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One exercise session can increase subsequent insulin-stimulated glucose uptake (ISGU) by skeletal muscle. Postexercise refeeding induces reversal of postexercise (PEX)-enhanced ISGU concomitant with attaining high muscle glycogen in rats. To test the relationship between high glycogen and reversal of PEX-ISGU, we injected one epitrochlearis muscle from each rat with adeno-associated virus (AAV) small hairpin RNA (shRNA) that targets glycogen synthase 1 (GS1) and injected contralateral muscles with AAV-shRNA-Scrambled (Scr). Muscles from PEX and sedentary rats were collected at 3-hour PEX (3hPEX) or 6-hour PEX (6hPEX). Rats were either not refed or refed rat-chow during the recovery period. Isolated muscles were incubated with [ 3 H]-3-O-methylglucose, with or without insulin. The results revealed: (1) GS1 abundance was substantially lower for AAV-shRNA-GS1-treated versus AAV-shRNA-Scr-treated muscles; (2) reduced GS1 abundance in refed-rats induced much lower glycogen in AAV-shRNA-GS1-treated versus AAV-shRNA-Scr-treated muscles at 3hPEX or 6hPEX; (3) PEX-ISGU was elevated in not refed-rats at either 3hPEX or 6hPEX versus sedentary controls, regardless of GS1 abundance; (4) PEX-ISGU was not reversed by 3 h of refeeding, regardless of GS1 abundance; (5) despite substantially lower glycogen in AAV-shRNA-GS1-treated versus AAV-shRNA-Scr-treated muscles, elevated PEX-ISGU was eliminated at 6hPEX in both of the paired muscles of refed-rats; and (6) 3hPEX versus sedentary non-refed rats had greater AMP-activated protein kinase-γ3 activity in both paired muscles, but this exercise effect was eliminated in both paired muscles by 3 h of refeeding. In conclusion, the results provided compelling evidence that the reversal of exercise-enhanced ISGU by refeeding was not attributable to the accumulation of high muscle glycogen concentration., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Glycogen, a branched polysaccharide organized into glycogen granules (GGs), is delivered from the cytoplasm to the lysosomes of hepatocytes by STBD1-driven selective autophagy (glycophagy). Recently, we developed Komagataella phaffii yeast as a simple model of GG autophagy and found that it proceeds non-selectively under nitrogen starvation conditions. However, another group, using Saccharomyces cerevisiae as a model, found that glycogen is a non-preferred cargo of nitrogen starvation-induced bulk autophagy. To clarify cargo characteristics of K. phaffii GGs, we used the same glycogen synthase-based reporter (Gsy1-GFP) of GG autophagy in K. phaffii as was used in S. cerevisiae . The K. phaffii Gsy1-GFP marked the GGs and reported on their autophagic degradation during nitrogen starvation, as expected. However, unlike in S. cerevisiae , glycogen synthase-marked GGs were delivered to the vacuole and degraded there with the same efficiency as a cytosolic glycogen synthase in glycogen-deficient cells, suggesting that glycogen is a neutral cargo of bulk autophagy in K. phaffii . We verified our findings with a new set of reporters based on the glycogen-binding CBM20 domain of human STBD1. The GFP-CBM20 and mCherry-CBM20 fusion proteins tagged GGs, reported about the autophagy of GGs, and confirmed that GGs in K. phaffii are neither preferred nor non-preferred substrates of bulk autophagy. They are its neutral substrates.

To adapt to various host microenvironments, the human fungal pathogen Candida albicans possesses the capacity to accumulate and store glycogen as an internal carbohydrate source. In the model yeast Saccharomyces cerevisiae , Sc Glc7p and Sc Gac1p are the serine/threonine type 1 protein phosphatase catalytic and regulatory subunits that control glycogen synthesis by altering the phosphorylation state of the glycogen synthase Gsy2p. Despite recent delineation of the glycogen synthesis pathway in C. albicans , the molecular events driving synthase activation are currently undefined. In this study, using a combination of microbiologic and genetic techniques, we determined that the protein encoded by uncharacterized gene C1_01140C , and not the currently annotated C. albicans Gac1p, is the major regulatory subunit involved in glycogen synthesis. C1_01140Cp contains a conserved GVNK motif observed across multiple starch/glycogen-binding proteins in various species, and alanine substitution of each residue in this motif significantly impaired glycogen accumulation in C. albicans . Fluorescent protein tagging and microscopy indicated that C1_01140Cp-GFPy colocalized with Ca Glc7p-tdTomato and Ca Gsy1p-tdTomato accordingly. Co-immunoprecipitation assays further confirmed that C1_01140Cp associates with Ca Glc7p and Ca Gsy1p during glycogen synthesis. Lastly, c1_01140c Δ/Δ exhibited colonization defects in a murine model of vulvovaginal candidiasis. Collectively, our data indicate that uncharacterized C1_01140Cp is the functional ortholog of the PPP1R subunit Sc Gac1p in C. albicans .IMPORTANCEThe capacity to synthesize glycogen offers microbes metabolic flexibility, including the fungal pathogen Candida albicans . In Saccharomyces cerevisiae , dephosphorylation of glycogen synthase by the Sc Glc7p-containing phosphatase is a critical rate-limiting step in glycogen synthesis. Subunits, including Sc Gac1p, target Sc Glc7p to α-1,4-glucosyl primers for efficient Sc Gsy2p synthase activation. However, this process in C. albicans had not been delineated. Here, we show that the C. albicans genome encodes for two homologous phosphatase-binding subunits, annotated Ca Gac1p and uncharacterized C1_01140Cp, both containing a GVNK motif required for polysaccharide affinity. Surprisingly, loss of Ca Gac1p only moderately reduced glycogen accumulation, whereas loss of C1_01140Cp ablated it. Fluorescence microscopy and co-immunoprecipitation approaches revealed that C1_01140Cp associates with Ca Glc7p and Ca Gsy1p during glycogen synthesis. Moreover, C1_01140Cp contributed to fungal fitness at the vaginal mucosa during murine vaginitis. Therefore, this work demonstrates that glycogen synthase regulation is conserved in C. albicans and C1_01140Cp is the functional ortholog of Sc Gac1p., Competing Interests: The authors declare no conflict of interest.

