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Dysregulated muscle glycogen metabolism preslaughter contributes to aberrant postmortem muscle pH (>5.8) in dark-cutting beef phenotypes. However, the underlying mechanisms have remained elusive. Herein, we examine the glycogen dependent regulation of postmortem muscle pH decline and darkening in beef. We show that supplementation of glycogen in vitro restores postmortem pH decline in dark-cutting beef by reverting the metabolome toward a typical postmortem muscle state characterized by increased activities of enzymes glycogen phosphorylase and lactate dehydrogenase ( p < 0.05) coupled with a pronounced abundance of glycolytic metabolites and reduced abundance of tricarboxylic acid cycle and amino acid metabolites. Furthermore, concurrent inhibition of mitochondrial respiration at complexes I, IV, and V with glycogen supplementation stimulates greater pH decline. Together, our findings show that supplementing glycogen at low concentrations (10 mM) can reprogram the dark-cutting beef muscle's metabolome toward typical postmortem state and promote muscle acidification. Thus, enhancing glycogen levels could represent a promising strategy for mitigating dark-cutting beef phenotypes and improving meat quality.

Glycogen phosphorylase (GP) is the rate-determining enzyme in glycogenolysis, and its druggability has been extensively studied over the years for the development of therapeutics against type 2 diabetes (T2D) and, more recently, cancer. However, the conservation of binding sites between the liver and muscle isoforms makes the inhibitor selectivity challenging. Using a combination of kinetic, crystallographic, modeling, and cellular studies, we have probed the binding of dietary flavonoids epigallocatechin gallate (EGCG) and epigallocatechin (EGC) to GP isoforms. The structures of rmGPb-EGCG and rmGPb-EGC complexes were determined by X-ray crystallography, showing binding at the quercetin binding site (QBS) in agreement with kinetic studies that revealed both compounds as noncompetitive inhibitors of GP, with EGCG also causing a significant reduction in cell viability and migration of U87-MG glioblastoma cells. Interestingly, EGCG exhibits different binding modes to GP isoforms, revealing QBS as a promising site for GP targeting, offering new opportunities for the design of liver-selective GP inhibitors.

Inhibition of glycogen phosphorylase (Pyg) - a regulatory enzyme of glycogen phosphorolysis - influences memory formation in rodents. We have previously shown that 2-week intraperitoneal administration of a Pyg inhibitor BAY U6751 stimulated the "rejuvenation" of the hippocampal proteome and dendritic spines morphology and improved cognitive skills of old mice. Given the tedious nature of daily intraperitoneal drug administration, in this study we investigated whether a single dose of BAY U6751 could induce enduring behavioral effects. Obtained results support the efficacy of such treatment in significantly improving the cognitive performance of 20-22-month-old mice. Metabolomic analysis of alterations observed in the hippocampus, cerebellum, and cortex reveal that the inhibition of glycogen phosphorolysis impacts not only glucose metabolism but also various other metabolic processes., (© 2024. The Author(s).)

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.)

The structure of glycogen α particles in healthy mouse liver has two states: stability and fragility. In contrast, glycogen α particles in diabetic liver present consistent fragility, which may exacerbate hyperglycemia. Currently, the molecular mechanism behind glycogen structural alteration is still unclear. In this study, we characterized the fine molecular structure of liver glycogen α particles in healthy mice under time-restricted feeding (TRF) mode during a 24-h cycle. Then, differentially expressed genes (DEGs) in the liver during daytime and nighttime were revealed via transcriptomics, which identified that the key downregulated DEGs were mainly related to insulin secretion in daytime. Furthermore, GO annotation and KEGG pathway enrichment found that negative regulation of the glycogen catabolic process and insulin secretion process were significantly downregulated in the daytime. Therefore, transcriptomic analyses indicated that the structural stability of glycogen α particles might be correlated with the glycogen degradation process via insulin secretion downregulation. Further molecular experiments confirmed the significant upregulation of glycogen phosphorylase (PYGL), phosphorylated PYGL (p-PYGL), and glycogen debranching enzyme (AGL) at the protein level during the daytime. Overall, we concluded that the downregulation of insulin secretion in the daytime under TRF mode facilitated glycogenolysis, contributing to the structural stability of glycogen α-particles., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Compound 1 was previously identified by our team as a glycogen phosphorylase (GP) inhibitor with glucose-lowering activity and demonstrated to have protective effects against myocardial and cerebral ischemia. However, its impact on muscle function has not been clarified. This study is the first to evaluate the long-term effects of GP inhibitors on muscle function and metabolism. After a 28-day administration of Compound 1 , we performed assays to assess muscle function and biochemical parameters in rats. We observed reductions in peak holding force, duration, tetanic contraction force, single-contraction force, and electromyographic signals under 20 s and 10 min contraction stimuli. The metabolic analysis showed no significant effects on muscle glycogen, ATP, lactic acid, and uric acid levels at low doses. In contrast, medium to high doses resulted in increased glycogen, decreased ATP, and reduced lactic acid (only at high doses), without affecting uric acid. These findings suggest that Compound 1 may adversely affect muscle function in rats, potentially due to the glycogen inhibition effects of GP inhibitors. This study provides crucial safety data and insights into the long-term effects of GP inhibitors on rat muscles, which will guide future developments and applications.

Loss of glycogen myophosphorylase (PYGM) expression results in an inability to break down muscle glycogen, leading to McArdle disease-an autosomal recessive metabolic disorder characterized by exercise intolerance and muscle cramps. While previously considered relatively benign, this condition has recently been associated with pattern dystrophy in the retina, accompanied by variable sight impairment, secondary to retinal pigment epithelial (RPE) cell involvement. However, the pathomechanism of this condition remains unclear. In this study, we generated a PYGM-null induced pluripotent stem cell line and differentiated it into mature RPE to examine structural and functional defects, along with metabolite release into apical and basal media. Mutant RPE exhibited normal photoreceptor outer segment phagocytosis but displayed elevated glycogen levels, reduced transepithelial resistance, and increased cytokine secretion across the epithelial layer compared to isogenic WT controls. Additionally, decreased expression of the visual cycle component, RDH11, encoding 11-cis-retinol dehydrogenase, was observed in PYGM-null RPE. While glycolytic flux and oxidative phosphorylation levels in PYGM-null RPE were near normal, the basal oxygen consumption rate was increased. Oxygen consumption rate in response to physiological levels of lactate was significantly greater in WT than PYGM-null RPE. Inefficient lactate utilization by mutant RPE resulted in higher glucose dependence and increased glucose uptake from the apical medium in the presence of lactate, suggesting a reduced capacity to spare glucose for photoreceptor use. Metabolic tracing confirmed slower 13 C-lactate utilization by PYGM-null RPE. These findings have key implications for retinal health since they likely underlie the vision impairment in individuals with McArdle disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: This study examined the impact of a 5-wk sprint interval training (SIT) intervention on time to task failure (TTF) during severe-intensity constant work rate (CWR) exercise, as well as in glycolytic enzymatic content and activity, and glycogen content., Methods: Fourteen active males were randomized into either a SIT group ( n = 8) composed of 15 SIT sessions over 5 wk, or a control group ( n = 6). At pretraining period, participants performed i) ramp incremental test to measure the cardiorespiratory function; ii) CWR cycling TTF at 150% of the power output (PO) at the respiratory compensation point (RCP-PO) with muscle biopsies at rest and immediately following task failure. After 5 wk, the same evaluations were repeated (i.e., exercise intensities matched to current training status), and an additional cycling CWR matched to pretraining 150% RCP-PO was performed only for TTF evaluation. The content and enzymatic activity of glycogen phosphorylase (GPhos), hexokinase (HK), phosphofructokinase (PFK), and lactate dehydrogenase (LDH), as well as the glycogen content, were analyzed. Content of monocarboxylate transporter isoform 4 (MCT4) and muscle buffering capacity were also measured., Results: Despite improvements in total work performed at CWR posttraining, no differences were observed for TTF. The GPhos, HK, PFK, and LDH content and activity, and glycogen content also improved after training only in the SIT group. Furthermore, the MCT4 concentrations and muscle buffering capacity were also improved only for the SIT group. However, no difference in glycogen depletion was observed between groups and time., Conclusions: Five weeks of SIT improved the glycolytic pathway parameters and total work performed; however, glycogen depletion was not altered during CWR severe-intensity exercise, and TTF remained similar., (Copyright © 2024 by the American College of Sports Medicine.)

