the anti-atherogenic M2 macrophage phenotype by modulating signal transducer and activator of transcription (STAT)-3 and STAT6 phosphorylation and activation. Together, the data presented in this thesis suggest; 1) GSK3α/β inhibition attenuates atherosclerosis in multiple mouse models, 2) PERK-GSK3α/β signaling regulates macrophage foam cell formation and 3) myeloid cell GSK3α mediates atherosclerosis and macrophage phenotype. / Thesis / Doctor of Philosophy (PhD)

In this investigation, skeletal muscle sarcoplasmic reticulum (SR) was purified from female Sprague Dawley rats (200-250 g). SR samples were subjected to two different biochemical glycogen-extraction protocols. The results suggest that both amylase and removal of EDTA (No-EDTA) from the homogenization and storage buffers reduced the amount of glycogen associated with the SR. Both of these treatments failed to impair SR calcium (Ca2+) handling when assayed under conditions where exogenous ATP was added and utilized for SR Ca2+ transport. In fact, these treatments seemed to cause a small increase in both SR Ca2+-uptake and release rates under these assay conditions. As expected, glycogen phosphorylase content was reduced as a result of glycogen extraction in the presence of amylase, however this was not the case for No-EDTA samples. Interestingly, many other proteins differed in content after glycogen extraction. These treatments resulted in a greater recovery of the sarco(endo)plasmic reticulum Ca2+ adenosine triphosphatase (SERCA) and a substantial loss of glycogen phosphorylase and glycogen debranching enzyme (AGL) in amylase-treated samples. Creatine kinase (CK) and pyruvate kinase (PK) contents were increased as a result of both glycogen-extraction conditions. It was imperative to consider these altered protein contents while analyzing the data and assessing the effects of glycogen extraction on SR Ca2+ handling. After normalizing to SERCA content, only No-EDTA samples had higher adenosine triphosphate (ATP)-supported SR Ca2+-uptake rates compared to control samples. For endogenously synthesized ATP-supported SR Ca2+-uptake experiments, normalizing data to protein content (either CK and SERCA or PK and SERCA) revealed that amylase-treated samples had lower SR Ca2+-uptake rates, compared to control samples. Although not significant, SR Ca2+-uptake rates for No-EDTA samples were also lower than control samples. These data suggest that changes in endogenously supported SR Ca2+-uptake due to glycogen extraction affected the source of ATP synthesis (either PK or CK), the effectiveness of energy utilization for Ca2+ transport (SERCA), or altered the metabolic channeling properties. / Ph. D.

The survival of 1,729 freshwater mussels (15 species) was monitored in ponds at study sites in Critz, Blacksburg and Marion, Virginia. Mussels were held within plastic and metal screen cages fastened to PVC float collars. Survival in the pond at Critz was 73% overall, with significant differences in survival among species after 26 months of captivity. Cyclonaias tub~X_~lJ.t~J_~ and three E11Jptt9 spp. exhibited the highest survivals (x=83%), whereas survival of Pleurobema cordatum and Lampsilis ovata was significantly lower, with 54% and 14%, respectively. Survival of five mussel species in a pond at Blacksburg was high (x=79%) after 7 months, but a nearly complete die-off of captive mussels occurred during hot weather in July, 1994. Survival of six mussel species at the Marion state Fish Hatchery was low for both the sleeve and unrestricted holding methods, 35% and 8%, respectively, after 14 months. It is suspected that excessive accumulation of particulates from pelleted feed contributed to the low survival observed at this site. Survival of four mussel species at Hoge's Pond in Blacksburg was 98% after 6 month. / Master of Science