Glycogen metabolism is an important biological process for organisms. In Caenorhabditis elegans, effect of 6-PPD quinone (6-PPDQ) on glycogen accumulation and underlying mechanism were examined. Exposure to 6-PPDQ (1 and 10 μg/L) increased glycogen accumulation. Meanwhile, exposure to 6-PPDQ (1 and 10 μg/L) increased expression of gsy-1 encoding glycogen synthase and decreased expression of pygl-1 encoding glycogen phosphorylase. In 6-PPDQ exposed animals, glycogen content and glycogen accumulation were inhibited by RNAi of gsy-1 and enhanced by RNAi of pygl-1. RNAi of gsy-1 increased pygl-1 expression, and RNAi of pygl-1 increased gsy-1 expression after 6-PPDQ exposure. In 6-PPDQ exposed nematodes, daf-16 and aak-2 expressions were decreased and glycogen accumulation was suppressed by RNAi of daf-16 and aak-2, suggesting alteration in daf-16 and aak-2 expressions did not mediate glycogen accumulation. Moreover, resistance to 6-PPDQ toxicity on locomotion and brood size was observed in gsy-1(RNAi) animals, and susceptibility to 6-PPDQ toxicity was found in pygl-1(RNAi) animals. Therefore, glycogen accumulation could be enhanced by exposure to 6-PPDQ in nematodes. In addition, alteration in expressions of gsy-1 and pygl-1 governing this enhancement in glycogen accumulation mediated induction of 6-PPDQ toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Astrocyte glycogenolysis shapes ventromedial hypothalamic nucleus (VMN) regulation of glucostasis in vivo. Glucose transporter-2 (GLUT2), a plasma membrane glucose sensor, controls hypothalamic primary astrocyte culture glycogen metabolism in vitro. In vivo gene silencing tools and single-cell laser-catapult-microdissection/multiplex qPCR techniques were used here to examine whether GLUT2 governs dorsomedial (VMNdm) and/or ventrolateral (VMNvl) VMN astrocyte metabolic sensor and glycogen metabolic enzyme gene profiles. GLUT2 gene knockdown diminished astrocyte GLUT2 mRNA in both VMN divisions. Hypoglycemia caused GLUT2 siRNA-reversible up-regulation of this gene profile in the VMNdm, but down-regulated VMNvl astrocyte GLUT2 transcription. GLUT2 augmented baseline VMNdm and VMNvl astrocyte glucokinase (GCK) gene expression, but increased (VMNdm) or reduced (VMNvl) GCK transcription during hypoglycemia. GLUT2 imposed opposite control, namely stimulation versus inhibition of VMNdm or VMNvl astrocyte 5'-AMP-activated protein kinase-alpha 1 and -alpha 2 gene expression, respectively. GLUT2 stimulated astrocyte glycogen synthase (GS) gene expression in each VMN division. GLUT2 inhibited transcription of the AMP-sensitive glycogen phosphorylase (GP) isoform GP-brain type (GPbb) in each site, yet diminished (VMNdm) or augmented (VMNvl) astrocyte GP-muscle type (GPmm) mRNA. GLUT2 enhanced VMNdm and VMNvl glycogen accumulation during euglycemia, and curbed hypoglycemia-associated VMNdm glycogen depletion. Results show that VMN astrocytes exhibit opposite, division-specific GLUT2 transcriptional responsiveness to hypoglycemia. Data document divergent GLUT2 control of GCK, AMPK catalytic subunit, and GPmm gene profiles in VMNdm versus VMNvl astrocytes. Ongoing studies seek to determine how differential GLUT2 regulation of glucose and energy sensor function and glycogenolysis in each VMN location may affect local neuron responses to hypoglycemia., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Glycogen is a form of energy storage for glucose in different tissues such as liver and skeletal muscle. It remains incompletely understood how glycogen impacts on adipose tissue functionality. Cold exposure elevated the expression of Gys1 that encodes glycogen synthase 1 in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT). The in vivo function of Gys1 was analyzed using a mouse model in which Gys1 was deleted specifically in adipose tissues. Under normal chow conditions, Gys1 deletion caused little changes to body weight and glucose metabolism. Deletion of Gys1 abrogated upregulation of UCP1 and other thermogenesis-related genes in iWAT upon prolonged cold exposure or treatment with β3-adrenergic receptor agonist CL-316,243. Stimulation of UCP1 by CL-316,243 in adipose-derived stromal cells (stromal vascular fractions, SVFs) was also reduced by Gys1 deletion. Both the basal glycogen content and CL-316,243-stimulated glycogen accumulation in adipose tissues were reduced by Gys1 deletion. High-fat diet-induced obesity and insulin resistance were aggravated in Gys1 -deleted mice. The loss of body weight upon CL-316,243 treatment was also abrogated by the loss of Gys1 . In conclusion, our results underscore the pivotal role of glycogen synthesis in adaptive thermogenesis in beige adipose tissue and its impact on diet-induced obesity in mice. NEW & NOTEWORTHY Glycogen is one of major types of fuel reserve in the body and its classical function is to maintain blood glucose level. This study uncovers that glycogen synthesis is required for beige fat tissue to generate heat upon cold exposure. Such a function of glycogen is linked to development of high-fat diet-induced obesity, thus extending our understanding about the physiological functions of glycogen.

Under normal physiological conditions, the mammalian brain contains very little glycogen, most of which is stored in astrocytes. However, the aging brain and the subareas of the brain in patients with neurodegenerative disorders tend to accumulate glycogen, the cause and significance of which remain largely unexplored. Using cellular models, we have recently demonstrated a neuroprotective role for neuronal glycogen and glycogen synthase in the context of Huntington's disease. To gain insight into the role of brain glycogen in regulating proteotoxicity, we utilized a Drosophila model of Huntington's disease, in which glycogen synthase is either knocked down or expressed ectopically. Enhancing glycogen synthesis in the brains of flies with Huntington's disease decreased mutant Huntingtin aggregation and reduced oxidative stress by activating auto-lysosomal functions. Further, overexpression of glycogen synthase in the brain rescues photoreceptor degeneration, improves locomotor deficits and increases fitness traits in this Huntington's disease model. We, thus, provide in vivo evidence for the neuroprotective functions of glycogen synthase and glycogen in neurodegenerative conditions, and their role in the neuronal autophagy process., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Patients with Lafora disease have a mutation in EPM2A or EPM2B, resulting in dysregulation of glycogen metabolism throughout the body and aberrant glycogen molecules that aggregate into Lafora bodies. Lafora bodies are particularly damaging in the brain, where the aggregation drives seizures with increasing severity and frequency, coupled with neurodegeneration. Previous work employed mouse genetic models to reduce glycogen synthesis by approximately 50%, and this strategy significantly reduced Lafora body formation and disease phenotypes. Therefore, an antisense oligonucleotide (ASO) was developed to reduce glycogen synthesis in the brain by targeting glycogen synthase 1 (Gys1). To test the distribution and efficacy of this drug, the Gys1-ASO was administered to Epm2b-/- mice via intracerebroventricular administration at 4, 7, and 10 months. The mice were then sacrificed at 13 months and their brains analyzed for Gys1 expression, glycogen aggregation, and neuronal excitability. The mice treated with Gys1-ASO exhibited decreased Gys1 protein levels, decreased glycogen aggregation, and reduced epileptiform discharges compared to untreated Epm2b-/- mice. This work provides proof of concept that a Gys1-ASO halts disease progression of EPM2B mutations of Lafora disease., (© 2023. The Author(s).)

Background: Carbamoyl phosphate synthetase I defect (CPS1D) is a rare disease with clinical case reports mainly in early neonates or adults, with few reports of first onset in late neonatal to childhood. We studied the clinical and genotypic characteristics of children with childhood onset CPS1D caused by two loci mutations (one of these is a rarely reported non-frame shift mutation) in the CPS1., Case Presentation: We present a rare case of adolescent-onset CPS1D that had been misdiagnosed due to atypical clinical features, and further investigations revealed severe hyperammonemia (287µmol/L; reference range 11.2 ~ 48.2umol/L). MRI of the brain showed diffuse white matter lesions. Blood genetic metabolic screening showed elevated blood alanine (757.06umol/L; reference range 148.8 ~ 739.74umol/L) and decreased blood citrulline (4.26umol/L; reference range 5.45 ~ 36.77umol/L). Urine metabolic screening showed normal whey acids and uracil. Whole-exome sequencing revealed compound heterozygous mutations in the CPS1, a missense mutation (c.1145 C > T) and an unreported de novo non-frame shift mutation (c.4080_c.4091delAGGCATCCTGAT), respectively, which provided a clinical diagnosis., Conclusion: A comprehensive description of the clinical and genetic features of this patient, who has a rare age of onset and a relatively atypical clinical presentation, will facilitate the early diagnosis and management of this type of late onset CPS1D and reduce misdiagnosis, thus helping to reduce mortality and improve prognosis. It also provides a preliminary understanding of the relationship between genotype and phenotype, based on a summary of previous studies, which reminds us that it may help to explore the pathogenesis of the disease and contribute to genetic counselling and prenatal diagnosis., (© 2023. The Author(s).)

Background: Hypoxia-induced glycogen turnover is implicated in cancer proliferation and therapy resistance. Triple-negative breast cancers (TNBCs), characterized by a hypoxic tumor microenvironment, respond poorly to therapy. We studied the expression of glycogen synthase 1 (GYS1), the key regulator of glycogenesis, and other glycogen-related enzymes in primary tumors of patients with breast cancer and evaluated the impact of GYS1 downregulation in preclinical models., Methods: mRNA expression of GYS1 and other glycogen-related enzymes in primary breast tumors and the correlation with patient survival were studied in the METABRIC dataset (n = 1904). Immunohistochemical staining of GYS1 and glycogen was performed on a tissue microarray of primary breast cancers (n = 337). In four breast cancer cell lines and a mouse xenograft model of triple-negative breast cancer, GYS1 was downregulated using small-interfering or stably expressed short-hairpin RNAs to study the effect of downregulation on breast cancer cell proliferation, glycogen content and sensitivity to various metabolically targeted drugs., Results: High GYS1 mRNA expression was associated with poor patient overall survival (HR 1.20, P = 0.009), especially in the TNBC subgroup (HR 1.52, P = 0.014). Immunohistochemical GYS1 expression in primary breast tumors was highest in TNBCs (median H-score 80, IQR 53-121) and other Ki67-high tumors (median H-score 85, IQR 57-124) (P < 0.0001). Knockdown of GYS1 impaired proliferation of breast cancer cells, depleted glycogen stores and delayed growth of MDA-MB-231 xenografts. Knockdown of GYS1 made breast cancer cells more vulnerable to inhibition of mitochondrial proteostasis., Conclusions: Our findings highlight GYS1 as potential therapeutic target in breast cancer, especially in TNBC and other highly proliferative subsets., (© 2023. The Author(s).)