The physiological function of muscle glycogen is to meet the energy demands of muscle contraction. The breakdown of glycogen occurs through two distinct pathways, primarily cytosolic and partially lysosomal. To obtain the necessary energy for their function, skeletal muscles utilise also fatty acids in the β-oxidation. Ketogenesis is an alternative metabolic pathway for fatty acids, which provides an energy source during fasting and starvation. Diseases arising from impaired glycogenolysis lead to muscle weakness and dysfunction. Here, we focused on the lack of muscle glycogen phosphorylase (PYGM), a rate-limiting enzyme for glycogenolysis in skeletal muscles, which leads to McArdle disease. Metabolic myopathies represent a group of genetic disorders characterised by the limited ability of skeletal muscles to generate energy. Here, we discuss the metabolic aspects of glycogenosis with a focus on McArdle disease, offering insights into its pathophysiology. Glycogen accumulation may influence the muscle metabolic dynamics in different ways. We emphasize that a proper treatment approach for such diseases requires addressing three important and interrelated aspects, which include: symptom relief therapy, elimination of the cause of the disease (lack of a functional enzyme) and effective and early diagnosis., Competing Interests: Declaration of competing interest None, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The three isoforms of glycogen phosphorylase - PYGM, PYGB, and PYGL - are expressed in glial cells. Unlike PYGB and PYGL, PYGM is the only isoform regulated by Rac1. This specific regulation may confer a differential functional role compared with the other glycogen phosphorylases-PYGB and PYGL. The involvement of muscle glycogen phosphorylase in glial cells and its association with post-translational modifications (PTMs) of proteins through O-glycosylation is indeed a fascinating and emerging area of research. The dual role it plays in metabolic processes and the regulation of PTMs within the brain presents intriguing implications for various neurological conditions. Disruptions in the O-GlcNAcylation cycle and neurodegenerative diseases like Alzheimer's disease (AD) is particularly noteworthy. The alterations in O-GlcNAcylation levels of specific proteins, such as APP, c-Fos, and tau protein, highlight the intricate relationship between PTMs and AD. Understanding these processes and the regulatory function of muscle glycogen phosphorylase sheds light on its impact on protein function, signaling pathways, cellular homeostasis, neurological health, and potential interventions for brain-related conditions., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Recently studied N -(β-d-glucopyranosyl)-3-aryl-1,2,4-triazole-5-carboxamides have proven to be low micromolar inhibitors of glycogen phosphorylase (GP), a validated target for the treatment of type 2 diabetes mellitus . Since in other settings, the bioisosteric replacement of the 1,2,4-triazole moiety with imidazole resulted in significantly more efficient GP inhibitors, in silico calculations using Glide molecular docking along with unbound state DFT calculations were performed on N -(β-d-glucopyranosyl)-arylimidazole-carboxamides, revealing their potential for strong GP inhibition. The syntheses of the target compounds involved the formation of an amide bond between per- O -acetylated β-d-glucopyranosylamine and the corresponding arylimidazole-carboxylic acids. Kinetics experiments on rabbit muscle GP b revealed low micromolar inhibitors, with the best inhibition constants ( K i s) of ~3-4 µM obtained for 1- and 2-naphthyl-substituted N -(β-d-glucopyranosyl)-imidazolecarboxamides, 2b - c . The predicted protein-ligand interactions responsible for the observed potencies are discussed and will facilitate the structure-based design of other inhibitors targeting this important therapeutic target. Meanwhile, the importance of the careful consideration of ligand tautomeric states in binding calculations is highlighted, with the usefulness of DFT calculations in this regard proposed.

Statin treatment may increase the risk of diabetes; there is insufficient data on how statins affect glucose regulation and glycemic control and the effects of statins on liver enzymes related to carbohydrate metabolism have not been fully studied. Therefore, we aimed to compare the effects of the statin derivatives, pravastatin, and rosuvastatin, on carbohydrate metabolism in an experimental diabetic rat model. Female Wistar albino rats were used and diabetes was induced by intraperitoneal injection of streptozotocin. Thereafter, 10 and 20 mg kg -1 day -1 doses of both pravastatin and rosuvastatin were administered by oral gavage to the diabetic rats for 8 weeks. At the end of the experiment, body masses, the levels of fasting blood glucose, serum insulin, insulin resistance (HOMA-IR), liver glycogen, and liver enzymes related to carbohydrate metabolism were measured. Both doses of pravastatin significantly in creased the body mass in diabetic rats, however, rosuvastatin, especially at the dose of 20 mg kg -1 day -1 reduced the body mass signi ficantly. Pravastatin, especially at a dose of 20 mg kg -1 day -1 , caused significant increases in liver glycogen synthase and glucose 6-phosphate dehydrogenase levels but significant decreases in the levels of glycogen phosphorylase, lactate dehydrogenase, and glucose-6-phosphatase. Hence, pravastatin partially ameliorated the adverse changes in liver enzymes caused by diabetes and, especially at the dose of 20 mg kg -1 day -1 , reduced the fasting blood glucose level and increased the liver glycogen content. However, rosuvastatin, especially at the dose of 20 mg kg -1 day -1 , significantly reduced the liver glycogen synthase and pyruvate kinase levels, but increased the glycogen phosphorylase level in diabetic rats. Rosuvastatin, 20 mg kg -1 day -1 dose, caused significant decreases in the body mass and the liver glycogen content of diabetic rats. It can be concluded that pravastatin, especially at the dose of 20 mg kg -1 day -1 is more effective in ameliorating the negative effects of diabetes by modulating carbohydrate metabolism., (© 2024 Hacer Kayhan Kaya et al., published by Sciendo.)

This study compared differences in exercise capacity as well as muscle glycogen content and degradation, and mitochondrial enzyme activity between C57BL/6J and BALB/cA mice. In exercise tests, grip strength was higher in BALB/cA mice. In Rotarod and Inverted screen test, C57BL/6J mice had significantly longer exercise durations and showed differences in motor function and muscle endurance time. Glycogen in the liver and muscle of C57BL/6J mice was significantly decreased after 20 min of swimming. Muscle glycogen content in BALB/cA mice was higher than in C57BL/6J, but swimming induced no decrease in glycogen content. Glycogen phosphorylase in muscle was inactive in the absence of AMP, and its activity increased in a concentration-dependent manner with the addition of AMP in C57BL/6J mice. In BALB/cA mice, phosphorylase activity was increased by AMP, but not further increased by higher concentrations of AMP. The citrate synthase activity in muscle did not differ between C57BL/6J and BALB/cA mice. The results of this study suggested that the reactivity of muscle glycogen phosphorylase to AMP differs among strains of mice and affects glycogen availability during exercise.

Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits, each activated by phosphorylation of the serine-14 residue. GP exists in three interconvertible forms, namely GPa (di-phosphorylated form), GPab (mono-phosphorylated form), and GPb (non-phosphorylated form); however, information on GPab remains scarce. Given the prevailing view that the two GP subunits collaboratively determine their catalytic characteristics, it is essential to conduct GPab characterization to gain a comprehensive understanding of glycogenolysis regulation. Thus, in the present study, we prepared rabbit muscle GPab from GPb, using phosphorylase kinase as the catalyst, and identified it using a nonradioactive phosphate-affinity gel electrophoresis method. Compared with the half-half GPa/GPb mixture, the as-prepared GPab showed a unique AMP-binding affinity. To further investigate the intersubunit communication in GP, its catalytic site was probed using pyridylaminated-maltohexaose (a maltooligosaccharide-based substrate comprising the essential dextrin structure for GP; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (substrate analogs lacking part of the essential dextrin structure). By comparing the initial reaction rates toward the PA-0 derivative (V derivative ) and PA-0 (V PA-0 ), we demonstrated that the V derivative /V PA-0 ratio for GPab was significantly different from that for the half-half GPa/GPb mixture. This result indicates that the interaction between the two GP subunits significantly influences substrate recognition at the catalytic sites, thereby providing GPab its unique substrate recognition profile., (© 2024. The Author(s).)