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: It is widely accepted that insufficient insulin-stimulated activation of muscle glycogen synthesis is one of the major components of non-insulin-dependent (type 2) diabetes mellitus. Glycogen synthase, a key enzyme in glycogen synthesis, is extensively regulated, both allosterically (by glucose-6-phosphate, ATP, and other ligands) and covalently (by phosphorylation). Although glycogen synthase has been a topic of intense study for more than 50 years, its kinetic characterization has been confounded by its large number of phosphorylation states. Questions remain regarding the function of glycogen synthase regulation and the relative importance of allosteric and covalent modification in fulfilling this function. The regulation of glycogen synthase and glycogen phosphorylase, the enzyme that catalyses the degradation of glycogen chains, are reciprocal in many respects. In the present research, using mathematical modelling, we aim to establish the function of the allosteric and covalent regulation of glycogen synthase and glycogen phosphorylase in muscle and, in the case of glycogen synthase, the relative importance of these two mechanisms in performing this function. In order to realize these aims it is essential that a detailed kinetic model of glycogen metabolism is constructed. We begin with a thorough review of the kinetics and regulation of glycogen synthase inwhich we propose that both allosteric and covalent modification of glycogen synthase can be described by a Monod-Wyman-Changeux model in terms of apparent changes to L0, the equilibrium constant between the T and R conformers. We then proceed to develop a rate equation according to the proposed Monod-Wyman-Changeux model and determine values for its kinetic parameters from published experimental data using non-linear least-squares regression. We show that the application of the Monod-Wyman-Changeux model to glycogen synthase kinetics also has important implications for the rate equations of enzymes that catalyse the phosphorylation and dephosphorylation of glycogen synthase. We formalize these implications for a generic protein that follows Monod-Wyman-Changeux-type conformational change and then also show how the findings apply to glycogen synthase. Taking into account the kinetic model of glycogen synthase and how it also influences the covalent regulation of the enzyme, we proceed to construct a detailed mathematical model of glycogen synthesis that includes the glycogen synthase phosphorylation cascade. A variation of this model in which glycogen synthase phosphorylation is described with a single parameter is also provided. We reuse an existing model of muscle glycogenolysis and also combine these models in an overall model of glycogen metabolism. Finally, we employ the theoretical frameworks of metabolic control analysis, supply-demand analysis, and co-response analysis to investigate the function of glycogen synthase and glycogen phosphorylase regulation. We show that the function of glycogen synthase regulation is not flux control, as assumed in the textbook view, but rather the maintenance of glucose-6-phosphate within a narrow range far from equilibrium. Similarly, we show that regulation of glycogen phosphorylase functions to minimize variation in cellular energy charge in the face of highly variable energy demand. We conclude with an appeal for a renewed interest in the enzyme kinetics of muscle glycogen metabolism. / AFRIKAANSE OPSOMMING: Daar word wyd aanvaar dat onvoldoende insulien-gestimuleerde aktivering van spierglikogeensintese een van die hoofkomponente van insulien-onafhanklike (tipe 2) diabetes mellitus is. Glikogeensintase, ’n sleutelensiem in glikogeensintese is onderworpe aan breedvoerige regulering, beide allosteries (deur glukose-6-fosfaat, ATP, en ander ligande) en kovalent (deur fosforilering). Alhoewel glikogeensintase reeds vir meer as 50 jaar deeglik bestudeer word, word die kinetiese karakterisering daarvan bemoeilik deur die groot aantal fosforilasiestate waarin die ensiem voorkom. Daar is steeds vrae betreffende die funksie van die regulering van glikogeensintase en die relatiewe bydrae van allosteriese en kovalente regulering in die vervulling van hierdie funksie. Die regulering van glikogeensintase en glikogeenfosforilase, die ensiem wat die afbraak van glikogeenkettings kataliseer, is in baie opsigte resiprook. In hierdie studie beoog ons om met die hulp van wiskundige modellering vas te stel watter funksie die regulering van glikogeensintase en glikogeenfosforilase vervul en, in die geval van glikogeensintase, wat die relatiewe belang is van allosteriese en kovalente regulering in die vervulling van hierdie funksie. Om hierdie oogmerke te verwesentlik is dit nodig dat ’n kinetiese model van glikogeenmetabolisme ontwikkel word. Ons begin met ’n omvattende oorsig van die kinetika en regulering van glikogeensintase waarin ons voorstel dat beide die allosteriese en kovalente regulering van glikogeensintase beskryf kan word met die Monod-Wyman-Changeux model in terme van oënskynlike veranderings aan L0, die ekwilibriumkonstante tussen die T en R