Glycogen Phosphorylase Inhibitors Research Articles

Human hair follicles operate an internal Cori cycle and modulate their growth via glycogen phosphorylase

Hair follicles (HFs) are unique, multi-compartment, mini-organs that cycle through phases of active hair growth and pigmentation (anagen), apoptosis-driven regression (catagen) and relative quiescence (telogen). Anagen HFs have high demands for energy and biosynthesis precursors mainly fulfilled by aerobic glycolysis. Histochemistry reports the outer root sheath (ORS) contains high levels of glycogen. To investigate a functional role for glycogen in the HF we quantified glycogen by Periodic-Acid Schiff (PAS) histomorphometry and colorimetric quantitative assay showing ORS of anagen VI HFs contained high levels of glycogen that decreased in catagen. qPCR and immunofluorescence microscopy showed the ORS expressed all enzymes for glycogen synthesis and metabolism. Using human ORS keratinocytes (ORS-KC) and ex vivo human HF organ culture we showed active glycogen metabolism by nutrient starvation and use of a specific glycogen phosphorylase (PYGL) inhibitor. Glycogen in ORS-KC was significantly increased by incubation with lactate demonstrating a functional Cori cycle. Inhibition of PYGL significantly stimulated the ex vivo growth of HFs and delayed onset of catagen. This study defines translationally relevant and therapeutically targetable new features of HF metabolism showing that human scalp HFs operate an internal Cori cycle, synthesize glycogen in the presence of lactate and modulate their growth via PYGL activity.

Exercise-Induced Lactate Release Mediates Mitochondrial Biogenesis in the Hippocampus of Mice via Monocarboxylate Transporters.

Regular exercise training induces mitochondrial biogenesis in the brain via activation of peroxisome proliferator-activated receptor gamma-coactivator 1α (PGC-1α). However, it remains unclear whether a single bout of exercise would increase mitochondrial biogenesis in the brain. Therefore, we first investigated whether mitochondrial biogenesis in the hippocampus is affected by a single bout of exercise in mice. A single bout of high-intensity exercise, but not low- or moderate-intensity, increased hippocampal PGC-1α mRNA and mitochondrial DNA (mtDNA) copy number at 12 and 48h. These results depended on exercise intensity, and blood lactate levels observed immediately after exercise. As lactate induces mitochondrial biogenesis in the brain, we examined the effects of acute lactate administration on blood and hippocampal extracellular lactate concentration by in vivo microdialysis. Intraperitoneal (I.P.) lactate injection increased hippocampal extracellular lactate concentration to the same as blood lactate level, promoting PGC-1α mRNA expression in the hippocampus. However, this was suppressed by administering UK5099, a lactate transporter inhibitor, before lactate injection. I.P. UK5099 administration did not affect running performance and blood lactate concentration immediately after exercise but attenuated exercise-induced hippocampal PGC-1α mRNA and mtDNA copy number. In addition, hippocampal monocarboxylate transporters (MCT)1, MCT2, and brain-derived neurotrophic factor (BDNF) mRNA expression, except MCT4, also increased after high-intensity exercise, which was abolished by UK5099 administration. Further, injection of 1,4-dideoxy-1,4-imino-D-arabinitol (glycogen phosphorylase inhibitor) into the hippocampus before high-intensity exercise suppressed glycogen consumption during exercise, but hippocampal lactate, PGC-1α, MCT1, and MCT2 mRNA concentrations were not altered after exercise. These results indicate that the increased blood lactate released from skeletal muscle may induce hippocampal mitochondrial biogenesis and BDNF expression by inducing MCT expression in mice, especially during short-term high-intensity exercise. Thus, a single bout of exercise above the lactate threshold could provide an effective strategy for increasing mitochondrial biogenesis in the hippocampus.

UHPLC\u2013electrospray ionization\u2013mass spectrometric analysis of brain cell-specific glucogenic and neurotransmitter amino acid content

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 decarboxylase65/67-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.

Mechanochemical Synthesis and Reactivity of 1,2,3-Triazole Carbohydrate Derivatives as Glycogen Phosphorylase Inhibitors.