Many lines of evidence demonstrate a correlation between liver glycogen content and food intake. We previously demonstrated that mice overexpressing protein targeting to glycogen (PTG) specifically in the liver-which have increased glycogen content in this organ-are protected from high-fat diet (HFD)-induced obesity by reduced food intake. However, the use of PTG to increase liver glycogen implies certain limitations. PTG stimulates glycogen synthesis but also inhibits the enzyme responsible for glycogen degradation. Furthermore, as PTG is a regulatory subunit of protein phosphatase 1 (PP1), which regulates many cellular functions, its overexpression could have side effects beyond the regulation of glycogen metabolism. Therefore, it is necessary to determine whether the direct activation of glycogen synthesis, without affecting its degradation or other cellular functions, has the same effects. To this end, we generated mice overexpressing a non-inactivatable form of glycogen synthase (GS) specifically in the liver (9A-MGS Alb mice). Control and 9a-MGS Alb mice were fed a standard diet (SD) or HFD for 16 weeks. Glucose tolerance and feeding behavior were analyzed. 9A-MGS Alb mice showed an increase in hepatic glycogen in fed and fasting conditions. When fed an HFD, these animals preserved their hepatic energy state, had a reduced food intake, and presented a lower body weight and fat mass than control animals, without changes in energy expenditure. Furthermore, 9A-MGS Alb animals showed improved glucose tolerance when fed an SD or HFD. Moreover, liver triacylglycerol levels that were increased after HFD feeding were lower in these mice. These results confirm that increased liver glycogen stores contribute to decreased appetite and improve glucose tolerance in mice fed an HFD. On the basis of our findings, strategies to preserve hepatic glycogen stores emerge as potential treatments for obesity and hyperglycemia.

Hepatic glycogen is the main source of blood glucose and controls the intervals between meals in mammals. Hepatic glycogen storage in mammalian pups is insufficient compared to their adult counterparts; however, the detailed molecular mechanism is poorly understood. Here, we show that, similar to glycogen storage pattern, N6-methyladenosine (m6A) modification in mRNAs gradually increases during the growth of mice in liver. Strikingly, in the hepatocyte-specific Mettl3 knockout mice, loss of m6A modification disrupts liver glycogen storage. On the mechanism, mRNA of Gys2, the liver-specific glycogen synthase, is a substrate of METTL3 and plays a critical role in m6A-mediated glycogenesis. Furthermore, IGF2BP2, a "reader" protein of m6A, stabilizes the mRNA of Gys2. More importantly, reconstitution of GYS2 almost rescues liver glycogenesis in Mettl3-cKO mice. Collectively, a METTL3-IGF2BP2-GYS2 axis, in which METTL3 and IGF2BP2 regulate glycogenesis as "writer" and "reader" proteins respectively, is essential on maintenance of liver glycogenesis in mammals., (© 2022. The Author(s).)

Motor neurons in the spinal cord are essential for movement. During the embryonic period, developing motor neurons store glycogen to protect against hypoglycemic and hypoxic stress. However, the mechanisms by which glycogen metabolism is regulated in motor neurons remain unclear. We herein investigated the transcriptional regulation of genes related to glycogen metabolism in the developing spinal cord. We focused on the regulatory mechanism of glycogen synthase (Gys1) and glycogen phosphorylase brain isoform (PygB), which play central roles in glycogen metabolism, and found that the transcription factor STAT3 regulated the expression of Gys1 and PygB via cis-regulatory promoter sequences in the developing spinal cord. These results suggest that STAT3 is important for the regulation of glycogen metabolism during motor neuron development., (© 2022 Federation of European Biochemical Societies.)

Grain size is a key agronomic trait that determines the yield in plants. Regulation of grain size by brassinosteroids (BRs) in rice has been widely reported. However, the relationship between the BR signaling pathway and grain size still requires further study. Here, we isolated a rice mutant, named small grain2 (sg2), which displayed smaller grain and a semi-dwarf phenotype. The decreased grain size was caused by repressed cell expansion in spikelet hulls of the sg2 mutant. Using map-based cloning combined with a MutMap approach, we cloned SG2, which encodes a plant-specific protein with a ribonuclease H-like domain. SG2 is a positive regulator downstream of GLYCOGEN SYNTHASE KINASE2 (GSK2) in response to BR signaling, and its mutation causes insensitivity to exogenous BR treatment. Genetical and biochemical analysis showed that GSK2 interacts with and phosphorylates SG2. We further found that BRs enhance the accumulation of SG2 in the nucleus, and subcellular distribution of SG2 is regulated by GSK2 kinase activity. In addition, Oryza sativa OVATE family protein 19 (OsOFP19), a negative regulator of grain shape, interacts with SG2 and plays an antagonistic role with SG2 in controlling gene expression and grain size. Our results indicated that SG2 is a new component of GSK2-related BR signaling response and regulates grain size by interacting with OsOFP19., (© 2022 Institute of Botany, Chinese Academy of Sciences.)

Longer glucan chains tend to precipitate. Glycogen, by far the largest mammalian glucan and the largest molecule in the cytosol with up to 55 000 glucoses, does not, due to a highly regularly branched spherical structure that allows it to be perfused with cytosol. Aberrant construction of glycogen leads it to precipitate, accumulate into polyglucosan bodies that resemble plant starch amylopectin and cause disease. This pathology, amylopectinosis, is caused by mutations in a series of single genes whose functions are under active study toward understanding the mechanisms of proper glycogen construction. Concurrently, we are characterizing the physicochemical particularities of glycogen and polyglucosans associated with each gene. These genes include GBE1, EPM2A and EPM2B, which respectively encode the glycogen branching enzyme, the glycogen phosphatase laforin and the laforin-interacting E3 ubiquitin ligase malin, for which an unequivocal function is not yet known. Mutations in GBE1 cause a motor neuron disease (adult polyglucosan body disease), and mutations in EPM2A or EPM2B a fatal progressive myoclonus epilepsy (Lafora disease). RBCK1 deficiency causes an amylopectinosis with fatal skeletal and cardiac myopathy (polyglucosan body myopathy 1, OMIM# 615895). RBCK1 is a component of the linear ubiquitin chain assembly complex, with unique functions including generating linear ubiquitin chains and ubiquitinating hydroxyl (versus canonical amine) residues, including of glycogen. In a mouse model we now show (i) that the amylopectinosis of RBCK1 deficiency, like in adult polyglucosan body disease and Lafora disease, affects the brain; (ii) that RBCK1 deficiency glycogen, like in adult polyglucosan body disease and Lafora disease, has overlong branches; (iii) that unlike adult polyglucosan body disease but like Lafora disease, RBCK1 deficiency glycogen is hyperphosphorylated; and finally (iv) that unlike laforin-deficient Lafora disease but like malin-deficient Lafora disease, RBCK1 deficiency's glycogen hyperphosphorylation is limited to precipitated polyglucosans. In summary, the fundamental glycogen pathology of RBCK1 deficiency recapitulates that of malin-deficient Lafora disease. Additionally, we uncover sex and genetic background effects in RBCK1 deficiency on organ- and brain-region specific amylopectinoses, and in the brain on consequent neuroinflammation and behavioural deficits. Finally, we exploit the portion of the basic glycogen pathology that is common to adult polyglucosan body disease, both forms of Lafora disease and RBCK1 deficiency, namely overlong branches, to show that a unified approach based on downregulating glycogen synthase, the enzyme that elongates glycogen branches, can rescue all four diseases., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glycogen is the major glucose reserve in eukaryotes, and defects in glycogen metabolism and structure lead to disease. Glycogenesis involves interaction of glycogenin (GN) with glycogen synthase (GS), where GS is activated by glucose-6-phosphate (G6P) and inactivated by phosphorylation. We describe the 2.6 Å resolution cryo-EM structure of phosphorylated human GS revealing an autoinhibited GS tetramer flanked by two GN dimers. Phosphorylated N- and C-termini from two GS protomers converge near the G6P-binding pocket and buttress against GS regulatory helices. This keeps GS in an inactive conformation mediated by phospho-Ser641 interactions with a composite "arginine cradle". Structure-guided mutagenesis perturbing interactions with phosphorylated tails led to increased basal/unstimulated GS activity. We propose that multivalent phosphorylation supports GS autoinhibition through interactions from a dynamic "spike" region, allowing a tuneable rheostat for regulating GS activity. This work therefore provides insights into glycogen synthesis regulation and facilitates studies of glycogen-related diseases., (© 2022. The Author(s).)