konformasies. Ons gaan dan voort om ’n snelheidsvergelyking te ontwikkel volgens die voorgestelde Monod-Wyman-Changuex-model en bepaal ook die waardes van hierdie vergelyking se parameters vanaf gepubliseerde eksperimentele data deur middel van nie-lineêre kleinste-vierkantsregressie. Ons wys dat die toepassing van die Monod-Wyman-Changuex-model op glikogeensintase-kinetika belangrike gevolge het vir die snelheidsvergelykings van die ensieme wat die fosforilering en defosforilering van glikogeensintase kataliseer. Ons formaliseer hierdie gevolge vir ’n generiese Monod-Wyman-Changeux-tipe proteïen en wys dan ook hoe die bevindings op glikogeensintase van toepassing is. Met inagneming van die kinetiese model vir glikogeensintase en hoe dit die kovalente regulering van die ensiem be¨ınvloed, gaan ons voort om ’n gedetaileerde wiskundige model van glikogeensintese, wat ook die glikogeensintase-fosforileringskaskade insluit, te ontwikkel. ’n Variasie op hierdie model waarin die fosforilering van glikogeensintase deur ’n enkele parameter beskryf word, word ook voorsien. Ons herbruik ’n bestaande model van spierglikogenolise en kombineer ook hierdie modelle in ’n oorkoepelende model van glikogeenmetabolisme. Uiteindelik span ons die teoretiese raamwerke van metaboliese kontrole-analise, vraag-aanbod-analise, en ko-responsanalise in om die funksie van die regulering van glikogeensintase en glikogeenfosforilase te ondersoek. Ons wys dat die funksie van die regulering van glikogeensintase nie fluksiekontrole, soos algemeen in handboeke aangeneem word, is nie, maar liewer dat dit glukose-6-fosfaat handhaaf binne ’n noue band ver vanaf ekwilibrium. Insgelyks wys ons dat die regulering van glikogeenfosforilase funksioneer om variasie in sellulˆere energielading te beperk ten spyte van hoogs wisselende vlakke van energie-aanvraag. Ons sluit af met ’n pleidooi vir hernieude belangstelling in die ensiemkinetika van glikogeenmetabolisme in die spier. / National Research Foundation

The purpose of this investigation was to determine the effect of passive and active recovery on the resynthesis of muscle glycogen after high intensity cycle ergometer exercise in untrained subjects. In a cross over design, six college-age males performed three, one min exercise bouts, at 130% V02max with a 4 min rest period between each work bout. Subjects refrained from exercise for two days prior to testing, and consumed a 15% carbohydrate solution (300g sugar in 2000ml of water) the day before each trial to help elevate glycogen concentration. The exercise protocol for each trial was identical, while the recovery following exercise was eitheractive (40-50% VO2max) or passive. The initial muscle glycogen values averaged 144.2 mmol•kg-1 w.w. for the active trial and 158.7 mmol•kg-1 w.w. for the passive trial. Corresponding post-exercise glycogen contents were 97.7 and 106.8 mmol•kg-1 w.w., respectively. These differences were not significant (P>0.05). However, the rate of muscle glycogen resynthesis during passive recovery increased 15 mmol•kg-1 w.w. whereas it decreased 6.27 mmol•kg-1 w.w. following active recovery (P<0.01). Also, the decrease in blood lactate concentration during active recovery was much faster than during passive recovery and significantly different at 10 and 30 min of the recovery period (P<0.01). The major finding of this investigation was that the rate of muscle glycogen resynthesis during passive recovery was significantly greater than that during active recovery. These data suggest that lactate can be used as an endogenous glycogenic precusor in muscle, and that glycogenesis was the prevalent pathway of lactate removal during passive recovery following high intensity cycle ergometer exercise. / Human Performance Laboratory

[Truncated abstract] It is well established that fish, amphibians and reptiles recovering from physical activity of near maximal intensity can replenish completely their muscle glycogen stores in the absence of food. In contrast, the extent to which these stores are replenished under these conditions in humans has been reported in all but one study to be partial. This implies that a few consecutive bouts of intense exercise might eventually lead to the sustained depletion of the muscle glycogen stores in humans if food is unavailable, thus limiting their capacity to engage in fight or flight behaviors unless mechanisms exist to protect muscle glycogen against sustained depletion. The objective of Study 1 was to test this prediction. Eight