Acetamiprid (ACMP) is a second-generation neonicotinoid that has been extensively used in the last few years. The present study examined the toxic effects of ACMP on the pancreas and glucose homeostasis through the evaluation of histological and biochemical changes and the possible ameliorative role of fenugreek seed extract (FG). Fifty adult albino rats were divided into 5 groups: negative control, positive control, FG-treated, ACMP-treated, and ACMP + FG-treated groups by oral gavage for 12 weeks. The ACMP-treated group highlighted significant elevations in plasma glucose, glycosylated haemoglobin levels (HbA1c), serum amylase, and serum lipase, along with a decrease in plasma insulin levels. In addition, significant increases in tumour necrosis factor- alpha (TNF-α) and malondialdehyde (MDA) were associated with reductions in the levels of interleukin 10 (IL-10), glutathione peroxidase, and catalase. Moreover, glucose-6-phosphatase and glycogen phosphorylase were significantly increased, with a significant reduction in hexokinase and liver glycogen stores. These biochemical changes were associated with histological changes in pancreatic sections stained by haematoxylin and eosin, Masson stain, and Orcein stain. ACMP-treated cells showed a marked reduction in β- cell immune reactivity to insulin, with pronounced p53, and beclin 1 immune expression. The use of FG with ACMP induced partial protection except for hexokinase and glycogen phosphorylase., 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 © 2023 Elsevier Ltd. All rights reserved.)

It remains a major challenge to ascertain the specific structurally dynamic changes that underpin protein functional switching. There is a growing need in molecular biology and drug discovery to complement structural models with the ability to determine the dynamic structural changes that occur as these proteins are regulated and function. The archetypal allosteric enzyme glycogen phosphorylase is a clinical target of great interest to treat type II diabetes and metastatic cancers. Here, we developed a time-resolved nonequilibrium millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS) approach capable of precisely locating dynamic structural changes during allosteric activation and inhibition of glycogen phosphorylase. We resolved obligate transient changes in the localized structure that are absent when directly comparing active/inactive states of the enzyme and show that they are common to allosteric activation by AMP and inhibition by caffeine, operating at different sites. This indicates that opposing allosteric regulation by inhibitor and activator ligands is mediated by pathways that intersect with a common structurally dynamic motif. This mass spectrometry approach uniquely stands to discover local transient structural dynamics and could be used broadly to identify features that influence the structural transitions of proteins.

Glycogen phosphorylase (GP) is the rate-determining enzyme in the glycogenolysis pathway. Glioblastoma (GBM) is amongst the most aggressive cancers of the central nervous system. The role of GP and glycogen metabolism in the context of cancer cell metabolic reprogramming is recognised, so that GP inhibitors may have potential treatment benefits. Here, baicalein (5,6,7-trihydroxyflavone) is studied as a GP inhibitor, and for its effects on glycogenolysis and GBM at the cellular level. The compound is revealed as a potent GP inhibitor against human brain GPa (K i  = 32.54 μM), human liver GPa (K i  = 8.77 μM) and rabbit muscle GPb (K i  = 5.66 μM) isoforms. It is also an effective inhibitor of glycogenolysis (IC 50  = 119.6 μM), measured in HepG2 cells. Most significantly, baicalein demonstrated anti-cancer potential through concentration- and time-dependent decrease in cell viability for three GBM cell-lines (U-251 MG, U-87 MG, T98-G) with IC 50 values of ∼20-55 μM (48- and 72-h). Its effectiveness against T98-G suggests potential against GBM with resistance to temozolomide (the first-line therapy) due to a positive O 6 -methylguanine-DNA methyltransferase (MGMT) status. The solved X-ray structure of rabbit muscle GP-baicalein complex will facilitate structure-based design of GP inhibitors. Further exploration of baicalein and other GP inhibitors with different isoform specificities against GBM is suggested., 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 © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Glucose accesses the brain primarily via the astrocyte cell compartment, where it passes through the glycogen shunt before catabolism to the oxidizable fuel L-lactate. Glycogen phosphorylase (GP) isoenzymes GPbb and GPmm impose distinctive control of ventromedial hypothalamic nucleus (VMN) glucose-regulatory neurotransmission during hypoglycemia, but lactate and/or gliotransmitter involvement in those actions is unknown. Lactate or the octadecaneuropeptide receptor antagonist cyclo(1-8)[DLeu 5 ] OP (LV-1075) did not affect gene product down-regulation caused by GPbb or GPmm siRNA, but suppressed non-targeted GP variant expression in a VMN region-specific manner. Hypoglycemic up-regulation of neuronal nitric oxide synthase was enhanced in rostral and caudal VMN by GPbb knockdown, yet attenuated by GPMM siRNA in the middle VMN; lactate or LV-1075 reversed these silencing effects. Hypoglycemic inhibition of glutamate decarboxylase65/67 was magnified by GPbb (middle and caudal VMN) or GPmm (middle VMN) knockdown, responses that were negated by lactate or LV-1075. GPbb or GPmm siRNA enlarged hypoglycemic VMN glycogen profiles in rostral and middle VMN. Lactate and LV-1075 elicited progressive rostral VMN glycogen augmentation in GPbb knockdown rats, but stepwise-diminution of rostral and middle VMN glycogen after GPmm silencing. GPbb, not GPmm, knockdown caused lactate or LV-1075 - reversible amplification of hypoglycemic hyperglucagonemia and hypercorticosteronemia. Results show that lactate and octadecaneuropeptide exert opposing control of GPbb protein in distinct VMN regions, while the latter stimulates GPmm. During hypoglycemia, GPbb and GPmm may respectively diminish (rostral, caudal VMN) or enhance (middle VMN) nitrergic transmission and each oppose GABAergic signaling (middle VMN) by lactate- and octadecaneuropeptide-dependent mechanisms., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Inhibition of glycogen breakdown blocks memory formation in young animals, but it stimulates the maintenance of the long-term potentiation, a cellular mechanism of memory formation, in hippocampal slices of old animals. Here, we report that a 2-week treatment with glycogen phosphorylase inhibitor BAY U6751 alleviated memory deficits and stimulated neuroplasticity in old mice. Using the 2-Novel Object Recognition and Novel Object Location tests, we discovered that the prolonged intraperitoneal administration of BAY U6751 improved memory formation in old mice. This was accompanied by changes in morphology of dendritic spines in hippocampal neurons, and by "rejuvenation" of hippocampal proteome. In contrast, in young animals, inhibition of glycogen degradation impaired memory formation; however, as in old mice, it did not alter significantly the morphology and density of cortical dendritic spines. Our findings provide evidence that prolonged inhibition of glycogen phosphorolysis improves memory formation of old animals. This could lead to the development of new strategies for treatment of age-related memory deficits., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Lactate is an important metabolic substrate for sustaining brain energy requirements when glucose supplies are limited. Recurring exposure to hypoglycemia (RH) raises lactate levels in the ventromedial hypothalamus (VMH), which contributes to counterregulatory failure. However, the source of this lactate remains unclear. The current study investigates whether astrocytic glycogen serves as the major source of lactate in the VMH of RH rats. By decreasing the expression of a key lactate transporter in VMH astrocytes of RH rats, we reduced extracellular lactate concentrations, suggesting excess lactate was locally produced from astrocytes. To determine whether astrocytic glycogen serves as the major source of lactate, we chronically delivered either artificial extracellular fluid or 1,4-dideoxy-1,4-imino-d-arabinitol to inhibit glycogen turnover in the VMH of RH animals. Inhibiting glycogen turnover in RH animals prevented the rise in VMH lactate and the development of counterregulatory failure. Lastly, we noted that RH led to an increase in glycogen shunt activity in response to hypoglycemia and elevated glycogen phosphorylase activity in the hours following a bout of hypoglycemia. Our data suggest that dysregulation of astrocytic glycogen metabolism following RH may be responsible, at least in part, for the rise in VMH lactate levels., Article Highlights: Astrocytic glycogen serves as the major source of elevated lactate levels in the ventromedial hypothalamus (VMH) of animals exposed to recurring episodes of hypoglycemia. Antecedent hypoglycemia alters VMH glycogen turnover. Antecedent exposure to hypoglycemia enhances glycogen shunt activity in the VMH during subsequent bouts of hypoglycemia. In the immediate hours following a bout of hypoglycemia, sustained elevations in glycogen phosphorylase activity in the VMH of recurrently hypoglycemic animals contribute to sustained elevations in local lactate levels., (© 2023 by the American Diabetes Association.)