Adult polyglucosan body disease (APBD) and Lafora disease (LD) are autosomal recessive glycogen storage neurological disorders. APBD is caused by mutations in the glycogen branching enzyme (GBE1) gene and is characterized by progressive upper and lower motor neuron dysfunction and premature death. LD is a fatal progressive myoclonus epilepsy caused by loss of function mutations in the EPM2A or EPM2B gene. These clinically distinct neurogenetic diseases share a common pathology. This consists of time-dependent formation, precipitation, and accumulation of an abnormal form of glycogen (polyglucosan) into gradually enlarging inclusions, polyglucosan bodies (PBs) in ever-increasing numbers of neurons and astrocytes. The growth and spread of PBs are followed by astrogliosis, microgliosis, and neurodegeneration. The key defect in polyglucosans is that their glucan branches are longer than those of normal glycogen, which prevents them from remaining in solution. Since the lengths of glycogen branches are determined by the enzyme glycogen synthase, we hypothesized that downregulating this enzyme could prevent or hinder the generation of the pathogenic PBs. Here, we pursued an adeno-associated virus vector (AAV) mediated RNA-interference (RNAi) strategy. This approach resulted in approximately 15% reduction of glycogen synthase mRNA and an approximately 40% reduction of PBs across the brain in the APBD and both LD mouse models. This was accompanied by improvements in early neuroinflammatory markers of disease. This work represents proof of principle toward developing a single lifetime dose therapy for two fatal neurological diseases: APBD and LD. The approach is likely applicable to other severe and common diseases of glycogen storage., (© 2022. The Author(s).)

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver tumor with increasing incidence worldwide. Metabolic reprogramming caused by metabolic related gene disorders is a prominent hallmark of tumors, among which Glycogen Synthase 2 (GYS2) is the key gene responsible for regulating cellular energy metabolism, and its expression disorders are closely related to various tumors and glycometabolic diseases. However, we still know nothing about its role in ICC. This study is intended to reveal the functional role of GYS2 in the ICC progress and explore the underlying mechanism. Based on the integrated pan-cancer analysis of GYS2 in the GEPIA database, the expression of GYS2 in paired ICC and adjacent non tumor tissues was detected by qPCR. It was found that the expression of GYS2 was significantly down-regulated in ICC. Further analysis showed that its low expression was not only associated with the degree of pathological differentiation, tumor size, microvascular invasion and lymph node metastasis, but also an independent risk factor for unfavorable prognosis. Functional studies have shown that GYS2 overexpression can significantly impair the proliferation, replication, cloning, migration and invasion of cholangiocarcinoma cells, while the silencing GYS2 dramatically promotes the development of the aforementioned phenotypes, the underlying mechanism may be that GYS2 activates the P53 pathway. In conclusions,low GYS2 expression in ICC predicted unfavorable patient outcomes; GYS2 overexpression could significantly impair the proliferation, migration and invasion of cholangiocarcinoma cells via activating the P53 pathway and GYS2 was expected to become a potential therapeutic target for such patients.

The hallmark of Lafora disease, a fatal neurodegenerative disorder, is the accumulation of intracellular glycogen aggregates called Lafora bodies. Until recently, it was widely believed that brain Lafora bodies were present exclusively in neurons and thus that Lafora disease pathology derived from their accumulation in this cell population. However, recent evidence indicates that Lafora bodies are also present in astrocytes. To define the role of astrocytic Lafora bodies in Lafora disease pathology, we deleted glycogen synthase specifically from astrocytes in a mouse model of the disease (malinKO). Strikingly, blocking glycogen synthesis in astrocytes-thus impeding Lafora bodies accumulation in this cell type-prevented the increase in neurodegeneration markers, autophagy impairment, and metabolic changes characteristic of the malinKO model. Conversely, mice that over-accumulate glycogen in astrocytes showed an increase in these markers. These results unveil the deleterious consequences of the deregulation of glycogen metabolism in astrocytes and change the perspective that Lafora disease is caused solely by alterations in neurons., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Glycosylation defects are a hallmark of many nervous system diseases. However, the molecular and metabolic basis for this pathology is not fully understood. In this study, we found that N-linked protein glycosylation in the brain is metabolically channeled to glucosamine metabolism through glycogenolysis. We discovered that glucosamine is an abundant constituent of brain glycogen, which functions as a glucosamine reservoir for multiple glycoconjugates. We demonstrated the enzymatic incorporation of glucosamine into glycogen by glycogen synthase, and the release by glycogen phosphorylase by biochemical and structural methodologies, in primary astrocytes, and in vivo by isotopic tracing and mass spectrometry. Using two mouse models of glycogen storage diseases, we showed that disruption of brain glycogen metabolism causes global decreases in free pools of UDP-N-acetylglucosamine and N-linked protein glycosylation. These findings revealed fundamental biological roles of brain glycogen in protein glycosylation with direct relevance to multiple human diseases of the central nervous system., Competing Interests: Declaration of interests M.S.G. is a consultant for Maze Therapeutics, Enable Therapeutics, Glut1-Deficiency Syndrome Foundation, and Chelsea's Hope. M.S.G., R.C.S., C.W.V.K., and R.C.B. are founders of Atterogen, LLC., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Caralluma tuberculata (C. tuberculata) has traditionally been used in Pakistan and other parts of the world as a folk treatment for diabetes mellitus. A few studies indicated its antihyperglycemic effect, however, the mystery remained unfolded as how did it modify the pathophysiological condition., Aim of Study: Hence, this study aimed to explore underlying mechanism(s) for its hypoglycemic activity at biochemical and molecular levels., Materials and Methods: Methanol extract (ME) of C. tuberculata as well as its hexane (HF) and aqueous (AF) fractions were explored for their effect on total glycogen in liver and skeletal muscle of alloxan-induced rats by spectroscopy. Moreover, the expression of genes related to hepatic carbohydrate metabolizing enzymes was quantified. At molecular level, mRNA expression of glucose transporter 2 (GLUT-2), glycogen synthase (GS), glucokinase (GK), hexokinase 1 (HK-1), pyruvate kinase (PK), glucose 6 phosphate dehydrogenase (G-6-PDH), pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6 phosphatase (G-6-Pase) was determined by using quantitative real time polymerase chain reaction (qRT-PCR) after administration of ME (350 mg), HF(3 mg), AF (10 mg) and metformin (500 mg). The doses were administered twice daily according to per kg of body weight., Results: A significant reduction in hepatic and skeletal muscle glycogen content was exhibited. The data of qRT-PCR revealed that gene's expression of GLUT-2 was significantly decreased after treatment with ME and HF, whilst it was unaltered by AF, however, a significant decrease was observed in genes corresponding to GS, GK and HK-1 after treatment with ME. Similarly, there was a significant decrease in expression of genes corresponding to GS, GK and HK-1 following treatment with HF. Surprisingly, post-treatment with AF didn't modify the gene's expression of GS and GK, whilst it caused a profound decrease in expression of HK-1 gene. Contrarily, the expression of gene related to PK was significantly up-regulated post-administration with ME, HF and AF. The expression levels of G-6-PDH, however, remained unaltered after treatment with the experimental extract and fractions of the plant. In addition, HF and AF did not cause any modification in PEPCK, whereas ME caused a significant down-regulation of the gene. Treatment with all the extract and fractions of the plant caused a substantial decrease in the gene's expression of PC, while there was a significant increase in the expression of gene related to G-6-Pase., Conclusion: The three experimental extract and fractions caused a substantial decrease in glycogen content in liver and skeletal muscle tissues. The analysis by qRT-PCR showed that glucose transport via GLUT-2 was profoundly declined by ME and HF. The expression of genes related to various metabolic pathways involved in metabolism of carbohydrate in hepatocytes revealed explicitly that the ME, HF and AF decreased the phenomena of glycogenesis and gluconeogenesis. Contrarily, all the extract and fractions of the plant activated glycogenolysis and glycolysis but did not modify the pentose phosphate shunt pathway., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Many adult and most childhood neurological diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency, and specificity hurdles. We applied CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric disease. Adult polyglucosan body disease is a neurodegenerative disease resembling amyotrophic lateral sclerosis. Lafora disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene, Gys1, responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body disease and two mouse models of Lafora disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, abnormal glycogen accumulation, and polyglucosan bodies, as well as ameliorations of neuroinflammatory markers in all three models. Our work represents proof of principle for virally delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological diseases., (© 2021. The American Society for Experimental NeuroTherapeutics, Inc.)

