Inhibition of glycogenolysis in primary rat hepatocytes by 1,4-dideoxy-1,4-imino-D-arabinitol

1,4-Dideoxy-1,4-imino-D-arabinitol (DAB) was identified previously as a potent inhibitor of both the phosphorylated and non-phosphorylated forms of glycogen phosphorylase (EC 2.4.1.1). In the present study, the effects of DAB were investigated in primary cultured rat hepatocytes. The transport of DAB into hepatocytes was dependent on time and DAB concentration. The rate of DAB transport was 192 pmol/min per mg of protein per mM DABmedium-concentration. In hepatocytes, DAB inhibited basal and glucagon-stimulated glycogenolysis with IC50 values of 1.0±0.3 and 1.1±0.2 μM, respectively. The primary inhibitory effect of DAB on glycogenolysis was shown to be due to inhibition of glycogen phosphorylase but, at higher concentrations of DAB, inhibition of the debranching enzyme (4-α-glucanotransferase, EC 2.4.1.25) may have an effect. No effects on glycogen synthesis were observed, demonstrating that glycogen recycling does not occur in cultured hepatocytes under the conditions tested. Furthermore, DAB had no effects on phosphorylase kinase, the enzyme responsible for phosphorylation and thereby activation of glycogen phosphorylase, or on protein phosphatase 1, the enzyme responsible for inactivation of glycogen phosphorylase through dephosphorylation.

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Inhibition of the interaction between protein phosphatase 1 glycogen-targeting subunit and glycogen phosphorylase increases glycogen synthesis in primary rat hepatocytes

Biochemical Journal ◽

10.1042/bj20071483 ◽

2008 ◽

Vol 412 (2) ◽

pp. 359-366 ◽

Cited By ~ 19

Author(s):

Darya Zibrova ◽

Rolf Grempler ◽

Rüdiger Streicher ◽

Stefan G. Kauschke

Keyword(s):

Protein Phosphatase ◽

Glycogen Phosphorylase ◽

Glycogen Synthesis ◽

Rat Hepatocytes ◽

Protein Phosphatase 1 ◽

Diabetic Patients ◽

Glycogen Accumulation ◽

Primary Rat Hepatocytes ◽

Glucose Levels ◽

Elevated Glucose

In Type 2 diabetes, increased glycogenolysis contributes to the hyperglycaemic state, therefore the inhibition of GP (glycogen phosphorylase), a key glycogenolytic enzyme, is one of the possibilities to lower plasma glucose levels. Following this strategy, a number of GPis (GP inhibitors) have been described. However, certain critical issues are associated with their mode of action, e.g. an impairment of muscle function. The interaction between GP and the liver glycogen targeting subunit (termed GL) of PP1 (protein phosphatase 1) has emerged as a new potential anti-diabetic target, as the disruption of this interaction should increase glycogen synthesis, potentially providing an alternative approach to counteract the enhanced glycogenolysis without inhibiting GP activity. We identified an inhibitor of the GL–GP interaction (termed GL–GPi) and characterized its mechanism of action in comparison with direct GPis. In primary rat hepatocytes, at elevated glucose levels, the GL–GPi increased glycogen synthesis similarly to direct GPis. Direct GPis significantly reduced the cellular GP activity, caused a dephosphorylation of the enzyme and decreased the amounts of GP in the glycogen-enriched fraction; the GL–GPi did not influence any of these parameters. Both mechanisms increased glycogen accumulation at elevated glucose levels. However, at low glucose levels, only direct GPis led to increased glycogen amounts, whereas the GL–GPi allowed the mobilization of glycogen because it did not block the activity of GP. Due to this characteristic, GL–GPi in comparison with GPis could offer an advantageous risk/benefit profile circumventing the potential downsides of a complete prevention of glycogen breakdown while retaining glucose- lowering efficacy, suggesting that inhibition of the GL–GP interaction may provide an attractive novel approach for rebalancing the disturbed glycogen metabolism in diabetic patients.