participants performed three intense exercise bouts each separated by a recovery period of 75 minutes. Although only 53% of muscle glycogen was replenished after the first exercise bout (postexercise and post-recovery glycogen levels of 246 ± 25 and 320 ± 36 mmol.kg-1 dry mass, respectively), all the glycogen mobilised during the second and third bouts was completely replenished during the respective recovery periods, with glycogen reaching levels of 319 ± 29 mmol.kg-1 dry mass after recovery from the third bout. These findings show that humans are not different from other vertebrate species in that there are conditions where humans have the ability to completely replenish without food intake the muscle glycogen mobilised during exercise. The results of our first study raise the intriguing possibility that humans have pre-set muscle glycogen levels that are protected against sustained depletion, with the extent to which muscle glycogen stores are replenished after exercise being dependent on the amount of glycogen required to attain those protected levels. ... During recovery, glycogen levels in the NORM group increased by more than ~50% and reached levels close to those alleged to be protected (189 ± 21 mmol.kg-1 dry mass), whereas no glycogen was deposited in the HCHO group. The sustained post-exercise activation of glycogen synthase, the transient fall in whole body carbohydrate oxidation rate, the increased mobilisation of body proteins, and the prolonged elevation in NEFA levels most probably played important roles in enabling glycogen synthesis in the NORM group. In conclusion, this thesis shows for the first time that there are some conditions (e.g. low pre-exercise muscle glycogen levels) where humans recovering from intense exercise have the capacity, like other species, to replenish completely their muscle glycogen stores from endogenous carbon sources. This study also suggests that humans protect preset levels of muscle glycogen against sustained depletion and at levels high enough to support at least one maximal sprint effort to exhaustion. Evidence is also provided for the existence of a feedback mechanism whereby glycogen below their protected levels mediate the activation of glycogen synthase to restore the depleted muscle glycogen stores back to their protected levels. Our findings, however, leave us with a number of novel unanswered questions which clearly show that the regulation of glycogen metabolism is far from the simple process generally depicted in most textbooks of biochemistry.

Orientadores: Carlos Eduardo Steiner, Carmen Silvia Bertuzzo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T18:18:42Z (GMT). No. of bitstreams: 1 Carlin_MarceloPaschoalete_M.pdf: 1614647 bytes, checksum: 62e5b428719069b2b173ab2baf51970f (MD5) Previous issue date: 2011 / Resumo: A transformação de glicogênio em glicose acontece através de reações químicas realizadas por enzimas específicas e uma deficiência em uma delas leva ao acúmulo de glicogênio, resultando em distúrbios hereditários conhecidos como doenças de depósito de glicogênio (GSD, da sigla em inglês), ou glicogenoses. A glicogenose tipo I (GSDI), responsável por mais de 90% dos casos, é causada pela deficiência de G6Pase, enzima chave na homeostase da glicose sanguínea. Seu complexo enzimático é constituído por duas subunidades (catalítica e translocase) que determinam os subtipos Ia e Ib. A GSDIa, também conhecida como doença de von Gierke, é a mais comum das GSDI com 80 a 90% dos casos e corresponde a uma deficiência na sub-unidade catalítica da G6Pase. A GSDIb é a segunda forma mais prevalente e mais grave. É resultante da deficiência da glicose-6-fosfato translocase que transporta a glicose-6-fosfato para o lúmen do retículo endoplasmático, onde a unidade catalítica da G6Pase está situada. Em ambas, a deficiência enzimática é o resultado de mutações genéticas nos genes que codificam estas enzimas, conhecidos respectivamente como G6PC e G6PT1. O diagnóstico bioquímico é recomendável caso se queira desvendar e recomendar modalidades de tratamento, porém não fornece informação suficiente para a determinação do subtipo envolvido. Para essa diferenciação é necessário análise enzimática da G6Pase. Como essa enzima não é expressa em tecidos como fibroblastos ou linfócitos, sua aferição só é possível por procedimento cirúrgico, através de biópsia hepática. Nesse contexto, a clonagem do cDNA do G6PC e G6PT1 possibilitou o rastreamento de mutações responsáveis pelos subtipos Ia e