Laforin is the only known glycogen phosphatase. Mutations in the laforin gene lead to the fatal childhood dementia and progressive myoclonic epilepsy known as Lafora disease (LD). A hallmark of LD is aberrant, cytoplasmic, glycogen-like aggregates known as Lafora bodies. Surprisingly, recent reports indicate that overexpression of a phosphatase-deficient laforin mutant, with the catalytic cysteine mutated to serine (LCS), prevented the formation of Lafora bodies in a laforin KO mouse model. This finding led to questions regarding the biological relevance of laforin phosphatase activity and its role in LD etiology. In this study, we defined the in vitro and in vivo effects of the LCS mutation. LCS protein lacks catalytic activity but exhibits significantly higher binding to phosphate and long glucan chains compared with WT laforin. In addition, LCS exhibits altered dynamics via hydrogen-deuterium exchange mass spectrometry and interacts more robustly with its binding partners malin and protein targeting to glycogen. We demonstrate that these altered dynamics result in aberrant retention of the LCS protein in the brain of the LCS knock-in mouse model, compared with laforin levels in WT mice. To examine the metabolic consequences of these biophysical changes, we compared the brain metabolomic phenotypes of LCS mice to WT and laforin KO mice. Furthermore, LCS mice display a distinct and significant global perturbation in metabolism. These results indicate a key signaling role for glycogen phosphorylation in glycogen metabolism, revealing an important biological role for laforin catalytic phosphatase activity.

Lafora disease (LD) is an autosomal recessive myoclonus epilepsy with onset in the teenage years leading to death within a decade of onset. LD is characterized by the overaccumulation of hyperphosphorylated, poorly branched, insoluble, glycogen-like polymers called Lafora bodies. The disease is caused by mutations in either EPM2A, encoding laforin, a dual specificity phosphatase that dephosphorylates glycogen, or EMP2B, encoding malin, an E3-ubiquitin ligase. While glycogen is a widely accepted laforin substrate, substrates for malin have been difficult to identify partly due to the lack of malin antibodies able to detect malin in vivo. Here we describe a mouse model in which the malin gene is modified at the C-terminus to contain the c-myc tag sequence, making expression of malin-myc readily detectable. Mass spectrometry analyses of immunoprecipitates using c-myc tag antibodies demonstrate that malin interacts with laforin and a number of glycogen-metabolizing enzymes. To investigate the role of laforin in these interactions we analyzed two additional mouse models: malin-myc/laforin knockout and malin-myc/LaforinCS, where laforin was either absent or the catalytic Cys was genomically mutated to Ser, respectively. The interaction of malin with partner proteins requires laforin, but is not dependent on its catalytic activity or the presence of glycogen. Overall, the results demonstrate that laforin and malin form a complex in vivo, which stabilizes malin and enhances interaction with partner proteins to facilitate normal glycogen metabolism. They also provide insights into the development of LD and the rescue of the disease by the catalytically inactive phosphatase.

Glycogen synthase 1 (GYS1), the rate-limiting enzyme in muscle glycogen synthesis, plays a central role in energy homeostasis and has been proposed as a therapeutic target in multiple glycogen storage diseases. Despite decades of investigation, there are no known potent, selective small-molecule inhibitors of this enzyme. Here, we report the preclinical characterization of MZ-101, a small molecule that potently inhibits GYS1 in vitro and in vivo without inhibiting GYS2, a related isoform essential for synthesizing liver glycogen. Chronic treatment with MZ-101 depleted muscle glycogen and was well tolerated in mice. Pompe disease, a glycogen storage disease caused by mutations in acid α glucosidase (GAA), results in pathological accumulation of glycogen and consequent autophagolysosomal abnormalities, metabolic dysregulation, and muscle atrophy. Enzyme replacement therapy (ERT) with recombinant GAA is the only approved treatment for Pompe disease, but it requires frequent infusions, and efficacy is limited by suboptimal skeletal muscle distribution. In a mouse model of Pompe disease, chronic oral administration of MZ-101 alone reduced glycogen buildup in skeletal muscle with comparable efficacy to ERT. In addition, treatment with MZ-101 in combination with ERT had an additive effect and could normalize muscle glycogen concentrations. Biochemical, metabolomic, and transcriptomic analyses of muscle tissue demonstrated that lowering of glycogen concentrations with MZ-101, alone or in combination with ERT, corrected the cellular pathology in this mouse model. These data suggest that substrate reduction therapy with GYS1 inhibition may be a promising therapeutic approach for Pompe disease and other glycogen storage diseases.