Lafora disease (LD) is a fatal adolescence-onset neurodegenerative condition. The hallmark of LD is the accumulation of aberrant glycogen aggregates called Lafora bodies (LBs) in the brain and other tissues. Impeding glycogen synthesis from early embryonic stages by genetic suppression of glycogen synthase (MGS) in an animal model of LD prevents LB formation and ultimately the pathological manifestations of LD thereby indicating that LBs are responsible for the pathophysiology of the disease. However, it is not clear whether eliminating glycogen synthesis in an adult animal after LBs have already formed would halt or reverse the progression of LD. Herein we generated a mouse model of LD with inducible MGS suppression. We evaluated the effect of MGS suppression at different time points on LB accumulation as well as on the appearance of neuroinflammation, a pathologic trait of LD models. In the skeletal muscle, MGS suppression in adult LD mice blocked the formation of new LBs and reduced the number of glycogen aggregates. In the brain, early but not late MGS suppression halted the accumulation of LBs. However, the neuroinflammatory response was still present, as shown by the levels of reactive astrocytes, microglia and inflammatory cytokines. Our results confirm that MGS as a promising therapeutic target for LD and highlight the importance of an early diagnosis for effective treatment of the disease., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Differential diagnosis of hypoglycemic syndrome remains an urgent problem in Pediatrics. In this article, a case of glycogen storage disease (BNG) type 0 is described in the boy, which is undoubtedly a rare pathology, which makes it difficult to diagnose this form of glycogenosis. In this description, the case of type 0 BNG is caused by a mutation in the GYS2 gene encoding the hepatic isoform of glycogen synthase. This form of the disease is usually asymptomatic in infancy. However, it can be suspected in the case when the refusal of night feeding causes certain difficulties due to the hungry ketotic hypoglycemia that occurs in the child. The first clinical symptoms appear in early childhood with the inclusion of a more varied diet in the child, increasing the intervals between meals. Symptoms of the disease are primarily caused by hypoglycemia. It should be noted that, despite the low levels of glycemia, most children do not have any mental development disorders, since the increase in the level of ketone bodies in the blood plasma provides the brain with an alternative source of energy.

Glycogen storage disease type 0 (GSD0) is an autosomal recessive disorder caused by a sequence variant in the GYS2 gene, leading to decreased or absent activity of hepatic glycogen synthase. With a frequency of less than 1 in 1,000,000 individuals, GSD0 represents only around 1% of all glycogen storage disease cases but it might be underrecognized. A 13 - month - old girl of reportedly unrelated parents presented with a decreased level of consciousness, twitching in her left cheek, and munching. During a fasting test, hyperketotic hypoglycemia was found. A novel homozygous GYS2 gene sequence variant p.Thr445Arg was later confirmed by next-generation gene sequencing. After establishing a cornstarch- and protein-rich diet, the hypoglycemic episodes subsided and the patient's neurocognitive development was normal. To date, only 39 patients with 24 disease-causing gene variants have been identified in GSD0, and we review their characteristics. Because of the heterogeneous phenotypes, GSD0 is an underdiagnosed disorder. In patients with hyperketotic hypoglycemia and postprandial hyperglycemia, GYS2 gene analysis should be performed.

Photosynthetic production of ethanol with cyanobacteria utilizing CO 2 and solar energy could serve as a promising route for providing green biofuels. Comparing with Synechocystis sp. PCC6803, the most widely utilized cyanobacteria chassis, a marine cyanobacterium Synechococcus sp. PCC7002 (hereafter termed as PCC7002 for short) displayed better tolerances to high temperature and strong illuminations. To engineer ethanol production in PCC7002, we designed a strategy by simultaneous inactivation of glycogen synthesis pathways and introduction of two ethanolgenic cassettes. Two glgA genes responsible for elongation reaction of glycogen was selected as targets for blocking glycogen synthesis, and the removal of glycogen synthesis in PCC7002 caused growth retardation, which could be rescued by introducing two copies of ethanolgenic pathways. In addition, the synergy effects of blocked glycogen synthesis and gene dosage effects for ethanol synthesis resulted in significantly improved performances for ethanol production in the engineered strain, which synthesized 2.2 g/L ethanol in 10 days. In outdoor conditions, the PCC7002 derived cell factory still maintained considerable ethanolgenic capacities, accumulating 0.8 g/L ethanol in 7 days, with an average productivity of over 100 mg/L/day., (Copyright © 2020 Elsevier B.V. All rights reserved.)

While glycogen synthase A deficiency can reduce the growth and proliferation of Chlamydia muridarum, the effect of glycogen synthase A on the pathogenic process of C. muridarum remains unclear. To characterize the effect of glycogen synthase A deficiency on the pathogenicity of C. muridarum in the genital tract, BALB/c mice were intravaginally inoculated with wild-type, plasmid-free and glycogen synthase A-deficient C. muridarum, and the genital tract tissue was isolated to assess the severity of hydrosalpinx and the levels of oviduct dilatation at day 60 after infection. The glycogen storage capacity and in vitro infection ability of different C. muridarum strains were analyzed by periodic acid-Schiff staining and quantification of progeny elementary body(EB) formation. The tissue homogenate was used to determine the recovery of different C. muridarum strains. The results show that glycogen synthase A-deficient C. muridarum induced reduction of hydrosalpinx and attenuated the extent of oviduct dilatation in mice, and exhibited reduced growth and proliferation in the mouse lower genital tract. In addition, glycogen synthase A point mutations at different sites reduced the glycogen storage capacity and in vitro infectivity of C. muridarum to different degrees. Glycogen synthase A deficiency also reduced the host inflammatory reaction and ascending infection of C. muridarum., (© FEMS 2020.)

Myokines, such as irisin, have been purported to exert physiological effects on skeletal muscle in an autocrine/paracrine fashion. In this study, we aimed to investigate the mechanistic role of in vivo fibronectin type III domain-containing 5 (Fndc5)/irisin upregulation in muscle. Overexpression (OE) of Fndc5 in rat hindlimb muscle was achieved by in vivo electrotransfer, i.e., bilateral injections of Fndc5 harboring vectors for OE rats ( n  = 8) and empty vector for control rats ( n  = 8). Seven days later, a bolus of D 2 O (7.2 mL/kg) was administered via oral gavage to quantify muscle protein synthesis. After an overnight fast, on day 9 , 2-deoxy-d-glucose-6-phosphate (2-DG6P; 6 mg/kg) was provided during an intraperitoneal glucose tolerance test (2 g/kg) to assess glucose handling. Animals were euthanized, musculus tibialis cranialis muscles and subcutaneous fat (inguinal) were harvested, and metabolic and molecular effects were evaluated. Muscle Fndc5 mRNA increased with OE (~2-fold; P  = 0.014), leading to increased circulating irisin (1.5 ± 0.9 to 3.5 ± 1.2 ng/mL; P  = 0.049). OE had no effect on protein anabolism or mitochondrial biogenesis; however, muscle glycogen was increased, along with glycogen synthase 1 gene expression ( P  = 0.04 and 0.02, respectively). In addition to an increase in glycogen synthase activation in OE ( P  = 0.03), there was a tendency toward increased glucose transporter 4 protein ( P  = 0.09). However, glucose uptake (accumulation of 2-DG6P) was identical. Irisin elicited no endocrine effect on mitochondrial biogenesis or uncoupling proteins in white adipose tissue. Hindlimb overexpression led to physiological increases in Fndc5/irisin. However, our data indicate limited short-term impacts of irisin in relation to muscle anabolism, mitochondrial biogenesis, glucose uptake, or adipose remodeling.

Impairment of glucose (Glu) uptake and storage by skeletal muscle is a prime risk factor for the development of metabolic diseases. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a highly abundant RNA-binding protein that has been implicated in diverse cellular functions. The aim of this study was to investigate the function of hnRNP A1 on muscle tissue insulin sensitivity and systemic Glu homeostasis. Our results showed that conditional deletion of hnRNP A1 in the muscle gave rise to a severe insulin resistance phenotype in mice fed a high-fat diet (HFD). Conditional knockout mice fed a HFD showed exacerbated obesity, insulin resistance, and hepatic steatosis. In vitro interference of hnRNP A1 in C2C12 myotubes impaired insulin signal transduction and inhibited Glu uptake, whereas hnRNP A1 overexpression in C2C12 myotubes protected against insulin resistance induced by supraphysiological concentrations of insulin. The expression and stability of glycogen synthase (gys1) mRNA were also decreased in the absence of hnRNP A1. Mechanistically, hnRNP A1 interacted with gys1 and stabilized its mRNA, thereby promoting glycogen synthesis and maintaining the insulin sensitivity in muscle tissue. Taken together, our findings are the first to show that reduced expression of hnRNP A1 in skeletal muscle affects the metabolic properties and systemic insulin sensitivity by inhibiting glycogen synthesis., (© The Author(s) (2019). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

Chlamydia trachomatis (C. trachomatis) is the leading cause of bacterial sexually transmitted diseases and infectious diseases that cause blindness. The pathophysiology of chlamydial infections is poorly understood, but secreted proteins have emerged as key virulence factors. C. trachomatis glycogen synthase (GlgA) is a chlamydial secretory protein, which localizes in the lumen of chlamydial inclusion bodies and the cytosol of host cells. In order to improve understanding of the roles of GlgA in chlamydial pathogenesis, four proteins that interact with GlgA, Homo sapiens CXXC finger protein 1, prohibitin (PHB), gelsolin‑like actin‑capping protein and apolipoprotein A‑I binding protein were identified using yeast two‑hybrid assays. The functions of these proteins are complex, and preliminary results suggested that PHB interacts with GlgA. However, further studies are required to determine the specific interactions of these proteins with GlgA. The findings of the present study may provide a direction and foundation for future studies focusing on the mechanism of GlgA in C. trachomatis infection.