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Inhibitory effect of Tumor Necrosis Factor α, Interleukin 2, and Interferon γ on CTGF expression in cultured rat hepatocytes

Zeitschrift für Gastroenterologie ◽

10.1055/s-0029-1191794 ◽

2009 ◽

Vol 47 (01) ◽

Author(s):

I Peredniene ◽

OA Gressner ◽

B Lahme ◽

K Rehbein ◽

AM Gressner

Keyword(s):

Tumor Necrosis Factor ◽

Tumor Necrosis ◽

Tumor Necrosis Factor Α ◽

Interleukin 2 ◽

Rat Hepatocytes ◽

Inhibitory Effect ◽

Interferon Γ ◽

Cultured Rat Hepatocytes ◽

Factor Α ◽

Necrosis Factor

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Novel therapeutics based on inhibiting the interaction of glycogen phosphorylase and GL-subunit of glycogen-associated protein phosphatase 1: WO2009127723

Expert Opinion on Therapeutic Patents ◽

10.1517/13543771003781923 ◽

2010 ◽

Vol 20 (7) ◽

pp. 969-973 ◽

Cited By ~ 6

Author(s):

Lei Zhang ◽

Hong Liu

Keyword(s):

Protein Phosphatase ◽

Glycogen Phosphorylase ◽

Protein Phosphatase 1 ◽

Novel Therapeutics

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N-Acetyl-β-D-glucopyranosylamine 6-phosphate is a specific inhibitor of glycogen-bound protein phosphatase 1

Biochemical Journal ◽

10.1042/bj3280695 ◽

1997 ◽

Vol 328 (2) ◽

pp. 695-700 ◽

Cited By ~ 3

Author(s):

Mary BOARD

Keyword(s):

Phosphatase Activity ◽

Protein Phosphatase ◽

Glycogen Phosphorylase ◽

Glycogen Synthase ◽

Specific Inhibitor ◽

Competitive Inhibitor ◽

Protein Phosphatase 1 ◽

P Concentration ◽

Glucose Analogue ◽

Stimulation Of

Previous work has shown that the C-1-substituted glucose-analogue N-acetyl-β-D-glucopyranosylamine (1-GlcNAc) is a competitive inhibitor of glycogen phosphorylase (GP) and stimulates the inactivation of this enzyme by GP phosphatase. In addition to its effects on GP, 1-GlcNAc also prevents the glucose-led activation of glycogen synthase (GS) in whole hepatocytes. Such an effect on GS was thought to be due to the formation of 1-GlcNAc-6-P by the action of glucokinase within the hepatocyte [Board, Bollen, Stalmans, Kim, Fleet and Johnson (1995) Biochem. J. 311, 845-852]. To investigate this possibility further, a pure preparation of 1-GlcNAc-6-P was synthesized. The effects of the phosphorylated glucose analogue on the activity of protein phosphatase 1 (PP1), the enzyme responsible for dephosphorylation and activation of GS, are reported. During the present study, 1-GlcNAc-6-P inhibited the activity of the glycogen-bound form of PP1, affecting both the GSb phosphatase and GPa phosphatase activities. A level of 50% inhibition of GSb phosphatase activity was achieved with 85 μM 1-GlcNAc-6-P in the absence of Glc-6-P and with 135 μM in the presence of 10 mM Glc-6-P. At either Glc-6-P concentration, 500 μM 1-GlcNAc-6-P completely inhibited activity. The Glc-6-P stimulation of the GPa phosphatase activity of PP1 was negated by 1-GlcNAc-6-P but there was no inhibition of the basal rate in the absence of Glc-6-P. 1-GlcNAc-6-P inhibition was specific for the glycogen-bound form of PP1 and did not inhibit the GSb phosphatase activity of the cytosolic form of the enzyme. The present work explains our previous observations on the inactivating effects on GS of incubating whole hepatocytes with 1-GlcNAc. These observations have their basis in the inhibition of glycogen-bound PP1 by 1-GlcNAc-6-P. A novel inhibitor of PP1, specific for the glycogen-bound form of the enzyme, is presented.