Considerable evidence confirms the importance of Cyp26a1 to all-trans-retinoic acid (RA) homeostasis during embryogenesis. In contrast, despite its presence in postnatal liver as a potential major RA catabolizing enzyme and its acute sensitivity to induction by RA, some data suggested that Cyp26a1 contributes only marginally to endogenous RA homeostasis postnatally. We report reevaluation of a conditional Cyp26a1 knockdown in the postnatal mouse. The current results show that Cyp26a1 mRNA in WT mouse liver increases 16-fold upon refeeding after a fast, accompanied by an increased rate of RA elimination and a 41% decrease in the RA concentration. In contrast, Cyp26a1 mRNA in the refed homozygotic knockdown reached only 2% of its extent in WT during refeeding, accompanied by a slower rate of RA catabolism and no decrease in liver RA, relative to fasting. Refed homozygous knockdown mice also had decreased Akt1 and 2 phosphorylation and pyruvate dehydrogenase kinase 4 (Pdk4) mRNA and increased glucokinase (Gck) mRNA, glycogen phosphorylase (Pygl) phosphorylation, and serum glucose, relative to WT. Fasted homozygous knockdown mice had increased glucagon/insulin relative to WT. These data indicate that Cyp26a1 participates prominently in moderating the postnatal liver concentration of endogenous RA and contributes essentially to glucoregulatory control., Competing Interests: Conflict of 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 © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Glycogen is a polymerized form of glucose that serves as an energy reserve in all types of organisms. In animals glycogen synthesis and degradation, especially in liver and skeletal muscle, are regulated by hormonal and physiological signals that reciprocally control the opposing activities of glycogen synthase and glycogen phosphorylase. These enzymes are under allosteric control by binding of metabolites (e.g., ATP, AMP, G6P) and covalent control by reversible phosphorylation by kinase and phosphatase all assembled together on glycogen. More than 50 years ago Edmond Fischer and colleagues showed "flash activation" of phosphorylase in glycogen particles. This involved transient and extensive inhibition of protein phosphatase but even today the phenomenon is not understood. Phosphatase regulation is known to rely on regulatory subunits including glycogen binding subunits that serve as scaffolds, binding catalytic subunit, glycogen, and substrates. This tribute article to Edmond Fischer highlights his thoughts and ideas about the transient inhibition of phosphorylase phosphatase during flash activation of phosphorylase and speculates that phosphatase regulation in glycogen particles might involve a/b hybrids of phosphorylase., (© 2022 The Author. IUBMB Life published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Glycogen phosphorylase (GP) is a key regulator of glucose levels and, with that, an important target for the discovery of novel treatments against type 2 diabetes. β-d-Glucopyranosyl derivatives have provided some of the most potent GP inhibitors discovered to date. In this regard, C -β-d-glucopyranosyl azole type inhibitors proved to be particularly effective, with 2- and 4-β-d-glucopyranosyl imidazoles among the most potent designed to date. His377 backbone C=O hydrogen bonding and ion-ion interactions of the protonated imidazole with Asp283 from the 280s loop, stabilizing the inactive state, were proposed as crucial to the observed potencies. Towards further exploring these features, 4-amino-3-(β-d-glucopyranosyl)-5-phenyl-1 H -pyrazole ( 3 ) and 3-(β-d-glucopyranosyl)-4-guanidino-5-phenyl-1 H -pyrazole ( 4 ) were designed and synthesized with the potential to exploit similar interactions. Binding assay experiments against rabbit muscle GPb revealed 3 as a moderate inhibitor (IC 50 = 565 µM), but 4 displayed no inhibition at 625 µM concentration. Towards understanding the observed inhibitions, docking and post-docking molecular mechanics-generalized Born surface area (MM-GBSA) binding free energy calculations were performed, together with Monte Carlo and density functional theory (DFT) calculations on the free unbound ligands. The computations revealed that while 3 was predicted to hydrogen bond with His377 C=O in its favoured tautomeric state, the interactions with Asp283 were not direct and there were no ion-ion interactions; for 4 , the most stable tautomer did not have the His377 backbone C=O interaction and while ion-ion interactions and direct hydrogen bonding with Asp283 were predicted, the conformational strain and entropy loss of the ligand in the bound state was significant. The importance of consideration of tautomeric states and ligand strain for glucose analogues in the confined space of the catalytic site with the 280s loop in the closed position was highlighted.

Brain-type glycogen phosphorylase (PYGB) inhibitors are recognized as prospective drugs for treating ischemic brain injury. We previously reported compound 1 as a novel glycogen phosphorylase inhibitor with brain-protective properties. In this study, we validated whether PYGB could be used as the therapeutic target for hypoxic-ischemic diseases and investigated whether compound 1 exerts a protective effect against astrocyte hypoxia/reoxygenation (H/R) injury by targeting PYGB. A gene-silencing strategy was initially applied to downregulate PYGB proteins in mouse astrocytes, which was followed by a series of cellular experiments with compound 1 . Next, we compared relevant indicators that could prove the protective effect of compound 1 on brain injury, finding that after PYGB knockdown, compound 1 could not obviously alleviate astrocytes H/R injury, as evidenced by cell viability, which was not significantly improved, and lactate dehydrogenase (LDH) leakage rate, intracellular glucose content, and post-ischemic reactive oxygen species (ROS) level, which were not remarkably reduced. At the same time, cellular energy metabolism did not improve, and the degree of extracellular acidification was not downregulated after administration of compound 1 after PYGB knockdown. In addition, it could neither significantly increase the level of mitochondrial aerobic energy metabolism nor inhibit the expression of apoptosis-associated proteins. The above results indicate that compound 1 could target PYGB to exert its protective effect against cellular H/R injury in mouse astrocytes. Simultaneously, we further demonstrated that PYGB could be an efficient therapeutic target for ischemic-hypoxic diseases. This study provides a new reference for further in-depth study of the action mechanism of the efficacy of compound 1 .

