PYGL-mediated glucose metabolism reprogramming promotes EMT phenotype and metastasis of pancreatic cancer.

Epithelial-mesenchymal transition (EMT) is closely associated with tumor invasion and metastasis. However, key regulators of EMT in pancreatic ductal adenocarcinoma (PDAC) need to be further studied. Bioinformatics analyses of pancreatic cancer public datasets showed that glycogen phosphorylase L (PYGL) expression is elevated in quasimesenchymal PDAC (QM-PDAC) and positively associated with EMT. In vitro cellular experiments further confirm PYGL as a crucial EMT regulator in PDAC cells. Functionally, PYGL overexpression promotes cell migration and invasion in vitro and facilitates liver metastasis in vivo, while PYGL knockdown has opposite effects. Mechanically, hypoxia induces PYGL expression in a hypoxia inducible factor 1α (HIF1α)-dependent manner and promotes glycogen accumulation. Elevated PYGL mobilizes accumulated glycogen to fuel glycolysis via its activity as a glycogen phosphorylase, thus inducing the EMT process, which could be suppressed by the glycolysis inhibitor 2-deoxy-D-glucose (2-DG). Clinically, PYGL expression is upregulated in PDAC and correlates with its malignant features and poor prognosis. Collectively, the data from our study reveal that the hypoxia/PYGL/glycolysis-induced EMT promotes PDAC metastasis, which establishes the rational for targeting hypoxia/PYGL/glycolysis/EMT signaling pathway against PDAC.

miR-155-5p regulates hypoxia-induced pulmonary artery smooth muscle cell function by targeting PYGL.

Pulmonary arterial hypertension (PAH) is a cardiovascular disease that has high incidence and causes massive deaths. miR-155-5p/PYGL pathway was revealed to play a crucial role in PAH by weighted gene co-expression network analysis (WGCNA). The potential mechanism of miR-155-5p in regulating hypoxia-induced pulmonary artery smooth muscle cell (PASMC) function was analyzed through in vitro experiments. Hypoxia treatment stimulated the proliferation of PASMCs and increased the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α). At the same time, revealed by qRT-PCR and western blot, the level of miR-155-5p was raised, and the level of PYGL was decreased in hypoxia-induced PASMCs. Through CCK-8 assay, transwell assay and flow cytometry, it was revealed that miR-155-5p inhibitor remarkably inhibited the cell proliferation and migration and decreased the proportion of hypoxia-stimulated PASMCs in S and G2/M phases. Dual-luciferase reporter system was subsequently applied to validate the straight regulation of miR-155-5p on PYGL based on the analysis of online database. Furthermore, siPYGL was revealed to reverse the influence of miR-155-5p inhibitor on hypoxia-induced PASMCs. These outcomes indicate that the increased level of miR-155-5p in hypoxia-stimulated PASMCs could enhance the cell proliferation, cell migration, and cell cycle progression by targeting PYGL directly. This study may supply novel treatment strategies for PAH.Abbreviations: PH, pulmonary hypertension; PAH, pulmonary arterial hypertension; WGCNA, weighted gene co-expression network analysis; PASMCs, pulmonary artery smooth muscle cells; VEGF, vascular endothelial growth factor; HIF-1α, hypoxia-inducible factor-1α; SMCs, smooth muscle cells; DEGs, differentially expressed genes; GEO, Gene Expression Omnibus; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; FBS, fetal bovine serum; OD, optical density; BCA, bicinchoninic acid; PVDF, polyvinylidene fluoride; PBS, phosphate-buffered saline; BP, biological process; MF, molecular function; CC, cell component.

Identification of PYGL as a key prognostic gene of glioma by integrated bioinformatics analysis.

Aim: PYGL has been reported to have carcinogenic effects in a variety of tumors. This study is the first to reveal the relationship between PYGL and the prognosis of glioma. Materials & methods: Analyzing the Chinese Glioma Genome Atlas database, the authors revealed the expression status and prognostic value of PYGL in gliomas and used quantitative real-time PCR to verify PYGL expression again. Subsequently, they used Gene Set Enrichment Analysis to explore the biological pathways that PYGL may participate in. The authors also used the tumor immune estimation resource database to explore the relationship between PYGL and tumor immune cells. Results: PYGL is involved in the malignant progression of glioma. Conclusions: PYGL can be used as a new biomarker and molecular target for evaluating the prognosis and immunotherapy of glioma.

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.

Long noncoding RNA KCNMB2-AS1 promotes the development of esophageal cancer by modulating the miR-3194-3p/PYGL axis.