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Inhibition of protein phosphatase 1 reverses alcohol-induced ciliary dysfunction

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00336.2014 ◽

2015 ◽

Vol 308 (6) ◽

pp. L577-L585 ◽

Cited By ~ 15

Author(s):

Michael E. Price ◽

Jacqueline A. Pavlik ◽

Joseph H. Sisson ◽

Todd A. Wyatt

Keyword(s):

Protein Phosphatase ◽

Mucociliary Clearance ◽

Specific Inhibitor ◽

Alcohol Exposure ◽

Inhibitory Effect ◽

Regulatory Proteins ◽

Protein Phosphatase 1 ◽

Tissue Cell ◽

Inhaled Particles ◽

Phosphatase Activation

Airway mucociliary clearance is a first-line defense of the lung against inhaled particles and debris. Among individuals with alcohol use disorders, there is an increase in lung diseases. We previously identified that prolonged alcohol exposure impairs mucociliary clearance, known as alcohol-induced ciliary dysfunction (AICD). Cilia-localized enzymes, known as the ciliary metabolon, are key to the pathogenesis of AICD. In AICD, cyclic nucleotide-dependent ciliary kinases, which modulate phosphorylation to regulate cilia beat, are desensitized. We hypothesized that alcohol activates cilia-associated protein phosphatase 1 (PP1) activity, driving phosphorylation changes of cilia motility regulatory proteins. To test this hypothesis we identified the effects of prolonged alcohol exposure on phosphatase activity, cilia beat, and kinase responsiveness and cilia-associated phosphorylation targets when stimulated by β-agonist or cAMP. Prolonged alcohol activated PP1 and blocked cAMP-dependent cilia beat and protein kinase A (PKA) responsiveness and phosphorylation of a 29-kDa substrate of PKA. Importantly, prolonged alcohol-induced phosphatase activation was inhibited by the PP1 specific inhibitor, inhibitor-2 (I-2), restoring cAMP-stimulated cilia beat and PKA responsiveness and phosphorylation of the 29-kDa substrate. The I-2 inhibitory effect persisted in tissue, cell, and isolated cilia-organelle models, highlighting the association of ciliary metabolon-localized enzymes to AICD. Prolonged alcohol exposure drives ciliary metabolon-localized PP1 activation. PP1 activation modifies phosphorylation of a 29-kDa protein related to PKA activity. These data reinforce our previous findings that alcohol is acting at the level of the ciliary metabolon to cause ciliary dysfunction and identifies PP1 as a therapeutic target to prevent or reverse AICD.

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The level of the glycogen targetting regulatory subunit R5 of protein phosphatase 1 is decreased in the livers of insulin-dependent diabetic rats and starved rats

Biochemical Journal ◽

10.1042/bj3600449 ◽

2001 ◽

Vol 360 (2) ◽

pp. 449-459 ◽

Cited By ~ 12

Author(s):

Gareth J. BROWNE ◽

Mirela DELIBEGOVIC ◽

Stefaan KEPPENS ◽

Willy STALMANS ◽

Patricia T. W. COHEN

Keyword(s):