Glucagon rapidly and profoundly stimulates hepatic glucose production (HGP), but for reasons that are unclear, this effect normally wanes after a few hours, despite sustained plasma glucagon levels. This study characterized the time course of glucagon-mediated molecular events and their relevance to metabolic flux in the livers of conscious dogs. Glucagon was either infused into the hepato-portal vein at a sixfold basal rate in the presence of somatostatin and basal insulin, or it was maintained at a basal level in control studies. In one control group, glucose remained at basal, whereas in the other, glucose was infused to match the hyperglycemia that occurred in the hyperglucagonemic group. Elevated glucagon caused a rapid (30 min) and largely sustained increase in hepatic cAMP over 4 h, a continued elevation in glucose-6-phosphate (G6P), and activation and deactivation of glycogen phosphorylase and synthase activities, respectively. Net hepatic glycogenolysis increased rapidly, peaking at 15 min due to activation of the cAMP/PKA pathway, then slowly returned to baseline over the next 3 h in line with allosteric inhibition by glucose and G6P. Glucagon's stimulatory effect on HGP was sustained relative to the hyperglycemic control group due to continued PKA activation. Hepatic gluconeogenic flux did not increase due to the lack of glucagon's effect on substrate supply to the liver. Global gene expression profiling highlighted glucagon-regulated activation of genes involved in cellular respiration, metabolic processes, and signaling, as well as downregulation of genes involved in extracellular matrix assembly and development.NEW & NOTEWORTHY Glucagon rapidly stimulates hepatic glucose production, but these effects are transient. This study links the molecular and metabolic flux changes that occur in the liver over time in response to a rise in glucagon, demonstrating the strength of the dog as a translational model to couple findings in small animals and humans. In addition, this study clarifies why the rapid effects of glucagon on liver glycogen metabolism are not sustained.

Glycogen is the primary energy reserve in mammals, and dysregulation of glycogen metabolism can result in glycogen storage diseases (GSDs). In muscle, glycogen synthesis is initiated by the enzymes glycogenin-1 (GYG1), which seeds the molecule by autoglucosylation, and glycogen synthase-1 (GYS1), which extends the glycogen chain. Although both enzymes are required for proper glycogen production, the nature of their interaction has been enigmatic. Here, we present the human GYS1:GYG1 complex in multiple conformations representing different functional states. We observe an asymmetric conformation of GYS1 that exposes an interface for close GYG1 association, and propose this state facilitates handoff of the GYG1-associated glycogen chain to a GYS1 subunit for elongation. Full activation of GYS1 widens the GYG1-binding groove, enabling GYG1 release concomitant with glycogen chain growth. This structural mechanism connecting chain nucleation and extension explains the apparent stepwise nature of glycogen synthesis and suggests distinct states to target for GSD-modifying therapeutics.

CONTEXT:Glycogenin is considered to be an essential primer for glycogen biosynthesis. Nevertheless, patients with glycogenin-1 deficiency due to biallelic GYG1 (NM_004130.3) mutations can store glycogen in muscle. Glycogenin-2 has been suggested as an alternative primer for glycogen synthesis in patients with glycogenin-1 deficiency.OBJECTIVE:The objective of this article is to investigate the importance of glycogenin-1 and glycogenin-2 for glycogen synthesis in skeletal and cardiac muscle.DESIGN, SETTING, AND PATIENTS:Glycogenin-1 and glycogenin-2 expression was analyzed by Western blot, mass spectrometry, and immunohistochemistry in liver, heart, and skeletal muscle from controls and in skeletal and cardiac muscle from patients with glycogenin-1 deficiency.RESULTS:Glycogenin-1 and glycogenin-2 both were found to be expressed in the liver, but only glycogenin-1 was identified in heart and skeletal muscle from controls. In patients with truncating GYG1 mutations, neither glycogenin-1 nor glycogenin-2 was expressed in skeletal muscle. However, nonfunctional glycogenin-1 but not glycogenin-2 was identified in cardiac muscle from patients with cardiomyopathy due to GYG1 missense mutations. By immunohistochemistry, the mutated glycogenin-1 colocalized with the storage of glycogen and polyglucosan in cardiomyocytes.CONCLUSIONS:Glycogen can be synthesized in the absence of glycogenin, and glycogenin-1 deficiency is not compensated for by upregulation of functional glycogenin-2. Absence of glycogenin-1 leads to the focal accumulation of glycogen and polyglucosan in skeletal muscle fibers. Expression of mutated glycogenin-1 in the heart is deleterious, and it leads to storage of abnormal glycogen and cardiomyopathy.