Brain-type glycogen phosphorylase inhibitors are potential new drugs for treating ischemic brain injury. In our previous study, we reported compound 1 as a novel brain-type glycogen phosphorylase inhibitor with cardioprotective properties. We also found that compound 1 has high blood-brain barrier permeability through the ADMET prediction website. In this study, we deeply analyzed the protective effect of compound 1 on hypoxic-ischemic brain injury, finding that compound 1 could alleviate the hypoxia/reoxygenation (H/R) injury of astrocytes by improving cell viability and reducing LDH leakage rate, intracellular glucose content, and post-ischemic ROS level. At the same time, compound 1 could reduce the level of ATP in brain cells after ischemia, improve cellular energy metabolism, downregulate the degree of extracellular acidification, and improve metabolic acidosis. It could also increase the level of mitochondrial aerobic energy metabolism during brain cell reperfusion, reduce anaerobic glycolysis, and inhibit apoptosis and the expression of apoptosis-related proteins. The above results indicated that compound 1 is involved in the regulation of glucose metabolism, can control cell apoptosis, and has protective and potential therapeutic effects on cerebral ischemia-reperfusion injury, which provides a new reference and possibility for the development of novel drugs for the treatment of ischemic brain injury.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths. There is an urgent need for new targets to treat HCC due to limited treatment options and drug resistance. Many cancer cells are known to have high amount of glycogen than their tissue of origin and inhibition of glycogen catabolism induces cancer cell death by apoptosis. To further understand the role of glycogen in HCC and target it for pharmacotherapy, we studied metabolic adaptations and mitochondrial function in HepG2 cells after pharmacological inhibition of glycogen phosphorylase (GP) by CP-91149 (CP). GP inhibition increased the glycogen levels in HepG2 cells without affecting overall glucose uptake. Glycolytic capacity and importantly glycolytic reserve decreased significantly. Electron microscopy revealed that CP treatment altered mitochondrial morphology leading to mitochondrial swelling with less defined cristae. A concomitant decrease in mitochondrial oxygen consumption and mitochondria-linked ATP generation was observed. Metabolomics and enzyme activity / expression studies showed a decrease in the pentose phosphate pathway. In addition, CP treatment decreased the growth of HepG2 3D tumor spheroids in a dose- and time-dependent manner. Taken together, our study provides insights into metabolic alterations and mitochondrial dysfunction accompanying apoptosis in HepG2 cells upon GP inhibition. Our study can aid in the understanding of the mechanism and development of metabolic inhibitors to treat HCC., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Astrocyte glycogen constitutes the primary energy fuel reserve in the brain. Current research investigated the novel premise that glycogen turnover governs astrocyte responsiveness to critical metabolic and neurotransmitter (norepinephrine) regulatory signals in a sex-dimorphic manner. Here, rat hypothalamic astrocyte glycogen phosphorylase (GP) gene expression was silenced by short-interfering RNA (siRNA) to investigate how glycogen metabolism controlled by GP-brain type (GPbb) or GP-muscle type (GPmm) activity affects glucose [glucose transporter-2 (GLUT2)] and energy [5'-AMP-activated protein kinase (AMPK)] sensor and adrenergic receptor (AR) proteins in each sex. Results show that in the presence of glucose, glycogen turnover is regulated by GPbb in the male or by GPmm in the female, yet in the absence of glucose, glycogen breakdown is controlled by GPbb in each sex. GLUT2 expression is governed by GPmm-mediated glycogen breakdown in glucose-supplied astrocytes of each sex, but glycogenolysis controls glucoprivic GLUT2 up-regulation in male only. GPbb-mediated glycogen disassembly causes divergent changes in total AMPK versus phosphoAMPK profiles in male. During glucoprivation, glycogenolysis up-regulates AMPK content in male astrocytes by GPbb- and GPmm-dependent mechanisms, whereas GPbb-mediated glycogen breakdown inhibits phosphoAMPK expression in female. GPbb and GPmm activity governs alpha 2 -AR and beta 1 -AR protein levels in male, but has no effect on these profiles in the female. Outcomes provide novel evidence for sex-specific glycogen regulation of glucose- and energy-sensory protein expression in hypothalamic astrocytes, and identify GP isoforms that mediate such control in each sex. Results also show that glycogen regulation of hypothalamic astrocyte receptivity to norepinephrine is male-specific. Further studies are needed to characterize the molecular mechanisms that underlie sex differences in glycogen control of astrocyte protein expression., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In the last few years, several efforts have been made to identify original strategies against glioblastoma multiforme (GBM): this requires a more detailed investigation of the molecular mechanism of GBM so that novel targets can be identified for new possible therapeutic agents. Here, using a combined biochemical and proteomic approach, we evaluated the ability of a blood-brain barrier-permeable 2,3-benzodiazepin-4-one, called 1g, to interfere with the activity and the expression of brain glycogen phosphorylase (PYGB) on U87MG cell line in parallel with the capability of this compound to inhibit the cell growth and cycle. Thus, our results highlighted PYGB as a potential therapeutic target in GBM prompting 1g as a capable anticancer drug thanks to its ability to negatively modulate the uptake and metabolism of glucose, the so-called "Warburg effect", whose increase is considered a common feature of cancer cells in respect of their normal counterparts.

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10-12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs., (© 2022. The Author(s).)

Astrocytic glycogen serves as an important glucose reserve, and its degradation provides extra support for neighboring neurons during energy deficiency. Salvianolic acid B (SAB) exerts a neuroprotective effect on reperfusion insult after cerebrovascular occlusion, but the effect of SAB on astrocytic glycogen and its relationship with neuroprotection are not completely understood. Here, we knocked down astrocyte-specific glycogen phosphorylase (GP, the rate-limiting enzyme in glycogenolysis) in vitro and in vivo and investigated the changes in key enzymes in glycogen metabolism by performing immunoblotting in vitro and immunofluorescence in vivo. Neurobehavioral and morphological assessments were conducted to uncover the outcomes during brain reperfusion. SAB accelerated astrocytic glycogenolysis by upregulating GP activity but not GP expression after reperfusion. Suppression of astrocytic glycogenolysis weakened SAB-mediated neuroprotection against the reperfusion insult. In addition, activation of glycogenolysis by SAB contributed to the survival of astrocytes and surrounding neurons by increasing antioxidant levels in astrocytes. Our data reveal that astrocytic GP represents an important metabolic target in SAB-induced protection against brain damage after cerebrovascular recanalization., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The release of extracellular vesicles (EVs) is increased under cellular stress and cardiomyocyte damaging conditions. However, whether the cardiomyocyte-derived EVs eventually reach the systemic circulation and whether their number in the bloodstream reflects cardiac injury, remains unknown. Wild type C57B/6 and conditional transgenic mice expressing green fluorescent protein (GFP) by cardiomyocytes were studied in lipopolysaccharide (LPS)-induced systemic inflammatory response syndrome (SIRS). EVs were separated both from platelet-free plasma and from the conditioned medium of isolated cardiomyocytes of the left ventricular wall. Size distribution and concentration of the released particles were determined by Nanoparticle Tracking Analysis. The presence of GFP + cardiomyocyte-derived circulating EVs was monitored by flow cytometry and cardiac function was assessed by echocardiography. In LPS-treated mice, systemic inflammation and the consequent cardiomyopathy were verified by elevated plasma levels of TNFα, GDF-15, and cardiac troponin I, and by a decrease in the ejection fraction. Furthermore, we demonstrated elevated levels of circulating small- and medium-sized EVs in the LPS-injected mice. Importantly, we detected GFP + cardiomyocyte-derived EVs in the circulation of control mice, and the number of these circulating GFP + vesicles increased significantly upon intraperitoneal LPS administration (P = 0.029). The cardiomyocyte-derived GFP + EVs were also positive for intravesicular troponin I (cTnI) and muscle-associated glycogen phosphorylase (PYGM). This is the first direct demonstration that cardiomyocyte-derived EVs are present in the circulation and that the increased number of cardiac-derived EVs in the blood reflects cardiac injury in LPS-induced systemic inflammation (SIRS)., (© 2022. The Author(s).)