Phosphatase Activity ◽

Protein Phosphatase ◽

Glycogen Synthase ◽

Glycogen Synthesis ◽

Diabetic Rats ◽

Protein Phosphatase 1 ◽

Regulatory Subunit ◽

Hepatic Glycogen ◽

Insulin Dependent ◽

Insulin Dependent Diabetic

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats owing to defective activation of glycogen synthase by glycogen-bound protein phosphatase 1 (PP1). The identification of three glycogen-targetting subunits in liver, GL, R5/PTG and R6, which form complexes with the catalytic subunit of PP1 (PP1c), raises the question of whether some or all of these PP1c complexes are subject to regulation by insulin. In liver lysates of control rats, R5 and R6 complexes with PP1c were found to contribute significantly (16 and 21% respectively) to the phosphorylase phosphatase activity associated with the glycogen-targetting subunits, GL–PP1c accounting for the remainder (63%). In liver lysates of insulin-dependent diabetic and of starved rats, the phosphorylase phosphatase activities of the R5 and GL complexes with PP1c were shown by specific immunoadsorption assays to be substantially decreased, and the levels of R5 and GL were shown by immunoblotting to be much lower than those in control extracts. The phosphorylase phosphatase activity of R6–PP1c and the concentration of R6 protein were unaffected by these treatments. Insulin administration to diabetic rats restored the levels of R5 and GL and their associated activities. The regulation of R5 protein levels by insulin was shown to correspond to changes in the level of the mRNA, as has been found for GL. The in vitro glycogen synthase phosphatase/phosphorylase phosphatase activity ratio of R5-PP1c was lower than that of GL–PP1c, suggesting that R5–PP1c may function as a hepatic phosphorylase phosphatase, whereas GL–PP1c may be the major hepatic glycogen synthase phosphatase. In hepatic lysates, more than half the R6 was present in the glycogen-free supernatant, suggesting that R6 may have lower affinity for glycogen than R5 and GL

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Metformin inhibits glycogen synthesis and gluconeogenesis in cultured rat hepatocytes

Diabetes Obesity and Metabolism ◽

10.1046/j.1463-1326.2003.00263.x ◽

2003 ◽

Vol 5 (3) ◽

pp. 189-194 ◽

Cited By ~ 24

Author(s):

M. Otto ◽

J. Breinholt ◽

N. Westergaard

Keyword(s):

Glycogen Synthesis ◽

Rat Hepatocytes ◽

Cultured Rat Hepatocytes

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Protein phosphatase 1 and 2A inhibitors activate acyl–CoA:cholesterol acyltransferase and cholesterol ester formation in isolated rat hepatocytes

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism ◽

10.1016/s0005-2760(97)00141-0 ◽

1997 ◽

Vol 1349 (3) ◽

pp. 233-241 ◽

Cited By ~ 8

Author(s):

Marı́a L Hernández ◽

Marı́a J Martı́nez ◽

Miguel López de Heredia ◽

Begoña Ochoa

Keyword(s):

Protein Phosphatase ◽

Cholesterol Ester ◽

Rat Hepatocytes ◽

Protein Phosphatase 1 ◽

Isolated Rat Hepatocytes ◽

Ester Formation ◽

Isolated Rat

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Disruption of the striated muscle glycogen-targeting subunit of protein phosphatase 1: influence of the genetic background

Journal of Molecular Endocrinology ◽

10.1677/jme-07-0120 ◽

2007 ◽

Vol 40 (2) ◽

pp. 47-59 ◽

Cited By ~ 7

Author(s):

James Paterson ◽

Ian R Kelsall ◽

Patricia T W Cohen

Keyword(s):

Skeletal Muscle ◽

Glucose Transport ◽

Muscle Glycogen ◽

Protein Phosphatase ◽

Striated Muscle ◽

Protein Phosphatase 1 ◽

Phosphorylase Kinase ◽

Donor Strain ◽

Key Factor

A prediabetic phenotype of glucose intolerance, insulin resistance and obesity was observed at ∼12 months of age in mice homozygous for a null allele of the major skeletal muscle glycogen-targeting subunit GM of protein phosphatase 1 (PP1) and derived from a 129/Ola donor strain. In this study, backcrossing of these mice (termed obese mice) onto two different genetic backgrounds gave rise to lean, glucose-tolerant, insulin-sensitive mice (termed lean mice), indicating that at least one variant gene in the 129/Ola background, not present in the C57BL/6 or 129s2/sV background, is required for the development of the prediabetic phenotype of obese mice. Slightly elevated AMP-activated protein kinase α2 activity in the skeletal muscle of lean C57BL/6 mice was also observed to a lesser extent in the obese mice. Normal or slightly raised in vivo glucose transport in lean C57BL/6 mice compared with decreased glucose transport in the obese mice supports the tenet that adequate transport of glucose may be a key factor in preventing the development of the prediabetic phenotype. The pH 6.8/pH 8.6 activity ratio of phosphorylase kinase was increased in lean C57BL/6 mice compared with controls indicating that phosphorylase kinase is an in vivo substrate of PP1-GM.

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