OBJECTIVE:Adult polyglucosan body disease (APBD) is an adult-onset neurological variant of glycogen storage disease type IV. APBD is caused by recessive mutations in the glycogen branching enzyme gene, and the consequent accumulation of poorly branched glycogen aggregates called polyglucosan bodies in the nervous system. There are presently no treatments for APBD. Here, we test whether downregulation of glycogen synthesis is therapeutic in a mouse model of the disease.METHODS:We characterized the effects of knocking out two pro-glycogenic proteins in an APBD mouse model. APBD mice were crossed with mice deficient in glycogen synthase (GYS1), or mice deficient in protein phosphatase 1 regulatory subunit 3C (PPP1R3C), a protein involved in the activation of GYS1. Phenotypic and histological parameters were analyzed and glycogen was quantified.RESULTS:APBD mice deficient in GYS1 or PPP1R3C demonstrated improvements in life span, morphology, and behavioral assays of neuromuscular function. Histological analysis revealed a reduction in polyglucosan body accumulation and of astro- and micro-gliosis in the brains of GYS1- and PPP1R3C-deficient APBD mice. Brain glycogen quantification confirmed the reduction in abnormal glycogen accumulation. Analysis of skeletal muscle, heart, and liver found that GYS1 deficiency reduced polyglucosan body accumulation in all three tissues and PPP1R3C knockout reduced skeletal muscle polyglucosan bodies.INTERPRETATION:GYS1 and PPP1R3C are effective therapeutic targets in the APBD mouse model. These findings represent a critical step toward the development of a treatment for APBD and potentially other glycogen storage disease type IV patients.

The overaccumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenyl-1H-imidazol-5-yl)phenol (H23), as a first-in-class inhibitor for yeast GS 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure-activity relationship profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyrogallol, with a 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.

The addition of phosphate groups into glycogen modulates its branching pattern and solubility which all impact its accessibility to glycogen interacting enzymes. As glycogen architecture modulates its metabolism, it is essential to accurately evaluate and quantify its phosphate content. Simultaneous direct quantitation of glucose and its phosphate esters requires an assay with high sensitivity and a robust dynamic range. Herein, we describe a highly-sensitive method for the accurate detection of both glycogen-derived glucose and glucose-phosphate esters utilizing gas-chromatography coupled mass spectrometry. Using this method, we observed higher glycogen levels in the liver compared to skeletal muscle, but skeletal muscle contained many more phosphate esters. Importantly, this method can detect femtomole levels of glucose and glucose phosphate esters within an extremely robust dynamic range with excellent accuracy and reproducibility. The method can also be easily adapted for the quantification of plant starch, amylopectin or other biopolymers.

The overaccumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)­ethyl)-4-phenyl-1H-imidazol-5-yl)­phenol (H23), as a first-in-class inhibitor for yeast GS 2 (yGsy2p). Data from X-ray crystallography at 2.85 A, as well as kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure–activity relationship profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)­pyrogallol, with a 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.

Browning induction or transplantation of brown adipose tissue (BAT) or brown/beige adipocytes derived from progenitor or induced pluripotent stem cells (iPSCs) can represent a powerful strategy to treat metabolic diseases. However, our poor understanding of the mechanisms that govern the differentiation and activation of brown adipocytes limits the development of such therapy. Various genetic factors controlling the differentiation of brown adipocytes have been identified, although most studies have been performed using in vitro cultured pre-adipocytes. We investigate here the differentiation of brown adipocytes from adipose progenitors in the mouse embryo. We demonstrate that the formation of multiple lipid droplets (LDs) is initiated within clusters of glycogen, which is degraded through glycophagy to provide the metabolic substrates essential for de novo lipogenesis and LD formation. Therefore, this study uncovers the role of glycogen in the generation of LDs.

Lafora disease (LD) is a fatal childhood epilepsy and a non-classical glycogen storage disorder with no effective therapy or cure. LD is caused by recessive mutations in the EPM2A or EPM2B genes that encode the glycogen phosphatase laforin and an E3 ubiquitin ligase malin, respectively. A hallmark of LD is the intracellular accumulation of abnormal and insoluble α-linked polysaccharide deposits known as Lafora bodies (LBs) in several tissues, including most regions of the brain. In mouse models of LD, genetic reduction of glycogen synthesis eliminates LB formation and rescues the neurological phenotype. Since multiple groups have confirmed that neurodegeneration and epilepsy result from LB accumulation, a major focus in the field has shifted toward the development of therapies that reduce glycogen synthesis or target LBs for degradation with the goal of treating LD. Herein, we identify the optimal enzymes for degrading LBs, and we develop a novel therapeutic agent by fusing human pancreatic α-amylase to a cellpenetrating antibody fragment. This antibody-enzyme fusion (VAL-0417) degrades LBs in vitro , shows robust cellular uptake, and significantly reduces the LB load in vivo in Epm2a -/- mice. VAL-0417 is a promising therapeutic for the treatment of LD and a putative precision therapy for an intractable epilepsy. Antibody-enzyme fusions represent a new class of antibody-based drugs that could be utilized to treat glycogen storage disorders and other diseases. One Sentence Summary An antibody-enzyme fusion delivering an amylase degrades the toxic polyglucosan bodies that cause Lafora disease, a fatal childhood epilepsy.