Nilaparvata lugens is a serious threat to rice growth. Glycogen metabolism is one of the important physiological processes of insects, which is mainly regulated by glycogen synthase (GS) and glycogen phosphorylase (GP). In the present study, trehalose content was significantly reduced at 72 h after NlGP and NlGS knockdown, whereas glucose content was significantly increased at both 48 h and 72 h after GS knockdown. RNAi combined with RNA-Seq was used to identify NlGP- and NlGS-related pathways and genes in N. lugens. A total of 593 genes were up-regulated and 5969 genes were down-regulated after NlGP and NlGS knockdown, respectively. Moreover, the NlGS-knockdown group was mapped to 10,967 pathways, whereas the NlGP-knockdown group was mapped to 7948 pathways, and the greatest differences between the groups were associated with carbohydrate, lipid, amino acid and energy metabolism. Meanwhile, 1800, 1217, and 1211 transcripts in the NlGP-knockdown group and 2511, 1666, and 1727 transcripts in the NlGS-knockdown group were involved in bioprocess, cellular ingredients and molecular function, respectively. Almost all these genes were down-regulated by either NlGP or NlGS knockdown, with significant down-regulation of the 6-trehalose phosphate synthase (TPS), trehalase (TRE), GS, GP, phosphoacetylglucosamine mutase (PGM, n = 2), Insulin receptors (InRs) and insulin-like peptides (Ilps) genes. These results have demonstrated that RNAi-mediated NlGP and NlGS knockdown could lead to content of trehalose and glucose out of balance, but have no obvious effect on glycogen content, and have suggested that GS plays more complex role in other metabolism pathway of N. lugens., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: In this study, we aimed to investigate the pathologic and ultrastructural changes in transplanted mouse livers after different durations of cold storage by testing indicators of liver function and energy metabolism. We aimed to describe the effects of cold storage on liver function and the mechanisms of cold storage damage., Materials and Methods: We randomly placed 8-weekold male C57BL/6 mice into the following 4 groups to establish a cold-preserved mouse model of liver transplant: a normal control group and 3 cold storage groups, in which livers were stored for 4, 12, and 24 hours. Hepatic morphology, ultrastructural changes, and glycogenolysis were observed by hematoxylin and eosin staining, periodic acid-Schiff staining, and transmission electron microscopy. After different durations of cold storage, livers were reperfused with 4°C University of Wisconsin solution to obtain perfusion fluid, and alanine and aspartate aminotransferase levels were measured. Glycogen synthase, hypoxia-inducible factor-1α, Krüppel-like factor 2, and endothelial nitric oxide synthase mRNA expression levels in liver tissues were detected by real-time polymerase chain reaction, and aquaporin 8 protein expression levels in liver tissues were detected by Western blot., Results: Hematoxylin and eosin staining and electron microscopy ofliver showed signs ofinjury after 12 hours of cold storage, which included mainly cytoplasmic edema characterized by loose liver cell arrangement, increased hepatic sinus fissure, mitochondrial swelling, and nuclear pyknosis. Periodic acid-Schiff staining showed that glycogen content was significantly reduced, with glycogen synthase levels also reduced. Alanine aminotransferase and aspartate aminotransferase levels gradually increasedwith cold storage. Glycogen synthase, Krüppel-like factor 2, endothelial nitric oxide synthase, and aquaporin 8 expression levels also gradually increased in liver tissue. These levels gradually decreased, but hypoxia-inducible factor-1α increased., Conclusions: Mouse livers showed progressive damage to structure and function during cold storage, with mitochondrial damage perhaps showing the earliest damage.

During diapause in mosquitoes, efficient storage and utilization of energy are crucial for surviving prolonged periods of developmental arrest and for maximizing reproductive success once diapause is terminated and development recommences. In Culex pipiens, glycogen rapidly accumulates during early diapause (7-10 days after adult eclosion) and it is used to maintain energy homeostasis during the first month of diapause. In this study, a gene encoding glycogen synthase, which converts glucose residues into a polymeric chain for storage as glycogen, was characterized. After dsi-RNA directed against glycogen synthase was injected into mosquitoes programmed for diapause (reared under short day lengths), Cx. pipiens were fed 1% d-[ 13 C 6 ]glucose, and the knockdown effects after 7-days were monitored by measuring 13 C-labeled carbohydrate accumulation using solid-state NMR. The use of 13 C cross-polarization magic-angle spinning spectrum showed a 46% reduction of 13 C-labeled glycogen and a 6% reduction in lipid accumulation in glycogen synthase knockdown adult females. In addition, the suppression of glycogen synthase dramatically increased the mortality rate of diapausing Cx. pipiens by 88% at 30-days post injection. These findings indicated that glycogen synthase plays a critical role in regulating glycogen and lipid storages during overwintering diapause, and its function is essential for successful overwintering and survival of Cx. pipiens., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Portal hypertension is the primary cause of complications in patients with chronic liver diseases, and markedly impacts metabolism within the nervous system. Until recently, the role of portal hypertension in hepatocellular metabolism was unclear. The present study demonstrated that an increase in extracellular pressure significantly decreased hepatocellular glycogen concentrations in HepG2 and HL‑7702 cells. In addition, it reduced glycogen synthase activity, by inhibiting the phosphorylation of glycogen synthase 1. RNA‑seq analysis revealed that mechanical pressure suppressed glycogen synthesis by activating the p53/phosphatase and tensin homolog pathway, further suppressing glycogen synthase activity. The present study revealed an association between mechanical pressure and hepatocellular glycogen metabolism, and identified the regulatory mechanism of glycogen synthesis under pressure.

This study aimed at investigating the structure, hypoglycemic activity and the underlying mechanism of a homogeneous polysaccharide (PSP-2) purified from Sargassum pallidum. Structural characterization revealed that PSP-2 with a molecular weight of 144.8 kDa was composed of fucose (21.6%), arabinose (2.5%), galactose (22.4%), glucose (2.2%), xylose (18.8%), mannose (1.2%), glucuronic acid (7.7%) and galacturonic acid (23.6%). The backbone chain of PSP-2 was composed of →1)-β-d-Xylp-(3→, →1,3)-β-l-Fucp-(4→, →1)-α-d-Galp-(6→, and →1)-α-d-GlcpNAc-(2→, and the side chains were composed of →1,3,6)-α-d-Galp-(2→, →3)-β-l-Fucp-(1,4→, β-d-GalpNAc-(1→, and α-d-Manp-(1→. In vitro hypoglycemic assays indicated that PSP-2 could significantly enhance glucose consumption, glycogen synthesis, and pyruvate kinase (PK) and hexokinase (HK) activities of insulin-resistant HepG2 cells. Furthermore, the underlying mechanistic studies revealed that PSP-2 could ameliorate insulin resistance by up-regulating the expression levels of insulin receptor substrate-1 (IRS-1), glycogen synthase (GS), phosphoinositide-3-kinase (PI3K) and glucose transporter-4 (GLUT4). These results suggested that PSP-2 may be a potential candidate for the prevention and treatment of Type 2 diabetes mellitus.