Objective: Accumulating evidence indicates that an adverse perinatal environment contributes to a higher risk of metabolic disorders in the later life of the offspring. However, the underlying molecular mechanisms remain largely unknown. Thus, we investigated the contribution of maternal high-calorie diet and osteocalcin to metabolic homeostasis in the offspring., Methods: Eight-week-old C57Bl/6N female mice were mated with age-matched males and allocated randomly to three groups: a normal-diet (ND) or a high-fat, high-sucrose diet group, which was administered either saline (control) or GluOC (10 ng/g body mass) from the day of mating to that of delivery, and the dams were fed a ND after the delivery. Pups weaned at 24 days after birth were analyzed., Results: A maternal high-fat, high-sucrose diet during pregnancy causes metabolic disorders in the liver of the offspring via hypermethylation of the Pygl gene, encoding glycogen phosphorylase L, which mediates hepatic glycogenolysis. The reduced expression of Pygl induced by the maternal diet causes the hepatic accumulation of glycogen and triglyceride in the offspring, which remains in adulthood. In addition, the administration of uncarboxylated osteocalcin during pregnancy upregulates Pygl expression via both direct CREBH and ATF4 and indirect epigenomic pathways, mitigating the maternal diet-induced obesity and abnormal glucose and lipid metabolism in adulthood., Conclusions: We propose that maternal energy status is reflected in the hepatic glycogenolysis capacity of the offspring via epigenetic modification of Pygl and uncarboxylated osteocalcin regulates glycogenolysis., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Glucose consumption is generally increased in tumor cells to support tumor growth. Interestingly, we report that glycogen accumulation is a key initiating oncogenic event during liver malignant transformation. We found that glucose-6-phosphatase (G6PC) catalyzing the last step of glycogenolysis is frequently downregulated to augment glucose storage in pre-malignant cells. Accumulated glycogen undergoes liquid-liquid phase separation, which results in the assembly of the Laforin-Mst1/2 complex and consequently sequesters Hippo kinases Mst1/2 in glycogen liquid droplets to relieve their inhibition on Yap. Moreover, G6PC or another glycogenolysis enzyme-liver glycogen phosphorylase (PYGL) deficiency in both human and mice results in glycogen storage disease along with liver enlargement and tumorigenesis in a Yap-dependent manner. Consistently, elimination of glycogen accumulation abrogates liver growth and cancer incidence, whereas increasing glycogen storage accelerates tumorigenesis. Thus, we concluded that cancer-initiating cells adapt a glycogen storing mode, which blocks Hippo signaling through glycogen phase separation to augment tumor incidence., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The crystal structures of free T-state and R-state glycogen phosphorylase (GP) and of R-state GP in complex with the allosteric activators IMP and AMP are reported at improved resolution. GP is a validated pharmaceutical target for the development of antihyperglycaemic agents, and the reported structures may have a significant impact on structure-based drug-design efforts. Comparisons with previously reported structures at lower resolution reveal the detailed conformation of important structural features in the allosteric transition of GP from the T-state to the R-state. The conformation of the N-terminal segment (residues 7-17), the position of which was not located in previous T-state structures, was revealed to form an α-helix (now termed α0). The conformation of this segment (which contains Ser14, phosphorylation of which leads to the activation of GP) is significantly different between the T-state and the R-state, pointing in opposite directions. In the T-state it is packed between helices α4 and α16 (residues 104-115 and 497-508, respectively), while in the R-state it is packed against helix α1 (residues 22'-38') and towards the loop connecting helices α4' and α5' of the neighbouring subunit. The allosteric binding site where AMP and IMP bind is formed by the ordering of a loop (residues 313-326) which is disordered in the free structure, and adopts a conformation dictated mainly by the type of nucleotide that binds at this site.

Repeated early environmental deprivation is regarded as a typical paradigm to mimic the behavioral abnormalities and brain dysfunction that occur in psychiatric disorders. Previously, we reported that social isolation could disrupt prepulse inhibition (PPI) in Sprague-Dawley (SD) rats, producing the typical characteristics of a schizophrenia animal model. Based on further analysis of previous proteomic and transcriptomic data, a disrupted balance of glucose metabolism was found in the prefrontal cortex (PFC) of isolated rats. Subsequently, in the first experiment of this study, we investigated the effects of juvenile social isolation (postnatal days (PND) 21-34) on PPI and lactate levels in PND56 rats. Compared with the social rearing group, rats in the isolated rearing group showed disrupted PPI and increased lactate levels in the PFC. In the second experiment, at PND55, the model rats were acutely injected with a glycogen phosphorylase inhibitor (4-dideoxy-1,4-imino-darabinitol, DAB) or control saline in the bilateral PFC. Our data showed that acute DAB administration (50 pmol, 0.5 μl) significantly improved the disrupted PPI and decreased the levels of oxidative phosphorylation (OXPHOS)-related mRNAs as well as lactate. In summary, our results suggested that excess astrocytic lactate production was involved in the impairment of auditory sensory gating of isolated rats, which may contribute to the metabolic pathogenesis of schizophrenia., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The objective of the present study was to compare skeletal muscle proteomic profiles, histochemical characteristics, and expression levels of myogenic regulatory factors (MRFs) between fast- versus slow-growing yellow perch Perca flavescens and identify the proteins/peptides that might play a crucial role in the muscle growth dynamic. Yellow perch were nursed in ponds for 6 weeks from larval stage and cultured in two meter diameter tanks thereafter. The fingerlings were graded to select the top 10% and bottom 10% fish which represented fast- and slow-growing groups (31 yellow perch per each group). Our statistical analyses showed 18 proteins that had different staining intensities between fast- and slow-growing yellow perch. From those proteins 10 showed higher expression in slow-growers, and 8 demonstrated higher expression in fast-growers. Fast-growing yellow perch with a greater body weight was influenced by both the muscle fiber hypertrophy and mosaic hyperplasia compared to slow-growing fish. These hyperplastic and hypertrophic growth in fast-grower were associated with not only metabolic enzymes, including creatine kinase, glycogen phosphorylase, and aldolase, but also myoD and myogenin as MRFs. Overall, the results of the present study contribute to the identification of different expression patterns of gene products in fast- and slow-growing fish associated with their muscle growth., (© 2021. The Author(s).)

Astrocyte glycogen, the primary energy reserve in brain, undergoes continuous remodeling by glucose passage through the glycogen shunt prior to conversion to the oxidizable energy fuel L-lactate. Glucogenic amino acids (GAAs) are a potential non-glucose energy source during neuro-metabolic instability. Current research investigated whether diminished glycogen metabolism affects GAA homeostasis in astrocyte and/or nerve cell compartments. The glycogen phosphorylase (GP) inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) was injected into the ventromedial hypothalamic nucleus (VMN), a key metabolic-sensing structure, before vehicle or L-lactate infusion. Pure VMN astrocyte and metabolic-sensory neuron samples were obtained by combinatory immunocytochemistry/laser-catapult-microdissection for UHPLC-electrospray ionization-mass spectrometry (LC-ESI-MS) GAA analysis. DAB inhibition of VMN astrocyte aspartate and glutamine (Gln) levels was prevented or exacerbated, respectively, by lactate. VMN gluco-stimulatory nitric oxide (NO; neuronal nitric oxide synthase-immunoreactive (ir)-positive) and gluco-inhibitory γ-aminobutyric acid (GABA; glutamate decarboxylase 65/67 -ir-positive) neurons exhibited lactate-reversible asparate and glutamate augmentation by DAB, but dissimilar Gln responses to DAB. GP inhibition elevated NO and GABA nerve cell GABA content, but diminished astrocyte GABA; these responses were averted by lactate in neuron, but not astrocyte samples. Outcomes provide proof-of-principle of requisite LC-ESI-MS sensitivity for GAA measurement in specific brain cell populations. Results document divergent effects of decreased VMN glycogen breakdown on astrocyte versus neuron GAAs excepting Gln. Lactate-reversible DAB up-regulation of metabolic-sensory neuron GABA signaling may reflect compensatory nerve cell energy stabilization upon decline in astrocyte-derived metabolic fuel., (© 2021. The Author(s).)

The purine alkaloid caffeine is the most widely consumed psychostimulant drug in the world and has multiple beneficial pharmacological activities, for example, in neurodegenerative diseases. However, despite being an extensively studied bioactive natural product, the mechanistic understanding of caffeine's pharmacological effects is incomplete. While several molecular targets of caffeine such as adenosine receptors and phosphodiesterases have been known for decades and inspired numerous medicinal chemistry programs, new protein interactions of the xanthine are continuously discovered providing potentially improved pharmacological understanding and a molecular basis for future medicinal chemistry. In this Perspective, we gather knowledge on the confirmed protein interactions, structure activity relationship, and chemical biology of caffeine on well-known and upcoming targets. The diversity of caffeine's molecular activities on receptors and enzymes, many of which are abundant in the CNS, indicates a complex interplay of several mechanisms contributing to neuroprotective effects and highlights new targets as attractive subjects for drug discovery.