Esophageal cancer (ESCA), as a common cancer worldwide, is a main cause of cancer-related mortality. Comprehensive studies on molecular mechanism of ESCA have been carried out. Though numerous long noncoding RNAs (lncRNAs) was reported to participate in the occurrence and development of ESCA, the potential role of lncRNA potassium calcium-activated channel subfamily M regulatory beta subunit 2 (KCNMB2) antisense RNA 1 (KCNMB2-AS1) in ESCA remains to be discovered. This study intends to investigate the detailed function and molecular mechanism of KCNMB2-AS1 in ESCA. Gene expression was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Cell proliferation was examined by Cell Counting Kit-8 (CCK-8) assay and colony formation assay. Cell invasion and migration were measured by wound healing assay and Transwell assay. Luciferase reporter assay was adopted to validate the interaction between KCNMB2-AS1 and miR-3194-3p. Western blotting was performed to assess protein levels. We discovered that KCNMB2-AS1 was significantly upregulated in ESCA. KCNMB2-AS1 downregulation suppressed the growth, invasion, migration and stemness of ESCA cells. KCNMB2-AS1 bound with miR-3194-3p, and glycogen phosphorylase L (PYGL) was a direct target of miR-3194-3p. KCNMB2-AS1 upregulated PYGL expression by directly binding with miR-3194-3p. Additionally, PYGL overexpression abolished the inhibitory influence of KCNMB2-AS1 depletion on ESCA cell behaviors. Collectively, lncRNA KCNMB2-AS1 promotes ESCA development through targeting the miR-3194-3p/ PYGL axis, which might provide theoretical basis to explore novel biomarkers for ESCA treatment.

The vitamin B6-regulated enzymes PYGL and G6PD fuel NADPH oxidases to promote skin inflammation.

Psoriasis is a skin inflammatory disorder that affects 3% of the human population. Although several therapies based on the neutralization of proinflammatory cytokines have been used with relative success, additional treatments are required. The in silico analysis of gene expression data of psoriasis lesional skin and an analysis of vitamin B6 metabolites in the sera of psoriasis patients point to altered vitamin B6 metabolism at both local and systemic levels. Functional studies showed that vitamin B6 vitamers reduced skin neutrophil infiltration, oxidative stress and Nfkb activity in two zebrafish models of skin inflammation. Strikingly, inhibition of glycogen phosphorylase L (Pygl) and glucose-6-phosphate dehydrogenase (G6pd), two vitamin B6-regulated enzymes, alleviated oxidative-stress induced inflammation in zebrafish skin inflammation models. Despite the central role of G6pd in antioxidant defenses, the results of the study demonstrate that glycogen stores and G6pd fuel NADPH oxidase to promote skin inflammation, revealing novel targets for the treatment of skin inflammatory disorders.

The architecture of hydrogen and sulfur σ-hole interactions explain differences in the inhibitory potency of C-ß-d-glucopyranosyl thiazoles, imidazoles and an N-ß-d glucopyranosyl tetrazole for human liver glycogen phosphorylase and offer new insights to structure-based design.

C-Glucopyranosyl imidazoles, thiazoles, and an N-glucopyranosyl tetrazole were assessed in vitro and ex vivo for their inhibitory efficiency against isoforms of glycogen phosphorylase (GP; a validated pharmacological target for the development of anti-hyperglycaemic agents). Imidazoles proved to be more potent inhibitors than the corresponding thiazoles or the tetrazole. The most potent derivative has a 2-naphthyl substituent, a Ki value of 3.2 µM for hepatic glycogen phosphorylase, displaying also 60% inhibition of GP activity in HepG2 cells, compared to control vehicle treated cells, at 100 µM. X-Ray crystallography studies of the protein - inhibitor complexes revealed the importance of the architecture of inhibitor associated hydrogen bonds or sulfur σ-hole bond interactions to Asn284 OD1, offering new insights to structure-based design efforts. Moreover, while the 2-glucopyranosyl-tetrazole seems to bind differently from the corresponding 1,2,3-triazole compound, the two inhibitors are equipotent.

1,2-Dichloroethane impairs glucose and lipid homeostasis in the livers of NIH Swiss mice.