We have developed this work to recommend an original route for the preparation of triazole derivatives. Carbohydrates containing 1,2,3-triazole derivatives have various biological activities. Due to their advantageous and biological property, they are eye-catching synthetic targets in the arsenal of organic chemistry. Thus, finding green and efficient methods, as well as using the ball milling procedure for the synthesis of these heterocycles, is of interest to organic chemistry researchers. The objective of this study was to synthesize carbohydrate-derived triazoles under high-speed vibration milling conditions and investigate their properties. A mixture of glycoside azide derivatives (1 mmol) and prop-2-yn-1-ol (1.5 mmol) in the presence of copper (I) was vigorously shaken under vibration milling conditions at 650 rpm with three balls for 15 min. The deprotection of the resulting triazole derivatives was affected by treatment with 4M hydrochloric acid in methanol under reflux. A short and convenient route to synthesize carbohydrate-derived triazoles, based on a ball-mill via 1,3-dipolar cycloaddition reactions to prop-2-yn-1-ol, was developed. Cleavage of the isopropylidene protecting group provided water-soluble triazoles, evaluated as glycogen phosphorylase inhibitors. 1-[6- (4-Hydroxymethyl-[1,2,3]triazol-1-yl)-2,2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxol-4-yl]-ethane-1,2-diol was the best inhibitor of rabbit muscle glycogen phosphorylase b (IC50 = 60 μM). In summary, we developed new, short and convenient routes to glucose-derived 1,2,3-triazole based on 1,3-dipolar cycloaddition reactions flowed by ball milling. The use of isopropylidene protective groups gave access to the analogous deprotected water-soluble motifs, analogous to known inhibitors of glycogen phosphorylase.

Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay.

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.

Juvenile social isolation leads to schizophrenia-like behaviors via excess lactate production by astrocytes

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.

Glycogen synthesis from dihydroxyacetone in isolated rat hepatocytes

Glycogenolysis and gluconeogenesis are sensitive to nutritional state and their balances are disrupted in the liver by different pathological states. The regulation of the balance between glucose production and synthesis of glycogen continues to be a matter of investigation because of its implications in diseases and its value for therapeutics. We used the gluconeogenic precursor dihydroxyacetone (DHA) to study glycogen synthesis in isolated rat hepatocytes under gluconeogenic conditions. We used a glycogen phosphorylase (GP) inhibitor, 2-deoxy-2-fluoro-α-D-glucopyranosyl fluoride (F2Glc) to prevented GP from breaking up glycogen into glucose subunits and had a pool of glucose in the medium. Therefore, we evaluated the contribution of DHA as a unique source of carbohydrates on glycogen metabolism. We showed that DHA increased G6P levels that induced both GS activation and its translocation and, thus, an increment of glycogen deposition in a similar way as glucose did.

Targeting Glycogen Metabolism as a Novel Therapeutic Approach in Anaplastic Thyroid Cancer

Effective treatment options for well-differentiated papillary (PTC) and follicular (FTC) thyroid cancers afford positive patient prognoses. The absence of effective interventions for the stem-like, dedifferentiated anaplastic thyroid cancer (ATC) results in poor patient outcomes with a mortality rate higher than all other endocrine cancers combined (1). While receptor tyrosine kinase inhibitors such as sorafenib can extend ATC patient survival, drug resistance and tumor reoccurrence often develop (2). Therefore, there is a critical need for more effective targeted therapies for ATC. Although the cell signaling landscape of ATC tumors is well described, very little is known about tumorigenic adaptations in ATC cellular metabolism. Tumors exhibit an increased consumption of glucose compared to normal tissues to fuel tumor progression. Some cancers meet this high glucose requirement by storing and breaking down glycogen. In our studies here, we show for the first time that normal thyroid, PTC, FTC, and ATC cells express genes necessary for glycogen metabolism. We confirm these observations in patient samples in normal thyroid and thyroid cancer patient samples via immunofluorescence in tissue microarrays. Furthermore, we detect intracellular glycogen stores in cell lines representing normal thyroid, PTC, FTC, and ATC cells. Importantly, we demonstrate that glycogen phosphorylase inhibitors result in accumulation of intracellular glycogen and induce subsequent apoptosis in ATC cells. We further show that glycogen phosphorylase inhibitors synergize with kinase inhibitors such as sorafenib and buparlisib to decrease ATC cell viability. Our work establishes glycogen metabolism as a novel metabolic process in thyroid cells that is associated with thyroid cancer dedifferentiation and provides insight to the effectiveness of inhibiting glycogen metabolism as a therapeutic strategy in ATC.