Brain glycogen has a long and versatile history: Primarily regarded as an evolutionary remnant, it was then thought of as an unspecific emergency fuel store. A dynamic role for glycogen in normal brain function has been proposed later but exclusively attributed to astrocytes, its main storage site. Neuronal glycogen had long been neglected, but came into focus when sensitive technical methods allowed quantification of glycogen at low concentration range and the detection of glycogen metabolizing enzymes in cells and cell lysates. Recently, an active role of neuronal glycogen and even its contribution to neuronal survival could be demonstrated. We used the neuronal cell lines NSC-34 and N18TG2 and could demonstrate that they express the key-enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase and contain glycogen which is mobilized on glucose deprivation and elevated potassium concentrations, but not by hormones stimulating cAMP formation. Conditions of metabolic stress, namely hypoxia, oxidative stress and pH lowering, induce glycogen degradation. Our studies revealed that glycogen can contribute to the energy supply of neuronal cell lines in situations of metabolic stress. These findings shed new light on the so far neglected role of neuronal glycogen. The key-enzyme in glycogen degradation is glycogen phosphorylase. Neurons express only the brain isoform of the enzyme that is supposed to be activated primarily by the allosteric activator AMP and less by covalent phosphorylation via the cAMP cascade. Our results indicate that neuronal glycogen is not degraded upon hormone action but by factors lowering the energy charge of the cells directly.

Glycogen phosphorylase (GP) is a key enzyme of glycogen catabolism, so it is significant to discover a new GP inhibitor. A series of benzazepinone derivatives were discovered as GP inhibitors with potent activity. Among these derivatives, compound 5d showed significant potential against rabbit muscle GPa (IC 50  = 0.25 ± 0.05 μM) and cellular efficacy. The in vivo study revealed that 5d significantly inhibited increases in fasting blood glucose level in two kinds of hyperglycemic mice models. The possible binding mode of compound 5d was explored based on molecular docking simulations. These results indicated that derivatives with benzazepinone were potential chemical entities against hyperglycemia.

Novel pharmacological strategies for the treatment of diabetic patients are now focusing on inhibiting glycogenolysis steps. In this regard, glycogen phosphorylase (GP) is a validated target for the discovery of innovative antihyperglycemic molecules. Natural products, and in particular flavonoids, have been reported as potent inhibitors of GP at the cellular level. Herein, free-energy calculations and microscale thermophoresis approaches were performed to get an in-depth assessment of the binding affinities and elucidate intermolecular interactions of several flavonoids at the inhibitor site of GP. To our knowledge, this is the first study indicating genistein, 8-prenylgenistein, apigenin, 8-prenylapigenin, 8-prenylnaringenin, galangin and valoneic acid dilactone as natural molecules with high inhibitory potency toward GP. We identified: i) the residues Phe285, Tyr613, Glu382 and/or Arg770 as the most relevant for the binding of the best flavonoids to the inhibitor site of GP, and ii) the 5-OH, 7-OH, 8-prenyl substitutions in ring A and the 4'-OH insertion in ring B to favor flavonoid binding at this site. Our results are invaluable to plan further structural modifications through organic synthesis approaches and develop more effective pharmaceuticals for Type 2 Diabetes treatment, and serve as the starting point for the exploration of food products for therapeutic usage, as well as for the development of novel bio-functional food and dietary supplements/herbal medicines., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Small GTPases, together with their regulatory and effector molecules, are key intermediaries in the complex signalling pathways that control almost all cellular processes, working as molecular switches to transduce extracellular cues into cellular responses that drive vital functions, such as intracellular transport, biomolecule synthesis, gene activation and cell survival. How all of these networks are linked to metabolic pathways is a subject of intensive study. Because any response to cellular action requires some form of energy input, elucidating how cells coordinate the signals that lead to a tangible response involving metabolism is central to understand cellular activities. In this review, we summarize recent advances in our understanding of the molecular basis of the crosstalk between small GTPases of the Ras superfamily, specifically Rac1 and Ras/Rap1, and glycogen phosphorylase in T lymphocytes. Abbreviations : ADCY : adenylyl cyclase; ADCY6 : adenylyl cyclase 6; BCR : B cell receptor; cAMP : 3',5'-cyclic adenosine monophosphate; CRIB : Cdc42/Rac binding domain; DLPFC : dysfunction of the dorsolateral prefrontal cortex; EGFR : epidermal growth factor receptor; Epac2 : exchange protein directly activated by cAMP; GDP : guanodine-5'-diphosphate; GPCRs : G protein-coupled receptors; GTP : guanodin-5'-triphosphate; IL2 : interleukin 2; IL2-R : interleukin 2 receptor; JAK : janus kinases; MAPK : mitogen-activated protein kinase; O-GlcNAc : O-glycosylation; PAK1 : p21 activated kinase 1; PI3K : phosphatidylinositol 3-kinase; PK : phosphorylase kinase; PKA : cAMP-dependent protein kinase A; PKCθ : protein kinase Cθ; PLCγ : phospholipase Cγ; Src : proto-oncogene tyrosine-protein kinase c; STAT : signal transducer and activator of transcription proteins.

Glycogen metabolism shapes ventromedial hypothalamic nucleus (VMN) control of glucose homeostasis. Brain glycogen mass undergoes compensatory expansion post‑recovery from insulin‑induced hypoglycemia (IIH). Current research utilized combinatory high‑resolution microdissection/high‑sensitivity Western blotting to investigate whether IIH causes residual adjustments in glycogen metabolism within the metabolic‑sensory ventrolateral VMN (VMNvl). Micropunch‑dissected tissue was collected from rostral, middle, and caudal levels of the VMNvl in each sex for analysis of glycogen synthase (GS) and glycogen phosphorylase (GP)‑muscle type (GPmm; norepinephrine‑sensitive) and GP‑brain type (GPbb; glucoprivic‑sensitive) isoform expression during and after IIH. Hypoglycemic suppression of VMNvl GS levels in males disappeared or continued after reestablishment of euglycemia, according to sampled segment. Yet, reductions in female VMNvl GS persisted after IIH. Males exhibited reductions in GPmm content in select rostro‑caudal VMNvl segments, but this protein declined in each segment post‑hypoglycemia. Females, rather, showed augmented or diminished GPmm levels during IIH, but no residual effects of IIH on this protein. In each sex, region‑specific up‑ or down‑regulation of VMNvl GPbb profiles during glucose decrements were undetected post‑recovery from IIH. Results provide novel proof of estradiol‑dependent sex‑dimorphic patterns of VMNvl GP variant expression at specific rostro‑caudal levels of this critical gluco‑regulatory structure. Sex differences in persistence of IIH‑associated GS and GPmm patterns of expression after restoration of euglycemia infer that VMNvl recovery from this metabolic stress may involve dissimilar glycogen accumulation in male versus female.