Excessive exposure to 1,2-Dichloroethane (1,2-DCE), a chlorinated organic toxicant, can lead to liver dysfunction. To fully explore the mechanism of 1,2-DCE-induced hepatic abnormalities, 30 male National Institutes of Health (NIH) Swiss mice were exposed to 0, 350, or 700mg/m3 of 1,2-DCE, via inhalation, 6h/day for 28days. Increased liver/body weight ratios, as well as serum AST and serum ALT activity were observed in the 350 and 700mg/m3 1,2-DCE exposure group mice, compared with the control group mice. In addition, decreased body weights were observed in mice exposed to 700mg/m3 1,2-DCE, compared with control mice. Exposure to 350 and 700mg/m3 1,2-DCE also led to significant accumulation of hepatic glycogen, free fatty acids (FFA) and triglycerides, elevation of blood triglyceride and FFA levels, and decreases in blood glucose levels. Results from microarray analysis indicated that the decreases in glucose-6-phosphatase catalytic subunit (G6PC) and liver glycogen phosphorylase (PYGL) expression, mediated by the activation of AKT serine/threonine kinase 1 (Akt1), might be responsible for the hepatic glycogen accumulation and steatosis. Further in vitro study demonstrated that 2-chloroacetic acid (1,2-DCE metabolite), rather than 1,2-DCE, up-regulated Akt1 phosphorylation and suppressed G6PC and PYGL expression, resulting in hepatocellular glycogen accumulation. These results suggest that hepatic glucose and lipid homeostasis are impaired by 1,2-DCE exposure via down-regulation of PYGL and G6PC expression, which may be primarily mediated by the 2-chloroacetic acid-activated Akt1 pathway.

Discovery of a Genetic Metabolic Cause for Mauriac Syndrome in Type 1 Diabetes.

A mechanistic cause for Mauriac syndrome, a syndrome of growth failure and delayed puberty associated with massive liver enlargement from glycogen deposition in children with poorly controlled type 1 diabetes, is unknown. We discovered a mutation in the catalytic subunit of liver glycogen phosphorylase kinase in a patient with Mauriac syndrome whose liver extended into his pelvis. Glycogen phosphorylase kinase activates glycogen phosphorylase, the enzyme that catalyzes the first step in glycogen breakdown. We show that the mutant subunit acts in a dominant manner to completely inhibit glycogen phosphorylase kinase enzyme activity and that this interferes with glycogenolysis causing increased levels of glycogen in human liver cells. It is known that even normal blood glucose levels physiologically inhibit glycogen phosphorylase to diminish glucose release from the liver when glycogenolysis is not needed. The patient's mother possessed the same mutant glycogen phosphorylase kinase subunit, but did not have diabetes or hepatomegaly. His father had childhood type 1 diabetes in poor glycemic control, but lacked the mutation and had neither hepatomegaly nor growth failure. This case proves that the effect of a mutant enzyme of glycogen metabolism can combine with hyperglycemia to directly hyperinhibit glycogen phosphorylase, in turn blocking glycogenolysis causing the massive liver in Mauriac disease.

Structure based inhibitor design targeting glycogen phosphorylase B. Virtual screening, synthesis, biochemical and biological assessment of novel N-acyl-ß-d-glucopyranosylamines.

Glycogen phosphorylase (GP) is a validated target for the development of new type 2 diabetes treatments. Exploiting the Zinc docking database, we report the in silico screening of 1888 N-acyl-ß-d-glucopyranosylamines putative GP inhibitors differing only in their R groups. CombiGlide and GOLD docking programs with different scoring functions were employed with the best performing methods combined in a 'consensus scoring' approach to ranking of ligand binding affinities for the active site. Six selected candidates from the screening were then synthesized and their inhibitory potency was assessed both in vitro and ex vivo. Their inhibition constants' values, in vitro, ranged from 5 to 377µM while two of them were effective at causing inactivation of GP in rat hepatocytes at low µM concentrations. The crystal structures of GP in complex with the inhibitors were defined and provided the structural basis for their inhibitory potency and data for further structure based design of more potent inhibitors.

Expression profile based gene clusters for ischemic stroke detection.

In microarray studies alterations in gene expression in circulating leukocytes have shown utility for ischemic stroke diagnosis. We studied forty candidate markers identified in three gene expression profiles to (1) quantitate individual transcript expression, (2) identify transcript clusters and (3) assess the clinical diagnostic utility of the clusters identified for ischemic stroke detection. Using high throughput next generation qPCR 16 of the 40 transcripts were significantly up-regulated in stroke patients relative to control subjects (p<0.05). Six clusters of between 5 and 7 transcripts were identified that discriminated between stroke and control (p values between 1.01e-9 and 0.03). A 7 transcript cluster containing PLBD1, PYGL, BST1, DUSP1, FOS, VCAN and FCGR1A showed high accuracy for stroke classification (AUC=0.854). These results validate and improve upon the diagnostic value of transcripts identified in microarray studies for ischemic stroke. The clusters identified show promise for acute ischemic stroke detection.