Discovery and evaluation of novel benzazepinone derivatives as glycogen phosphorylase inhibitors with potent activity.

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 (IC50=0.25±0.05μM) 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.

Dual-Target Compounds against Type 2 Diabetes Mellitus: Proof of Concept for Sodium Dependent Glucose Transporter (SGLT) and Glycogen Phosphorylase (GP) Inhibitors.

A current trend in the quest for new therapies for complex, multifactorial diseases, such as diabetes mellitus (DM), is to find dual or even multi-target inhibitors. In DM, the sodium dependent glucose cotransporter 2 (SGLT2) in the kidneys and the glycogen phosphorylase (GP) in the liver are validated targets. Several (β-D-glucopyranosylaryl)methyl (het)arene type compounds, called gliflozins, are marketed drugs that target SGLT2. For GP, low nanomolar glucose analogue inhibitors exist. The purpose of this study was to identify dual acting compounds which inhibit both SGLTs and GP. To this end, we have extended the structure-activity relationships of SGLT2 and GP inhibitors to scarcely known (C-β-D-glucopyranosylhetaryl)methyl arene type compounds and studied several (C-β-D-glucopyranosylhetaryl)arene type GP inhibitors against SGLT. New compounds, such as 5-arylmethyl-3-(β-D-glucopyranosyl)-1,2,4-oxadiazoles, 5-arylmethyl-2-(β-D-glucopyranosyl)-1,3,4-oxadiazoles, 4-arylmethyl-2-(β-D-glucopyranosyl)pyrimidines and 4(5)-benzyl-2-(β-D-glucopyranosyl)imidazole were prepared by adapting our previous synthetic methods. None of the studied compounds exhibited cytotoxicity and all of them were assayed for their SGLT1 and 2 inhibitory potentials in a SGLT-overexpressing TSA201 cell system. GP inhibition was also determined by known methods. Several newly synthesized (C-β-D-glucopyranosylhetaryl)methyl arene derivatives had low micromolar SGLT2 inhibitory activity; however, none of these compounds inhibited GP. On the other hand, several (C-β-D-glucopyranosylhetaryl)arene type GP inhibitor compounds with low micromolar efficacy against SGLT2 were identified. The best dual inhibitor, 2-(β-D-glucopyranosyl)-4(5)-(2-naphthyl)-imidazole, had a Ki of 31 nM for GP and IC50 of 3.5 μM for SGLT2. This first example of an SGLT-GP dual inhibitor can prospectively be developed into even more efficient dual-target compounds with potential applications in future antidiabetic therapy.

Ventromedial hypothalamic nucleus glycogen regulation of metabolic-sensory neuron AMPK and neurotransmitter expression: role of lactate.