Ib, o que permite a alternativa de um diagnóstico menos invasivo baseado em técnicas de biologia molecular através de amostras de sangue. No presente estudo, treze pacientes com sintomas clínicos sugestivos de GSDIa e Ib foram investigados através do sequenciamento genético. Foram detectadas para o gene G6PC cinco alterações, incluindo, três mutações de ponto (G68R, R83C e Q347X) e dois polimorfismos (c.511G>A e c.1176T>C), todos previamente descritos. Já para o gene G6PT1 foram encontradas quatro alterações: uma mutação de ponto conhecida (G149E), uma inserção do tipo frameshift inédita na literatura especializada (c.1338_1339insT) e dois polimorfismos (c.1287G>A e c.1076-28C>T). A frequência das mutações em nosso meio é semelhante à observada na literatura, na qual a mutação R83C também é a mais frequente. Além disso, o presente estudo acrescentou a descrição de uma nova mutação. A pesquisa de ambos os genes deve ser considerado na investigação dessa condição para definir os subtipos envolvidos, pois no caso de ausência de alterações no gene 6PC, sugere-se o rastreamento no gene G6PT1. O estudo molecular dessa condição abre a possibilidade do diagnóstico precoce que é importante para estabelecer um tratamento correto aos pacientes, evitando o surgimento de complicações tardias e melhorando a qualidade de vida. Além disso, contribui para o aconselhamento genético adequado do casal podendo confirmar a estimativa do isco entre os próximos filhos e, eventualmente, permitir diagnóstico pré-natal por nálise de mutação / Abstract: Glucose transformation into glycogen is mediated by specific enzymes and a deficiency in one of them may cause glycogen accumulation, resulting in hereditary disorders known as glycogen storage diseases (GSD), or glycogenosis. Glycogenosis type I (GSDI), responsible for more than 90% of cases, is caused by deficiency of the glucose-6-phosphatase (G6Pase), the key enzyme in blood glucose homeostasis. Its enzyme complex consists of two subunits (catalytic and transporter) that determine subtypes Ia and Ib. GSDla, also known as von Gierke disease, is the most common GSDI responsible for 80 to 90% of cases and corresponds to a deficiency in the catalytic subunit of G6Pase. GSDIb is the second most prevalent but also the most severe, resulting from deficiency of lucose-6- phosphate translocase that transports glucose-6-phosphate into the lumen of the endoplasmic reticulum, where the catalytic unit of G6Pase is located. In both types, enzymatic deficiency results from genetic mutations in the genes that codify these enzymes, known as G6PC and G6PT1. Biochemical essay for GSDI is useful to confirm the diagnosis and to recommend treatment, however it does not allow the determination of the disease subtype. For this differentiation, enzymatic analysis of G6Pase is necessary. Since this enzyme is not expressed in tissues such as fibroblasts or lymphocytes, activity determination is only possible by liver biopsy. In this context, the cDNA cloning of G6PC and G6PT1 allowed the screening of mutations responsible for subtypes Ia and Ib, which gives the alternative of a less invasive diagnosis based on molecular biology techniques, using blood samples. In this study, thirteen patients with clinical symptoms suggestive of GSDIa and Ib were investigated through genetic sequencing. Five changes were detected in G6PC, including three known point mutations (G68R, R83C and Q347X) and two polymorphisms (c.511G> A and c.1176T>C). Concerning the G6PT1 gene, four changes were found: a known point mutation (G149E), a novel frameshift insertion (c.1338_1339insT) and two polymorphisms (c.1287G>A and c.1076-28C>T). The frequency of mutations in this population is similar to that observed in the literature, in which R83C is also the most frequent. Additionally, this study added a description of a new mutation. As result of this study, molecular analysis of both genes should be considered in he investigation of individuals with this GSDI in order to define the subtypes involved. Molecular analysis of G6PC and G6PT1 genes enable the achievement of positive diagnosis of GSDIa and Ib, securely without the need for liver biopsy. It also allows the differentiation of types and subtypes, which is not possible by the biochemical diagnosis. Finally, the identification of the mutation provides an additional tool for the genetic counseling (and eventually prenatal diagnosis) of the parents and other family members / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Indiana