Effects of celiac superior mesenteric ganglionectomy on glucose homeostasis and hormonal changes during oral glucose tolerance testing in rats.

The liver plays an important role in maintaining glucose homeostasis in the body. In the prandial state, some of the glucose which is absorbed by the gastrointestinal tract is converted into glycogen and stored in the liver. In contrast, the liver produces glucose by glycogenolysis and gluconeogenesis while fasting. Thus, the liver contributes to maintaining blood glucose level within normoglycemic range. Glycogenesis and glycogenolysis are regulated by various mechanisms including hormones, the sympathetic and parasympathetic nervous systems and the hepatic glucose content. In this study, we examined a rat model in which the celiac superior mesenteric ganglion (CSMG) was resected. We attempted to elucidate how the celiac sympathetic nervous system is involved in regulating glucose homeostasis by assessing the effects of CSMG resection on glucose excursion during an oral glucose tolerance test, and by examining hepatic glycogen content and hepatic glycogen phosphorylase (GP) activity. On the oral glucose tolerance test, CSMG-resected rats demonstrated improved glucose tolerance and significantly increased GP activity compared with sham-operated rats, whereas there were no significant differences in insulin, glucagon or catecholamine levels between the 2 groups. These results suggest that the celiac sympathetic nervous system is involved in regulating the rate of glycogen consumption through GP activity. In conclusion, the examined rat model showed that the celiac sympathetic nervous system regulates hepatic glucose metabolism in conjunction with vagal nerve innervations and is a critical component in the maintenance of blood glucose homeostasis.

Hepatic glucose metabolism in late pregnancy: normal versus high-fat and -fructose diet.

Net hepatic glucose uptake (NHGU) is an important contributor to postprandial glycemic control. We hypothesized that NHGU is reduced during normal pregnancy and in a pregnant diet-induced model of impaired glucose intolerance/gestational diabetes mellitus (IGT/GDM). Dogs (n = 7 per group) that were nonpregnant (N), normal pregnant (P), or pregnant with IGT/GDM (pregnant dogs fed a high-fat and -fructose diet [P-HFF]) underwent a hyperinsulinemic-hyperglycemic clamp with intraportal glucose infusion. Clamp period insulin, glucagon, and glucose concentrations and hepatic glucose loads did not differ among groups. The N dogs reached near-maximal NHGU rates within 30 min; mean ± SEM NHGU was 105 ± 9 µmol·100 g liver⁻¹·min⁻¹. The P and P-HFF dogs reached maximal NHGU in 90-120 min; their NHGU was blunted (68 ± 9 and 16 ± 17 µmol·100 g liver⁻¹·min⁻¹, respectively). Hepatic glycogen synthesis was reduced 20% in P versus N and 40% in P-HFF versus P dogs. This was associated with a reduction (>70%) in glycogen synthase activity in P-HFF versus P and increased glycogen phosphorylase (GP) activity in both P (1.7-fold greater than N) and P-HFF (1.8-fold greater than P) dogs. Thus, NHGU under conditions mimicking the postprandial state is delayed and suppressed in normal pregnancy, with concomitant reduction in glycogen storage. NHGU is further blunted in IGT/GDM. This likely contributes to postprandial hyperglycemia during pregnancy, with potential adverse outcomes for the fetus and mother.

Impact of a glycogen phosphorylase inhibitor and metformin on basal and glucagon-stimulated hepatic glucose flux in conscious dogs.