Astrocyte glycogen is dynamically remodeled during metabolic stability and provides oxidizable l-lactate equivalents during neuroglucopenia. Current research investigated the hypothesis that ventromedial hypothalamic nucleus (VMN) glycogen metabolism controls glucostimulatory nitric oxide (NO) and/or glucoinhibitory gamma-aminobutyric acid (GABA) neuron 5'-AMP-activated protein kinase (AMPK) and transmitter marker, e.g., neuronal nitric oxide synthase (nNOS), and glutamate decarboxylase65/67 (GAD) protein expression. Adult ovariectomized estradiol-implanted female rats were injected into the VMN with the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) before vehicle or l-lactate infusion. Western blot analysis of laser-catapult-microdissected nitrergic and GABAergic neurons showed that DAB caused lactate-reversible upregulation of nNOS and GAD proteins. DAB suppressed or increased total AMPK content of NO and GABA neurons, respectively, by lactate-independent mechanisms, but lactate prevented drug enhancement of pAMPK expression in nitrergic neurons. Inhibition of VMN glycogen disassembly caused divergent changes in counter-regulatory hormone, e.g. corticosterone (increased) and glucagon (decreased) secretion. Outcomes show that VMN glycogen metabolism controls local glucoregulatory transmission by means of lactate signal volume. Results implicate glycogen-derived lactate deficiency as a physiological stimulus of corticosterone release. Concurrent normalization of nitrergic neuron nNOS and pAMPK protein and corticosterone secretory response to DAB by lactate infers that the hypothalamic-pituitary-adrenal axis may be activated by VMN NO-mediated signals of cellular energy imbalance.

Noradrenaline‐induced l‐lactate production requires d‐glucose entry and transit through the glycogen shunt in single‐cultured rat astrocytes

During cognitive efforts mediated by local neuronal networks, approximately 20% of additional energy is required; this is mediated by chemical messengers such as noradrenaline (NA). NA targets astroglial aerobic glycolysis, the hallmark of which is the end product l-lactate, a fuel for neurons. Biochemical studies have revealed that astrocytes exhibit a prominent glycogen shunt, in which a portion of d-glucose molecules entering the cytoplasm is transiently incorporated into glycogen, a buffer and source of d-glucose during increased energy demand. Here, we studied single astrocytes by measuring cytosolic L-lactate ([lac]i ) with the FRET nanosensor Laconic. We examined whether NA-induced increase in [lac]i is influenced by: (a) 2-deoxy-d-glucose (2-DG, 3mM), a molecule that enters the cytosol and inhibits the glycolytic pathway; (b) 1,4-dideoxy-1,4-imino-d-arabinitol (DAB, 300µM), a potent inhibitor of glycogen phosphorylase and glycogen degradation; and (c) 3-nitropropionic acid (3-NPA, 1mM), an inhibitor of the Krebs cycle. The results of these pharmacological experiments revealed that d-glucose uptake is essential for the NA-induced increase in [lac]i , and that this exclusively arises from glycogen degradation, indicating that most, if not all, d-glucose molecules in NA-stimulated cells transit the glycogen shunt during glycolysis. Moreover, under the defined transmembrane d-glucose gradient, the glycolytic intermediates were not only used to produce l-lactate, but also to significantly support oxidative phosphorylation, as demonstrated by an elevation in [lac]i when Krebs cycle was inhibited. We conclude that l-lactate production via aerobic glycolysis is an essential energy pathway in NA-stimulated astrocytes; however, oxidative metabolism is important at rest.

On the promise of glycogen phosphorylase inhibition in acute inflammation

How glycogen metabolism directly regulates macrophages in the acute inflammatory state is not well understood. In the recent issue of Nature Communications, Ma et al. provide new insight into this process, demonstrating that glycogenolysis-driven pentose phosphate pathway and UDP-glucose-driven P2Y14 receptor promote an inflammatory phenotype in macrophages. They show that in vivo blockade of glycogenolysis is sufficient to rescue survival in peritonitis, hepatitis, and sepsis. Their results hold implications for the treatment of acute inflammatory disorders at large.

Combined in silico and in vitro studies to identify novel antidiabetic flavonoids targeting glycogen phosphorylase

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.

Synthesis, Kinetic and Conformational Studies of 2-Substituted-5-(β-d-glucopyranosyl)-pyrimidin-4-ones as Potential Inhibitors of Glycogen Phosphorylase.