Circadian clocks generate rhythms in cellular functions, including metabolism, to align biological processes with the 24-hour environment. Disruption of this alignment by shift work alters glucose homeostasis. Glucose homeostasis depends on signaling and allosteric control; however, the molecular mechanisms linking the clock to glucose homeostasis remain largely unknown. We investigated the molecular links between the clock and glycogen metabolism, a conserved glucose homeostatic process, in Neurospora crassa We find that glycogen synthase ( gsn ) mRNA, glycogen phosphorylase ( gpn ) mRNA, and glycogen levels, accumulate with a daily rhythm controlled by the circadian clock. Because the synthase and phosphorylase are critical to homeostasis, their roles in generating glycogen rhythms were investigated. We demonstrate that while gsn was necessary for glycogen production, constitutive gsn expression resulted in high and arrhythmic glycogen levels, and deletion of gpn abolished gsn mRNA rhythms and rhythmic glycogen accumulation. Furthermore, we show that gsn promoter activity is rhythmic and is directly controlled by core clock component white collar complex (WCC). We also discovered that WCC-regulated transcription factors, VOS-1 and CSP-1, modulate the phase and amplitude of rhythmic gsn mRNA, and these changes are similarly reflected in glycogen oscillations. Together, these data indicate the importance of clock-regulated gsn transcription over signaling or allosteric control of glycogen rhythms, a mechanism that is potentially conserved in mammals and critical to metabolic homeostasis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Polyhydroxybutyrate (PHB) is a polymer of great interest as a substitute for conventional plastics, which are becoming an enormous environmental problem. PHB can be produced directly from CO 2 in photoautotrophic cyanobacteria. The model cyanobacterium Synechocystis sp. PCC 6803 produces PHB under conditions of nitrogen starvation. However, it is so far unclear which metabolic pathways provide the precursor molecules for PHB synthesis during nitrogen starvation. In this study, we investigated if PHB could be derived from the main intracellular carbon pool, glycogen. A mutant of the major glycogen phosphorylase, GlgP2 ( slr1367 product), was almost completely impaired in PHB synthesis. Conversely, in the absence of glycogen synthase GlgA1 ( sll0945 product), cells not only produced less PHB, but were also impaired in acclimation to nitrogen depletion. To analyze the role of the various carbon catabolic pathways (EMP, ED and OPP pathways) for PHB production, mutants of key enzymes of these pathways were analyzed, showing different impact on PHB synthesis. Together, this study clearly indicates that PHB in glycogen-producing Synechocystis sp. PCC 6803 cells is produced from this carbon-pool during nitrogen starvation periods. This knowledge can be used for metabolic engineering to get closer to the overall goal of a sustainable, carbon-neutral bioplastic production.

The polysaccharide glycogen is an evolutionarily conserved storage form of glucose. However, the physiological significance of glycogen metabolism on homeostatic control throughout the animal life cycle remains incomplete. Here, we describe Drosophila mutants that have defective glycogen metabolism. Null mutants of glycogen synthase ( GlyS ) and glycogen phosphorylase ( GlyP ) displayed growth defects and larval lethality, indicating that glycogen plays a crucial role in larval development. Unexpectedly, however, a certain population of larvae developed into adults with normal morphology. Semi-lethality in glycogen mutants during the larval period can be attributed to the presence of circulating sugar trehalose. Homozygous glycogen mutants produced offspring, indicating that glycogen stored in oocytes is dispensable for embryogenesis. GlyS and GlyP mutants showed distinct metabolic defects in the levels of circulating sugars and triglycerides in a life stage-specific manner. In adults, glycogen as an energy reserve is not crucial for physical fitness and lifespan under nourished conditions, but glycogen becomes important under energy stress conditions. This study provides a fundamental understanding of the stage-specific requirements for glycogen metabolism in the fruit fly., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Cyanobacteria are oxygenic photosynthetic prokaryotes with important roles in the global carbon and nitrogen cycles. Unicellular nitrogen-fixing cyanobacteria are known to be ubiquitous, contributing to the nitrogen budget in diverse ecosystems. In the unicellular cyanobacterium Cyanothece sp. strain ATCC 51142, carbon assimilation and carbohydrate storage are crucial processes that occur as part of a robust diurnal cycle of photosynthesis and nitrogen fixation. During the light period, cells accumulate fixed carbon in glycogen granules to use as stored energy to power nitrogen fixation in the dark. These processes have not been thoroughly investigated, due to the lack of a genetic modification system in this organism. In bacterial glycogen metabolism, the glgX gene encodes a debranching enzyme that functions in storage polysaccharide catabolism. To probe the consequences of modifying the cycle of glycogen accumulation and subsequent mobilization, we engineered a strain of Cyanothece 51142 in which the glgX gene was genetically disrupted. We found that the Δ glgX strain exhibited a higher growth rate than the wild-type strain and displayed a higher rate of nitrogen fixation. Glycogen accumulated to higher levels at the end of the light period in the Δ glgX strain, compared to the wild-type strain. These data suggest that the larger glycogen pool maintained by the Δ glgX mutant is able to fuel greater growth and nitrogen fixation ability. IMPORTANCE Cyanobacteria are oxygenic photosynthetic bacteria that are found in a wide variety of ecological environments, where they are important contributors to global carbon and nitrogen cycles. Genetic manipulation systems have been developed in a number of cyanobacterial strains, allowing both the interruption of endogenous genes and the introduction of new genes and entire pathways. However, unicellular diazotrophic cyanobacteria have been generally recalcitrant to genetic transformation. These cyanobacteria are becoming important model systems to study diurnally regulated processes. Strains of the Cyanothece genus have been characterized as displaying robust growth and high rates of nitrogen fixation. The significance of our study is in the establishment of a genetic modification system in a unicellular diazotrophic cyanobacterium, the demonstration of the interruption of the glgX gene in Cyanothece sp. strain ATCC 51142, and the characterization of the increased nitrogen-fixing ability of this strain., (Copyright © 2019 American Society for Microbiology.)

MicroRNAs (miRNAs) are small noncoding RNAs that have been reported to play important roles in virus replication. Snakehead vesiculovirus (SHVV) is a new rhabdovirus isolated from diseased hybrid snakehead and has caused heavy economical losses in cultured snakehead fish in China. Our previous study has revealed that miR-214 inhibited SHVV replication, but the underline mechanism was not completely understood. In this study, glycogen synthase (GS) gene was identified as a target gene of miR-214. Overexpression of miR-214 reduced cellular GS gene expression. Knockdown of GS by siRNA, similar to the overexpression of miR-214, inhibited SHVV replication. Moreover, we found that siGS-mediated inhibition of SHVV replication could be restored by reducing cellular miR-214 level via using miR-214 inhibitor, indicating that miR-214 inhibited SHVV replication at least partially via targeting GS. This study provided information for understanding the molecular mechanism of SHVV pathogenicity and a potential antiviral strategy against SHVV infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The effects of heating on glycogen synthesis (incorporation of [ 14 C]glucose into glycogen) and accumulation after intense repeated contractions were investigated. Isolated mouse extensor digitorum longus muscle (type II) was stimulated electrically to perform intense tetanic contractions at 25°C. After 120 min recovery at 25°C, glycogen accumulated to almost 80% of basal, whereas after recovery at 35°C, glycogen remained low (~25% of basal). Glycogen synthesis averaged 0.97 ± 0.07 µmol·30 min -1 ·g wet wt -1 during recovery at 25°C and 1.48 ± 0.08 during recovery at 35°C ( P < 0.001). There were no differences in phosphorylase and glycogen synthase total activities nor in phosphorylase fractional activity, whereas glycogen synthase fractional activity was increased by ~50% after recovery at 35°C vs. 25°C. Inorganic phosphate (P i , substrate for phosphorylase) was markedly increased (~300% of basal) following contraction but returned to control levels after 120 min recovery at 25°C. In contrast, P i remained elevated after recovery at 35°C (>2-fold higher than recovery at 25°C). Estimates of glycogen breakdown indicated that phosphorylase activity (either via inhibition at 25°C or activation at 35°C) was responsible for ~60% of glycogen accumulation during recovery at 25°C and ~45% during recovery at 35°C. These data demonstrate that despite the enhancing effect of heating on glycogen synthesis during recovery from intense contractions, glycogen accumulation is inhibited owing to P i -mediated activation of phosphorylase. Thus phosphorylase can play a quantitatively important role in glycogen biogenesis during recovery from repeated contractions in isolated type II muscle.

The operations involved in preimplantation genetic testing (PGT) occur during the key stages of gametogenesis and early embryonic development, and the health of progeny following PGT (PGT-born) is worthy of attention. In order to fully assess the potential risk of abnormal glucose metabolism in adult PGT-born offspring and to evaluate possible mechanisms, we compared a mouse model of PGT (in vitro cultured embryos with biopsy, hereafter "PTG-born mice"), an in vitro embryo manipulation mouse model (in vitro cultured embryos without biopsy), and normal mice. PGT-born mice displayed increased fasting glucose, and decreased glycogen synthesis and glucose oxidative utilization in the liver. Moreover, PGT-born mice also displayed reduced expression of insulin receptor, AKT, and insulin-stimulated Akt phosphorylation (pAkt) in the liver. These results suggest a potential risk of insulin resistance in adult PGT-born mice. By analyzing the DNA methylation profiles of 7.5 days postconception (dpc) embryos, we identified differentially methylated genes associated with liver development between PGT-born and control groups; some of these genes are associated with glucose homeostasis and insulin response. These results suggest that abnormal methylation in embryos that develop after PGT may be a potential mechanism occurring during embryonic development that can influence the risk of liver-derived insulin resistance in adulthood., (© 2018 New York Academy of Sciences.)