The effects of a glycogen phosphorylase inhibitor (GPI) and metformin (MT) on hepatic glucose fluxes (µmol · kg(-1) · min(-1)) in the presence of basal and 4-fold basal levels of plasma glucagon were investigated in 18-h fasted conscious dogs. Compared with the vehicle treatment, GPI infusion suppressed net hepatic glucose output (NHGO) completely (-3.8 ± 1.3 versus 9.9 ± 2.8) despite increased glucose 6-phosphate (G-6-P) neogenesis from gluconeogenic precursors (8.1 ± 1.1 versus 5.5 ± 1.1). MT infusion did not alter those parameters. In response to a 4-fold rise in plasma glucagon levels, in the vehicle group, plasma glucose levels were increased 2-fold, and NHGO was increased (43.9 ± 5.7 at 10 min and 22.7 ± 3.4 at steady state) without altering G-6-P neogenesis (3.7 ± 1.5 and 5.5 ± 0.5, respectively). In the GPI group, there was no increase in NHGO due to decreased glucose-6-phosphatase flux associated with reduced G-6-P concentration. A lower G-6-P concentration was the result of increased net glycogenesis without altering G-6-P neogenesis. In the MT group, the increment in NHGO (22.2 ± 4.4 at 10 min and 12.1 ± 3.6 at steady state) was approximately half of that of the vehicle group. The lesser NHGO was associated with reduced glucose-6-phosphatase flux but a rise in G-6-P concentration and only a small incorporation of plasma glucose into glycogen. In conclusion, the inhibition of glycogen phosphorylase a activity decreases basal and glucagon-induced NHGO via redirecting glucose 6-phosphate flux from glucose toward glycogen, and MT decreases glucagon-induced NHGO by inhibiting glucose-6-phosphatase flux and thereby reducing glycogen breakdown.

Disruption of the allosteric phosphorylase a regulation of the hepatic glycogen-targeted protein phosphatase 1 improves glucose tolerance in vivo.

Type 2 diabetes is characterised by elevated blood glucose concentrations, which potentially could be normalised by stimulation of hepatic glycogen synthesis. Under glycogenolytic conditions, the interaction of hepatic glycogen-associated protein phosphatase-1 (PP1-G(L)) with glycogen phosphorylase a is believed to inhibit the dephosphorylation and activation of glycogen synthase (GS) by the PP1-G(L) complex, suppressing glycogen synthesis. Consequently, the interaction of G(L) with phosphorylase a has emerged as an attractive anti-diabetic target, pharmacological disruption of which could provide a novel mechanism to lower blood glucose levels by increasing hepatic glycogen synthesis. Here we report for the first time the in vivo consequences of disrupting the G(L)-phosphorylase a interaction, using a mouse model containing a Tyr284Phe substitution in the phosphorylase a-binding region of the G(L) protein. The resulting G(L)(Y284F/Y284F) mice display hepatic PP1-G(L) activity that is no longer sensitive to allosteric inhibition by phosphorylase a, resulting in increased GS activity under glycogenolytic conditions, demonstrating that regulation of G(L) by phosphorylase a operates in vivo. G(L)(Y284F/Y284F) and G(L)(Y284F/+) mice display improved glucose tolerance compared with G(L)(+/+) littermates, without significant accumulation of hepatic glycogen. The data provide the first in vivo evidence in support of targeting the G(L)-phosphorylase a interaction for treatment of hyperglycaemia. During prolonged fasting the G(L)(Y284F/Y284F) mice lose more body weight and display decreased blood glucose levels in comparison with their G(L)(+/+) littermates. These results suggest that, during periods of food deprivation, the phosphorylase a regulation of G(L) may prevent futile glucose-glycogen cycling, preserving energy and thus providing a selective biological advantage that may explain the observed conservation of the allosteric regulation of PP1-G(L) by phosphorylase a in mammals.

Changes in carbohydrate metabolism during acclimation to a moderate hyperthermic environment in rats.

We studied the influence of heat acclimation (1 to 48 h and 4 to 60 d at 35 +/- 1 degrees C) on certain hepatic carbohydrate-related enzymes and substrates in rats. The results showed a decrease of liver glycogen content and GPho-ase a activity during the period of short-term exposure, followed by normalization to the control level and stabilization to the new level in the period of long-term heat acclimation. Conversely, G-6-P-ase and F-1,6-BP-ase activities increased during the short-term period, followed by a decrease and stabilization to a new, lower level in the prolonged acclimation. The blood glucose level decreased during whole period of acclimation, whereas intermediate substrates increased during the short-term and stabilized at a new, higher level during prolonged acclimation. The time-dependent changes of duration of heat acclimation could be summarized in three phases: short-term heat exposure (1 to 24 h) with intensive glycogenolysis and gluconeogenesis to glucose; a period with temporary changes (24 h to 7 d) with tendency of normalization to control level, and prolonged heat acclimation (7 d to 60 d), which favors both direct and indirect glycogen synthesis.

Synthesis of a C-glucosylated cyclopropylamide and evaluation as a glycogen phosphorylase inhibitor.