Dysregulation of glycogen phosphorylase, an enzyme involved in glucose homeostasis, may lead to a number of pathological states such as type 2 diabetes and cancer, making it an important molecular target for the development of new forms of pharmaceutical intervention. Based on our previous work on the design and synthesis of 4-arylamino-1-(β-d-glucopyranosyl)pyrimidin-2-ones, which inhibit the activity of glycogen phosphorylase by binding at its catalytic site, we report herein a general synthesis of 2-substituted-5-(β-d-glucopyranosyl)pyrimidin-4-ones, a related class of metabolically stable, C-glucosyl-based, analogues. The synthetic development consists of a metallated heterocycle, produced from 5-bromo-2-methylthiouracil, in addition to protected d-gluconolactone, followed by organosilane reduction. The methylthio handle allowed derivatization through hydrolysis, ammonolysis and arylamine substitution, and the new compounds were found to be potent (μM) inhibitors of rabbit muscle glycogen phosphorylase. The results were interpreted with the help of density functional theory calculations and conformational analysis and were compared with previous findings.

Glycogen phosphorylase inhibitor, 2,3-bis[(2E)-3-(4-hydroxyphenyl)prop-2-enamido] butanedioic acid (BF142), improves baseline insulin secretion of MIN6 insulinoma cells.

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.

Affinity Crystallography Reveals Binding of Pomegranate Juice Anthocyanins at the Inhibitor Site of Glycogen Phosphorylase: The Contribution of a Sugar Moiety to Potency and Its Implications to the Binding Mode.

Anthocyanins (ACNs) are dietary phytochemicals with an acknowledged therapeutic significance. Pomegranate juice (PJ) is a rich source of ACNs with potential applications in nutraceutical development. Glycogen phosphorylase (GP) catalyzes the first step of glycogenolysis and is a molecular target for the development of antihyperglycemics. The inhibitory potential of the ACN fraction of PJ is assessed through a combination of in vitro assays, ex vivo investigation in hepatic cells, and X-ray crystallography studies. The ACN extract potently inhibits muscle and liver isoforms of GP. Affinity crystallography reveals the structural basis of inhibition through the binding of pelargonidin-3-O-glucoside at the GP inhibitor site. The glucopyranose moiety is revealed as a major determinant of potency as it promotes a structural binding mode different from that observed for other flavonoids. This inhibitory effect of the ACN scaffold and its binding mode at the GP inhibitor binding site may have significant implications for future structure-based drug design endeavors.

Sex differences in glucoprivic regulation of glycogen metabolism in hypothalamic primary astrocyte cultures: Role of estrogen receptor signaling

Hypoglycemia causes sex-reliant changes in hypothalamic astrocyte glycogen metabolism in vivo. The role of nuclear versus membrane astrocyte estrogen receptors (ER) in glucoprivic regulation of glycogen is unclear. Here, primary hypothalamic astrocyte cultures were treated with selective ER antagonists during glucoprivation to investigate the hypothesis that ER mediate sex-specific glycogen responses to glucoprivation. Results show that glucoprivic down-regulation of glycogen synthase expression is mediated by transmembrane G protein-coupled ER-1 (GPER) signaling in each sex and estrogen receptor (ER)-beta (ERβ) activity in females. Glucoprivic inhibition of glycogen phosphorylase involves GPER and ERβ in females, but ER-independent mechanisms in males. GPER, ERβ, and ER-alpha (ERα) inhibit or stimulate AMPK protein expression in male versus female astrocytes, respectively. Glucoprivic augmentation of phospho-AMPK profiles in male glia was opposed by GPER activation, whereas GPER and ERβ suppress this protein in females. Astrocyte ERα and GPER content was down-regulated in each sex during glucose deficiency, whereas ERβ levels was unaltered (males) or increased (females). Glucoprivation correspondingly elevated or diminished male versus female astrocyte glycogen content; ER antagonism reversed this response in males, but not females. Results identify distinctive ER variants involved in sex-similar versus sex-specific astrocyte protein responses to withdrawal of this substrate fuel. Notably, glucoprivation elicits a directional switch or gain-of-effect of GPER and ERβ on specific glial protein profiles. Outcomes infer that ERs are crucial for glucoprivic regulation of astrocyte glycogen accumulation in males. Alternatively, estradiol may act independently of ER signaling to disassemble this reserve in females.