Context: Glycogen synthesis is a critical metabolic function of the endometrium to prepare for successful implantation and sustain embryo development. Yet, regulation of endometrial carbohydrate metabolism is poorly characterized. Whereas glycogen synthesis is attributed to progesterone, we previously found that the metabolic B isoform of the insulin receptor is maximally expressed in secretory-phase endometrium, indicating a potential role of insulin in glucose metabolism., Objective: We sought to determine whether insulin or progesterone regulates glycogen synthesis in human endometrium., Design, Participants, Outcome Measurements: Endometrial epithelial cells were isolated from 28 healthy women and treated with insulin, medroxyprogesterone (MPA), or vehicle. Intracellular glycogen and the activation of key enzymes were quantified., Results: In epithelia, insulin induced a 4.4-fold increase in glycogen, whereas MPA did not alter glycogen content. Insulin inactivated glycogen synthase (GS) kinase 3α/β (GSK3α/β), relieving inhibition of GS. In a regulatory mechanism, distinct from liver and muscle, insulin also increased GS by 3.7-fold through increased GS 2 (GYS2) gene expression., Conclusions: We demonstrate that insulin, not progesterone, directly regulates glycogen synthesis through canonical acute inactivation of GSK3α/β and noncanonical stimulation of GYS2 transcription. Persistently elevated GS enables endometrium to synthesize glycogen constitutively, independent of short-term nutrient flux, during implantation and early pregnancy. This suggests that insulin plays a key, physiological role in endometrial glucose metabolism and underlines the need to delineate the effect of maternal obesity and hyperinsulinemia on fertility and fetal development.

Objective: To investigate the role of glycogen metabolism in regulating rheumatoid fibroblast-like synoviocyte (FLS)-mediated synovial inflammation and its underlying mechanism., Methods: FLSs were separated from synovial tissues (STs) obtained from rheumatoid arthritis (RA) patients. Glycogen content was determined by periodic acid Schiff staining. Protein expression was analyzed by Western blot or immunohistochemistry. Gene expression of cytokines and matrix metalloproteinases (MMPs) was evaluated by quantitative real-time PCR. FLS proliferation was detected by EdU incorporation. Migration and invasion were measured by Boyden chamber assay., Results: Glycogen levels and glycogen synthase 1 (GYS1) expression were significantly increased in the ST and FLSs of RA patients. TNF-α or hypoxia induced GYS1 expression and glycogen synthesis in RA FLSs. GYS1 knockdown by shRNA decreased the expression of IL-1β, IL-6, CCL-2, MMP-1, and MMP-9 and proliferation and migration by increasing AMP-activated protein kinase (AMPK) activity in RA FLS. AMPK inhibitor or knockdown AMPK could reverse the inhibitory effect of GYS1 knockdown on the inflammatory response in RA FLSs; however, an AMPK agonist blocked RA FLS activity. We further determined that hypoxia-inducible factor-1α mediates TNF-α- or hypoxia-induced GYS1 expression and glycogen levels. Local joint depletion of GYS1 or intraperitoneal administration with an AMPK agonist ameliorated the severity of arthritis in rats with collagen-induced arthritis., Conclusion: Our data demonstrate that GYS1-mediated glycogen accumulation contributes to FLS-mediated synovial inflammation in RA by blocking AMPK activation. In our knowledge, this is a first study linking glycogen metabolism to chronic inflammation. Inhibition of GYS1 might be a novel therapeutic strategy for chronic inflammatory arthritis, including RA.

Glycogen storage diseases (GSDs) of the liver are devastating disorders presenting with fasting hypoglycemia as well as hepatic glycogen and lipid accumulation, which could lead to long-term liver damage. Diet control is frequently utilized to manage the potentially dangerous hypoglycemia, but there is currently no effective pharmacological treatment for preventing hepatomegaly and concurrent liver metabolic abnormalities, which could lead to fibrosis, cirrhosis, and hepatocellular adenoma or carcinoma. In this study, we demonstrate that inhibition of glycogen synthesis using an RNAi approach to silence hepatic Gys2 expression effectively prevents glycogen synthesis, glycogen accumulation, hepatomegaly, fibrosis, and nodule development in a mouse model of GSD III. Mechanistically, reduction of accumulated abnormally structured glycogen prevents proliferation of hepatocytes and activation of myofibroblasts as well as infiltration of mononuclear cells. Additionally, we show that silencing Gys2 expression reduces hepatic steatosis in a mouse model of GSD type Ia, where we hypothesize that the reduction of glycogen also reduces the production of excess glucose-6-phosphate and its subsequent diversion to lipid synthesis. Our results support therapeutic silencing of GYS2 expression to prevent glycogen and lipid accumulation, which mediate initial signals that subsequently trigger cascades of long-term liver injury in GSDs., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

GLYCOGEN SYNTHASE KINASE3/Shaggy-like kinases (GSKs) represent a highly conserved group of proteins found in all eukaryotes. In plants they are encoded by multigene families and integrate signaling of brassinosteroids, auxin and abscisic acid in wide range of physiological and developmental processes with a strong impact on plant responses to environmental and biotic factors. Based on comprehensively studied structures of 10 Arabidopsis thaliana GSK genes and encoded proteins we report identification and phylogenetic reconstruction of 7 transcriptionally active GSK genes in barley. We re-evaluated annotation of the GSK genes in the current barley genome (Hv&#95;IBSC&#95;PGSB&#95;v2) and provided data that a single gene annotated in the previous barley genome ensemble should be retained in the current one. The novel structure of another GSK, predicted in Hv&#95;IBSC&#95;PGSB&#95;v2 to encode both GSK and amine oxidase domains, was proposed and experimentally confirmed based on the syntenic region in Brachypodium distachyon. The genes were assigned to 4 groups based on their encoded amino acid sequences and protein kinase domains. The analysis confirmed high level of conservation of functional protein domains and motifs among plant GSKs and the identified barley orthologs. Each of the seven identified HvGSK genes was expressed indicating semi-constitutive regulation in all tested organs and developmental stages. Regulation patterns of GSKs from the indicated groups showed a shift in organ-preferential expression in A. thaliana and barley illustrating diversification of biological roles of individual HvGSKs in different plant species., Competing Interests: The authors have declared that no competing interests exist.

Surface Pre-reacted Glass-ionomer (S-PRG) filler is a bioactive filler produced by PRG technology, which has been applied to various dental materials. A S-PRG filler can release multiple ions from a glass-ionomer phase formed in the filler. In the present study, detailed inhibitory effects induced by S-PRG eluate (prepared with S-PRG filler) against Streptococcus mutans, a major pathogen of dental caries, were investigated. S-PRG eluate effectively inhibited S. mutans growth especially in the bacterium before the logarithmic growth phase. Microarray analysis was performed to identify changes in S. mutans gene expression in the presence of the S-PRG eluate. The S-PRG eluate prominently downregulated operons related to S. mutans sugar metabolism, such as the pdh operon encoding the pyruvate dehydrogenase complex and the glg operon encoding a putative glycogen synthase. The S-PRG eluate inhibited several in vitro properties of S. mutans relative to the development of dental caries especially prior to active growth. These results suggest that the S-PRG eluate may effectively inhibit the bacterial growth of S. mutans following downregulation of operons involved in sugar metabolism resulting in attenuation of the cariogenicity of S. mutans, especially before the active growth phase.

Hacıhamdioğlu B, Özgürhan G, Çaran B, Meydan-Aksanlı E, Keskin E. Glycogen storage disease type 0 due to a novel frameshift mutation in glycogen synthase 2 (GYS2) gene in a child presenting with fasting hypoglycemia and postprandial hyperglycemia. Turk J Pediatr 2018; 60: 581-583. Glycogen storage disease type 0 (GSD0) has been considered a rare disorder, it is characterized with ketotic hypoglycemia after prolonged fasting and postprandial hyperglycemia. Herein, we report a novel mutation in the glycogen synthase 2 gene in a Turkish child, as well as her clinical characteristics and 12-month follow-up. We evaluated a 5-year-old girl for asymptomatic fasting ketotic hypoglycemia with postprandial hyperglycemia diagnosed with glycogen storage disease type 0. We identified a novel frameshift mutation, c.1081delA (p.Thr361Glnfs*2) in exon 8 on glycogen synthase 2 gene. Children with GSD0 may have a mild phenotype and GSD0 may be underdiagnosed due to subclinical or asymptomatic hypoglycemia. The diagnosis of GSD0 should be considered in a child with ketotic fasting hypoglycemia with postprandial hyperglycemia but without hepatomegaly.