Lafora disease (LD) is a fatal, autosomal recessive, glycogen-storage disorder that manifests as severe epilepsy. LD results from mutations in the gene encoding either the glycogen phosphatase laforin or the E3 ubiquitin ligase malin. Individuals with LD develop cytoplasmic, aberrant glycogen inclusions in nearly all tissues that more closely resemble plant starch than human glycogen. This Minireview discusses the unique window into glycogen metabolism that LD research offers. It also highlights recent discoveries, including that glycogen contains covalently bound phosphate and that neurons synthesize glycogen and express both glycogen synthase and glycogen phosphorylase.

Age‐related skeletal muscle wasting is associated with elevated 5′‐AMP‐Activated Protein Kinase (AMPK) activity, which inhibits overload‐induced skeletal muscle protein synthesis (MPS) and growth. Glycogen, an inhibitor of AMPK, is reduced in aged skeletal muscle. We recently found that glycogen enhancement augments 21‐day overload‐induced MPS and growth in aged rat skeletal muscle. Unexpectedly, these findings were largely independent of AMPK and other signaling regulating MPS. However, the effects of glycogen enhancement on the skeletal muscle response to other anabolic stimuli, such as leucine, are largely unknown. Therefore, the purpose of this investigation was to examine the effects of enhanced glycogen on AMPK phosphorylation status and activity, MPS, and MPS‐related signaling in response to leucine in C2C12 myotubes. We hypothesized that glycogen enhancement in myotubes would enhance the anabolic effect of leucine by inhibiting AMPK and increasing anabolic signaling and MPS, and that this effect would be reduced by rapamycin‐induced mechanistic target of rapamycin (mTOR) blockade. Four days after the onset of differentiation, myotubes were transfected (via Lipofectamine 3000) for 48 hrs with a mutant glycogen synthase plasmid (mutant‐GS; designed to enhance skeletal muscle glycogen content) or an empty‐vector plasmid. At six days post‐differentiation, transfected myotubes were serum‐starved for 4 hrs prior to an amino acid starvation for 1 hr. Myotubes were then treated with either a vehicle control (CT), 10 mM leucine (LEU), 100 nM rapamycin (RAP), or leucine/rapamycin co‐treatment (LEU+RAP) for 30 min. All treatment conditions contained 100 μM puromycin for measurement of MPS rate. All values were normalized to the empty‐vector CT condition. As expected, mutant‐GS expression and glycogen content were significantly (p ≤ 0.05) higher in all four mutant‐GS myotube conditions vs. corresponding empty‐vector conditions. Within both the mutant‐GS conditions and the empty‐vector conditions, MPS was stimulated with LEU treatment and suppressed with RAP treatment as expected; however, MPS was significantly (p ≤ 0.05) higher in all four mutant‐GS conditions vs. corresponding empty‐vector conditions. LEU treatment increased the phosphorylation status of mTOR (Ser2448), p70S6K (Thr389), and 4EBP1 (Thr37/46), and this effect was blocked with RAP treatment within both the mutant‐GS myotubes or the empty‐vector myotubes. However, there were no significant (p > 0.05) differences between mutant‐GS or empty‐vector myotubes in this signaling. Additionally, markers of AMPK phosphorylation status (Thr172) and activity (ACC at Ser79) and other MPS signaling intermediates (TSC2 Ser1387 & Thr1462) were not significantly different between mutant‐GS vs. empty‐vector conditions and were unaffected by LEU or RAP treatments. Although the stimulation of MPS by leucine is mTOR‐dependent regardless of glycogen enhancement in myotubes, glycogen enhancement augments MPS independent of changes in AMPK, mTOR, or other MPS‐related signaling markers assessed. Even though the signaling mechanism(s) remain to be identified, these findings along with our recent in vivo data support the translatable potential to enhance human skeletal muscle glycogen content to augment leucine‐stimulated MPS or augment MPS and growth under conditions of chronic overload (such as resistance exercise training).Support or Funding InformationThis research was partially supported by an ACSM Foundation Doctoral Student Research Grant from the American College of Sports Medicine Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Age-related skeletal muscle (SkM) wasting is associated with elevated 5’-AMP-Activated Protein Kinase (AMPK) activity, which inhibits overload-induced (OI) SkM protein synthesis (MPS) and growth. Glycogen, an inhibitor of AMPK, is reduced in aged SkM. PURPOSE: To examine the effects of manipulating glycogen on AMPK, MPS and related signaling, and OI-growth in aged SkM. METHODS: Mutant glycogen synthase (GS; designed to enhance SkM glycogen content [GC]) or empty-vector plasmids were electrotransferred into fast-twitch plantaris muscles prior to 21-day synergist ablation-induced unilateral overload in young adult (8 mo.; empty vector; YE, n=9) and old (33 mo.; empty vector, OE, n=11; or mutant GS, OM, n=13) male FBN rats. Contralateral limbs underwent SHAM ablations with no plasmid. RESULTS: As expected, mutant GS expression and GC were significantly higher in OM overloaded muscles (the only muscles receiving the mutant GS plasmid) vs SHAM OM muscles or vs both SHAM or overloaded YE and OE muscles. There were significant increases in OI-(all vs SHAM) MPS and hypertrophy in all groups and OM was greater than OE. Markers of AMPK activity and other signaling intermediates affecting MPS were largely unaltered by glycogen enhancement. However, there was a strong and significant effect of enhancing GC (via mutant GS vs empty vector plasmid) on myogenic regulatory factors MyoD and myogenin, embryonic myosin heavy chain-positive fibers, and total fiber number in aged muscle under conditions of overload. CONCLUSIONS: Thus, enhancing GC may lead to enhanced MPS and OI growth in aged SkM. This effect may be due, in part, to an enhanced myogenesis.