Synthetic flavonoid derivatives targeting the glycogen phosphorylase inhibitor site: QM/MM-PBSA motivated synthesis of substituted 5,7-dihydroxyflavones, crystallography, in vitro kinetics and ex-vivo cellular experiments reveal novel potent inhibitors

Glycogen phosphorylase (GP) is an important target for the development of new anti-hyperglycaemic agents. Flavonoids are novel inhibitors of GP, but their mode of action is unspecific in terms of the GP binding sites involved. Towards design of synthetic flavonoid analogues acting specifically at the inhibitor site and to exploit the site’s hydrophobic pocket, chrysin has been employed as a lead compound for the in silico screening of 1169 new analogues with different B ring substitutions. QM/MM-PBSA binding free energy calculations guided the final selection of eight compounds, subsequently synthesised using a Baker-Venkataraman rearrangement-cyclisation approach. Kinetics experiments against rabbit muscle GPa and GPb together with human liver GPa, revealed three of these compounds (11, 20 and 43) among the most potent that bind at the site (Ki s < 4 µM for all three isoforms), and more potent than previously reported natural flavonoid inhibitors. Multiple inhibition studies revealed binding exclusively at the inhibitor site. The binding is synergistic with glucose suggesting that inhibition could be regulated by blood glucose levels and would decrease as normoglycaemia is achieved. Compound 43 was an effective inhibitor of glycogenolysis in hepatocytes (IC50 = 70 µM), further promoting these compounds for optimization of their drug-like potential. X-ray crystallography studies revealed the B-ring interactions responsible for the observed potencies.

Contraction‐regulated mTORC1 and protein synthesis: Influence of AMPK and glycogen

AMP-activated protein kinase (AMPK)-dependent Raptor Ser792 phosphorylation does not influence mechanistic target of rapamycin complex 1 (mTORC1)-S6K1 activation by intense muscle contraction. α2 -AMPK activity-deficient mice have lower contraction-stimulated protein synthesis. Increasing glycogen activates mTORC1-S6K1. Normalizing muscle glycogen content rescues reduced protein synthesis in AMPK-deficient mice. The mechansitic target of rapamycin complex 1 (mTORC1)-S6K1 signalling pathway regulates muscle growth-related protein synthesis and is antagonized by AMP-activated protein kinase (AMPK) in multiple cell types. Resistance exercise stimulates skeletal muscle mTORC1-S6K1 and AMPK signalling and post-contraction protein synthesis. Glycogen inhibits AMPK and has been proposed as a pro-anabolic stimulus. The present study aimed to investigate how muscle mTORC1-S6K1 signalling and protein synthesis respond to resistance exercise-mimicking contraction in the absence of AMPK and with glycogen manipulation. Resistance exercise-mimicking unilateral in situ contraction of musculus quadriceps femoris in anaesthetized wild-type and dominant negative α2 AMPK kinase dead transgenic (KD-AMPK) mice, measuring muscle mTORC1 and AMPK signalling immediately (0h) and 4h post-contraction, and protein-synthesis at 4h. Muscle glycogen manipulation by 5day oral gavage of the glycogen phosphorylase inhibitor CP316819 and sucrose (80gL-1 ) in the drinking water prior to in situ contraction. The mTORC1-S6K1 and AMPK signalling axes were coactivated immediately post-contraction, despite potent AMPK-dependent Ser792 phosphorylation on the mTORC1subunit raptor. KD-AMPK muscles displayed normal mTORC1-S6K1 activation at 0h and 4h post-exercise, although there was impaired contraction-stimulated protein synthesis 4h post-contraction. Pharmacological/dietary elevation of muscle glycogen content augmented contraction-stimulated mTORC1-S6K1-S6 signalling and rescued the reduced protein synthesis-response in KD-AMPK to wild-type levels. mTORC-S6K1 signalling is not influenced by α2 -AMPK during or after intense muscle contraction. Elevated glycogen augments mTORC1-S6K1 signalling. α2 -AMPK-deficient KD-AMPK mice display impaired contraction-induced muscle protein synthesis, which can be rescued by normalizing muscle glycogen content.