University-Purdue University Indianapolis (IUPUI) / Glycogen is a highly branched polymer of glucose that functions to store glucose residues for future metabolic use. Skeletal muscle and liver comprise the largest glycogen reserves and play critical roles in maintaining whole body glucose homeostasis. In addition to glucose, glycogen contains small amounts of covalent phosphate of unknown function, origin and structure. Evidence to support the involvement of glycogen associated phosphate in glycogen metabolism comes from patients with Lafora Disease. Lafora disease is an autosomal recessive, fatal form of progressive myoclonus epilepsy. Approximately 90% of cases of Lafora disease are caused by mutations in either the EPM2A or EPM2B genes that encode, respectively, a dual specificity phosphatase called laforin and an E3 ubiquitin ligase called malin. Lafora patients accumulate intracellular inclusion bodies, known as Lafora bodies that are primarily composed of poorly branched, insoluble glycogen-like polymers. We have shown that laforin is a glycogen phosphatase capable of releasing phosphate from glycogen in vitro and that this activity is dependent on a functional carbohydrate binding domain. In studies of laforin knockout mice, we observed a progressive change in the properties and structure of glycogen that paralleled the formation of Lafora bodies. Glycogen isolated from these mice showed increased glycogen phosphate, up to 6-fold (p< 0.001) compared to WT, providing strong evidence that laforin acts as a glycogen phosphatase in vivo. Furthermore we have demonstrated that glycogen synthase introduces phosphate into glycogen during synthesis by transferring the beta-phosphate of UDP-glucose into the polymer and that laforin is capable of releasing the phosphate incorporated by glycogen synthase. Analysis of mammalian glycogen revealed the presence of covalently linked phosphate at the 2 hydroxyl and the 3 hydroxyl of glucose residues in the polysaccharide, providing the first direct evidence of the chemical nature of the phosphate linkage. We envision a glycogen damage/repair process, analogous to errors during DNA synthesis that are subsequently repaired. We propose that laforin action parallels that of DNA repair enzymes and Lafora disease results from the inability of the phosphatase to repair damaged glycogen, adding another biological polymer to the list of those prone to errors by their respective polymerizing enzymes.

Metabolic reprogramming in cancer cells provides energy and important metabolites required to sustain tumour proliferation. Hypoxia represents a hostile environment that can encourage these transformations and other adaptive responses that contribute to poor prognosis and resistance to radiation and chemotherapy. Hypoxic signatures associated with worse prognosis were previously derived in different cancer types, and led to the selection of two candidates with potential metabolic implications, namely the mir210-putuative target iron-sulfur scaffold protein ISCU and glycogen phosphorylase (PYGL). Firstly, it was verified that the hypoxia-induced miR-210 targets ISCU. Iron-sulfur clusters represent cofactors for key enzymes involved in Krebs cycle and electron transport chain. Downregulation of ISCU was associated with the induction of reactive oxygen species (ROS) and reduced mitochondrial complex I and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. This indicates that the induction of a single microRNA, miR-210, can mediate a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation. Secondly, it was found that changes in PYGL expression reflect a characteristic upregulation of glycogen metabolism in hypoxia in both tumour xenografts and in cancer cell lines. More specifically, hypoxia stimulates glycogen accumulation and its utilisation, as well as the concurrent upregulation of several glycogen-metabolizing enzymes such as glycogen synthase (GYS1) and PYGL. PYGL depletion led to glycogen accumulation in hypoxic cells, increased intracellular levels of ROS, and a reduction in proliferation due to a p53-dependent induction of senescence. Furthermore, depletion of PYGL was associated with markedly impaired tumorigenesis in vivo. Finally, metabolic analyses indicated that glycogen degradation by PYGL is important for the optimal functioning of the pentose phosphate pathway. Collectively, this study shows the contribution of two important pathways to the metabolic adaptations induced by hypoxia.