Background: Pyranoid spirofused sugar derivatives represent a class of compounds with a significant impact in the literature. From the structural point of view, the rigidity inferred by the spirofused entity has made these compounds object of interest mainly as enzymatic inhibitors, in particular, carbohydrate processing enzymes. Among them glycogen phosphorylase and sodium glucose co-transporter 2 are important target enzymes for diverse pathological states. Most of the developed compounds present the spirofused entity at the C1 position of the sugar moiety; nevertheless, spirofused entities can also be found at other sugar ring positions. The main spirofused entities encountered are spiroacetals/thioacetals, spiro-hydantoin and derivatives, spiro-isoxazolines, spiro-aminals, spiro-lactams, spiro-oxathiazole and spiro-oxazinanone, but also others are present., Objectives: The present review focuses on the most explored synthetic strategies for the preparation of this class of compounds, classified according to the position and structure of the spirofused moiety on the pyranoid scaffold. Moreover, the structures are correlated to their main biological activities or to their role as chiral auxiliaries., Conclusion: It is clear from the review that, among the different derivatives, the spirofused structures at position C1 of the pyranoid scaffold are the most represented and possess the most relevant enzymatic inhibitor activities. Nevertheless, great efforts have been devoted to the introduction of the spirofused entity also in the other positions, mainly for the preparation of biologically active compounds but also for the synthesis of chiral auxiliaries useful in asymmetric reactions; examples of such auxiliaries are the spirofused chiral 1,3-oxazolidin-2-ones and 1,3-oxazolidine-2-thiones., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

General anesthesia severely affects the metabolites in the brain. Glycogen, principally stored in astrocytes and providing the short-term delivery of substrates to neurons, has been implicated as an affected molecule. However, whether glycogen plays a pivotal role in modulating anesthesia-arousal remains unclear. Here, we demonstrated that isoflurane-anesthetized mice exhibited dynamic changes in the glycogen levels in various brain regions. Glycogen synthase (GS) and glycogen phosphorylase (GP), key enzymes of glycogen metabolism, showed increased activity after isoflurane exposure. Upon blocking glycogenolysis with 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), a GP antagonist, we found a prolonged time of emergence from anesthesia and an enhanced δ frequency in the EEG (electroencephalogram). In addition, augmented expression of glycogenolysis genes in glycogen phosphorylase, brain (Pygb) knock-in (Pygb H11/H11 ) mice resulted in delayed induction of anesthesia, a shortened emergence time, and a lower ratio of EEG-δ. Our findings revealed a role of brain glycogen in regulating anesthesia-arousal, providing a potential target for modulating anesthesia.

We have analyzed protein expression and enzyme activity of the sarcoplasmic reticulum Ca 2+ -transporting ATPase (SERCA) in horse gluteal muscle. Horses exhibit a high incidence of recurrent exertional rhabdomyolysis, with myosolic Ca 2+ proposed, but yet to be established, as the underlying cause. To better assess Ca 2+ regulatory mechanisms, we developed an improved protocol for isolating sarcoplasmic reticulum (SR) vesicles from horse skeletal muscle, based on mechanical homogenization and optimized parameters for differential centrifugation. Immunoblotting identified the peak subcellular fraction containing the SERCA1 protein (fast-twitch isoform). Gel analysis using the Stains-all dye demonstrated that calsequestrin (CASQ) and phospholipids are highly enriched in the SERCA-containing subcellular fraction isolated from horse gluteus. Immunoblotting also demonstrated that these horse SR vesicles show low content of glycogen phosphorylase (GP), which is likely an abundant contaminating protein of traditional horse SR preps. The maximal Ca 2+ -activated ATPase activity (V max ) of SERCA in horse SR vesicles isolated using this protocol is 5‒25-fold greater than previously-reported SERCA activity in SR preps from horse skeletal muscle. We propose that this new protocol for isolating SR vesicles will be useful for determining enzymatic parameters of horse SERCA with high fidelity, plus assessing regulatory effect of SERCA peptide subunit(s) expressed in horse muscle., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Brain-type glycogen phosphorylase (PYGB) has been correlated with the progression of various human malignancies; however, its effects and regulatory mechanisms in non-small cell lung cancer (NSCLC) are still unclear. We used Western blotting, immunohistochemistry, and qRT-PCR to verify that the protein and mRNA expression levels of PYGB are up-regulated in both NSCLC cell lines and tissues. The expression of PYGB was positively related to TNM stage, positive lymph node metastasis, and poor prognosis in patients with NSCLC. Moreover, overexpression of PYGB promoted cell proliferation, migration, and invasiveness, but inhibited apoptosis, in vitro. Immunofluorescence assays showed that overexpression of PYGB promoted the nuclear import and accumulation of β-catenin. By comparison, silencing PYGB produced the opposite effects. Further, overexpression of PYGB resulted in activation of the Wnt signaling pathway, and transfection with Sh-PYGB produced the opposite effect, and these effects were abrogated by XAV-939 (a β-catenin inhibitor) or overexpression of β-catenin, respectively. Finally, knockdown of PYGB inhibited tumor growth in a mouse model of xenograft tumors. These findings highlight the role of PYGB in the progression of NSCLC, and reveal a link between PYGB and the Wnt-β-catenin signaling pathway, thus providing a new potential target for treatment of NSCLC.

Type 2 diabetes mellitus (T2DM), one of the most common metabolic diseases, is characterized by insulin resistance and inadequate insulin secretion of β cells. Glycogen phosphorylase (GP) is the key enzyme in glycogen breakdown, and contributes to hepatic glucose production during fasting or during insulin resistance. Pharmacological GP inhibitors are potential glucose lowering agents, which may be used in T2DM therapy. A natural product isolated from the cultured broth of the fungal strain No. 138354, called 2,3-bis(4-hydroxycinnamoyloxy)glutaric acid (FR258900), was discovered a decade ago. In vivo studies showed that FR258900 significantly reduced blood glucose levels in diabetic mice. We previously showed that GP inhibitors can potently enhance the function of β cells. The purpose of this study was to assess whether an analogue of FR258900 can influence β cell function. BF142 (Meso-Dimethyl 2,3-bis[(E)-3-(4-acetoxyphenyl)prop-2-enamido]butanedioate) treatment activated the glucose-stimulated insulin secretion pathway, as indicated by enhanced glycolysis, increased mitochondrial oxidation, significantly increased ATP production, and elevated calcium influx in MIN6 cells. Furthermore, BF142 induced mTORC1-specific phosphorylation of S6K, increased levels of PDX1 and insulin protein, and increased insulin secretion. Our data suggest that BF142 can influence β cell function and can support the insulin producing ability of β cells., Competing Interests: The authors have declared that no competing interests exist.

McArdle disease results from a lack of muscle glycogen phosphorylase in skeletal muscle tissue. Regenerating skeletal muscle fibres can express the brain glycogen phosphorylase isoenzyme. Stimulating expression of this enzyme could be a therapeutic strategy. Animal model studies indicate that sodium valproate (VPA) can increase expression of phosphorylase in skeletal muscle affected with McArdle disease. This study was designed to assess whether VPA can modify expression of brain phosphorylase isoenzyme in people with McArdle disease. This phase II, open label, feasibility pilot study to assess efficacy of six months treatment with VPA (20 mg/kg/day) included 16 people with McArdle disease. Primary outcome assessed changes in VO 2 peak during an incremental cycle test. Secondary outcomes included: phosphorylase enzyme expression in post-treatment muscle biopsy, total distance walked in 12 min, plasma lactate change (forearm exercise test) and quality of life (SF36). Safety parameters. 14 participants completed the trial, VPA treatment was well tolerated; weight gain was the most frequently reported drug-related adverse event. There was no clinically meaningful change in any of the primary or secondary outcome measures including: VO 2 peak, 12 min walk test and muscle biopsy to look for a change in the number of phosphorylase positive fibres between baseline and 6 months of treatment. Although this was a small open label feasibility study, it suggests that a larger randomised controlled study of VPA, may not be worthwhile., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A small set of indole-2-carboxamide derivatives identified from a high-throughput screening campaign has been described as a novel, potent, and glucose-sensitive inhibitors of glycogen phosphorylase a (GPa). Among this series of compounds, compound 2 exhibited moderate GP inhibitory activity (IC 50  = 0.29 μM), good cellular efficacy (IC 50  = 3.24 μM for HepG2 cells and IC 50  = 7.15 μM for isolated rat hepatocytes), together with good absorption, distribution, metabolism, and elimination (ADME) profiles. The in vivo animal study revealed that compound 2 significantly inhibited an increase of fasting blood glucose level in adrenaline-induced diabetic mice., 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 © 2020. Published by Elsevier Ltd.)