Disruption of the Gys2 gene encoding the liver isoform of glycogen synthase generates a mouse strain (LGSKO) that almost completely lacks hepatic glycogen, has impaired glucose disposal, and is pre-disposed to entering the fasted state. This study investigated how the lack of liver glycogen increases fat accumulation and the development of liver insulin resistance. Insulin signaling in LGSKO mice was reduced in liver, but not muscle, suggesting an organ-specific defect. Phosphorylation of components of the hepatic insulin-signaling pathway, namely IRS1, Akt, and GSK3, was decreased in LGSKO mice. Moreover, insulin stimulation of their phosphorylation was significantly suppressed, both temporally and in an insulin dose response. Phosphorylation of the insulin-regulated transcription factor FoxO1 was somewhat reduced and insulin treatment did not elicit normal translocation of FoxO1 out of the nucleus. Fat overaccumulated in LGSKO livers, showing an aberrant distribution in the acinus, an increase not explained by a reduction in hepatic triglyceride export. Rather, when administered orally to fasted mice, glucose was directed toward hepatic lipogenesis as judged by the activity, protein levels, and expression of several fatty acid synthesis genes, namely, acetyl-CoA carboxylase, fatty acid synthase, SREBP1c, chREBP, glucokinase, and pyruvate kinase. Furthermore, using cultured primary hepatocytes, we found that lipogenesis was increased by 40% in LGSKO cells compared with controls. Of note, the hepatic insulin resistance was not associated with increased levels of pro-inflammatory markers. Our results suggest that loss of liver glycogen synthesis diverts glucose toward fat synthesis, correlating with impaired hepatic insulin signaling and glucose disposal.

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

Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G-6-P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G-6-P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. On the basis of these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation.

The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab 13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab 17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of P-32 from [beta-P-22]UDP-glucose to glycogen by glycogen synthase. The P-32 associated with the glycogen fraction was stable to ethanol precipitation, SOS-PAGE and gel filtration on Sephadex G50. The P-32-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [beta-P-32]UDP generated in the reaction. Furthermore, [beta-P-32]UDP did not bind non-covalently to glycogen. The P-32 associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation. (C) 2016 Elsevier Inc. All rights reserved.

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 AMPK gamma 3 subunit (AMPK gamma 3(R200Q)); this may decrease the glycogen storage capacity of AMPK gamma 3(R200Q) + RyR1(R615C) muscle. Therefore, our aim in this study was to utilize our pig model to understand how AMPK gamma 3(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 AMPK gamma 3(R200Q), RyR1(R615C), and AMPK gamma 3(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), AMPK gamma 3(R200Q) increased the glycogen content by approximately 70%. Activity of glycogen synthase (GS) without the allosteric activator glucose 6-phosphate (G6P) was decreased in AMPK gamma 3(R200Q) relative to all other genotypes, whereas both AMPK gamma 3(R200Q) and AMPK gamma 3(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, AMPK gamma 3(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 AMPK gamma 3(R200Q) mutation than activity.