The synthesis of carbohydrate-based glycogen phosphorylase inhibitors is attractive for potential applications in the treatment of type 2 diabetes. A titanium-mediated synthesis led to a benzoylated C-glucosylated cyclopropylamine intermediate, which underwent a benzoyl migration to afford the corresponding 2-hydroxy-C-glycoside. X-ray crystallographic studies revealed a unit cell composed of four molecules as pairs of dimers connected through two hydrogen bonds. The deprotection of the benzoate esters under Zemplén conditions afforded a glycogen phosphorylase inhibitor candidate displaying weak inhibition toward glycogen phosphorylase (16% at 2.5mM).

Molecular recognition of the protein phosphatase 1 glycogen targeting subunit by glycogen phosphorylase.

Disrupting the interaction between glycogen phosphorylase and the glycogen targeting subunit (G(L)) of protein phosphatase 1 is emerging as a novel target for the treatment of type 2 diabetes. To elucidate the molecular basis of binding, we have determined the crystal structure of liver phosphorylase bound to a G(L)-derived peptide. The structure reveals the C terminus of G(L) binding in a hydrophobically collapsed conformation to the allosteric regulator-binding site at the phosphorylase dimer interface. G(L) mimics interactions that are otherwise employed by the activator AMP. Functional studies show that G(L) binds tighter than AMP and confirm that the C-terminal Tyr-Tyr motif is the major determinant for G(L) binding potency. Our study validates the G(L)-phosphorylase interface as a novel target for small molecule interaction.

The hepatic PP1 glycogen-targeting subunit interaction with phosphorylase a can be blocked by C-terminal tyrosine deletion or an indole drug.

The inhibition of hepatic glycogen-associated protein phosphatase-1 (PP1-G(L)) by glycogen phosphorylase a prevents the dephosphorylation and activation of glycogen synthase, suppressing glycogen synthesis when glycogenolysis is activated. Here, we show that a peptide ((280)LGPYY(284)) comprising the last five amino acids of G(L) retains high-affinity interaction with phosphorylase a and that the two tyrosines play crucial roles. Tyr284 deletion abolishes binding of phosphorylase a to G(L) and replacement by phenylalanine is insufficient to restore high-affinity binding. We show that a phosphorylase inhibitor blocks the interaction of phosphorylase a with the G(L) C-terminus, suggesting that the latter interaction could be targeted to develop an anti-diabetic drug.

Glutathione-dependent reduction of arsenate by glycogen phosphorylase responsiveness to endogenous and xenobiotic inhibitors.

Rabbit muscle glycogen phosphorylase-a (GPa) reduces arsenate (As(V)) to the more toxic arsenite (As(III)) in a glutathione (GSH)-dependent fashion. To determine whether reduction of As(V) by GPa is countered by compounds known to inhibit GP-catalyzed glycogenolysis, the effects of thiol reagents, endogenous compounds (glucose, ATP, ADP) as well as nonspecific glycogen phosphorylase inhibitors (GPIs; caffeine, quercetin, flavopiridol [FP]), and specific GPIs (1,4-dideoxy-1,4-imino-D-arabinitol [DAB], BAY U6751, CP320626) were tested on reduction of As(V) by rabbit muscle GPa in the presence of glycogen (substrate), AMP (activator), and GSH, and the As(III) formed from As(V) was quantified by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry. The As(V)-reducing activity of GPa was moderately sensitive to thiol reagents. Glucose above 5mM and ADP or ATP at physiological levels diminished GPa-catalyzed As(V) reduction. All GPIs inhibited As(V) reduction by GPa in a concentration-dependent fashion; however, their effects were differentially affected by glucose (10mM) or AMP (200microM instead of 25microM), known modulators of the action of some GPIs on the GP-catalyzed glycogenolysis. Inhibition of As(V) reduction by DAB and quercetin was not influenced by glucose or AMP. Glucose that potentiates the inhibitory effects of caffeine, BAY U6751, and CP320626 on the glycogenolytic activity of GPa also enhanced the inhibitory effects of these GPIs on GPa-catalyzed As(V) reduction. AMP at high concentration alleviated the inhibition by BAY U6751 and CP320626 (whose antagonistic effect on GP-catalyzed glycogen breakdown is also AMP sensitive), whereas the inhibition in As(V) reduction by FP or caffeine was little affected by AMP. Thus, GPIs inhibit both the glycogenolytic and As(V)-reducing activities of GP, supporting that the latter is coupled to glycogenolysis. It was also shown that a GPa-rich extract of rat liver contained GSH-dependent As(V)-reducing activity that was inhibited by specific GPIs, suggesting that the liver-type GPa can also catalyze reduction of As(V).