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

2008 ◽  
Vol 412 (2) ◽  
pp. 359-366 ◽  
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.

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

1999 ◽  
Vol 342 (3) ◽  
pp. 545-550 ◽  
Author(s):  
Birgitte ANDERSEN ◽  
Andreas RASSOV ◽  
Niels WESTERGAARD ◽  
Karsten LUNDGREN
Keyword(s):  
Protein Phosphatase ◽  
Glycogen Phosphorylase ◽  
Glycogen Synthesis ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Protein Phosphatase 1 ◽  
Phosphorylase Kinase ◽  
Debranching Enzyme ◽  
Ic50 Values ◽  
Cultured Rat Hepatocytes

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.

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

1997 ◽  
Vol 328 (2) ◽  
pp. 695-700 ◽  
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.

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

2001 ◽  
Vol 360 (2) ◽  
pp. 449-459 ◽  
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

scholarly journals Elevated glucose acts directly on osteocytes to increase sclerostin expression in diabetes

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Donna M. Pacicca ◽  
Tammy Brown ◽  
Dara Watkins ◽  
Karen Kover ◽  
Yun Yan ◽  
...  
Keyword(s):  
Bone Quality ◽  
High Glucose ◽  
Negative Impact ◽  
Negative Regulator ◽  
Diabetic Rat ◽  
Diabetic Patients ◽  
Bone Homeostasis ◽  
Glucose Levels ◽  
Elevated Glucose

AbstractBone quality in diabetic patients is compromised, leading to weaker bones and increased fracture risk. However, the mechanism by which this occurs in diabetic bone remains to be fully elucidated. We hypothesized that elevated glucose and glucose variation would affect the function of osteocytes, essential regulators of bone homeostasis and quality. To first test this hypothesis, we used the IDG-SW3 osteocyte-like cell line to examine the effects of glucose levels on osteocyte function and viability in vitro. We confirmed our in vitro findings using the in vivo streptozotocin-induced (STZ) diabetic rat model and ex-vivo cultured osteocytes from these rats. IDG-SW3 cells cultured under high glucose conditions displayed significantly increased Sost mRNA(100-fold) and sclerostin protein, a negative regulator of bone formation(5000-fold), compared to cells in control media. mRNA expression of osteoblast markers such as Osx, Ocn and Col1a1 was unaffected by glucose. Factors associated with osteoclast activation were affected by glucose, with Rankl being upregulated by low glucose. Opg was also transiently upregulated by high glucose in mature IDG-SW3 cells. Induction of diabetes in Sprague-Dawley rats via a single dose of STZ (70 mg/kg) resulted in elevated maximum glucose and increased variability compared to control animals (670/796 vs. 102/142 mg/dL). This was accompanied by increased Sost/sclerostin expression in the osteocytes of these animals. These results show that glucose levels directly regulate osteocyte function through sclerostin expression and suggest a potential mechanism for the negative impact of diabetes on bone quality.

The effect of high glucose levels on the hypermethylation of protein phosphatase 1 regulatory subunit 3C (PPP1R3C) gene in colorectal cancer

2015 ◽  
Vol 94 (1) ◽  
pp. 75-85 ◽  
Author(s):  
SOO KYUNG LEE ◽  
JI WOOK MOON ◽  
YONG WOO LEE ◽  
JUNG OK LEE ◽  
SU JIN KIM ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
High Glucose ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Glucose Levels

scholarly journals Substrate-induced Nuclear Export and Peripheral Compartmentalization of Hepatic Glucokinase Correlates with Glycogen Deposition

2001 ◽  
Vol 2 (3) ◽  
pp. 173-186 ◽  
Author(s):  
Thomas L. Jetton ◽  
Masa Shiota ◽  
Susan M. Knobel ◽  
David W. Piston ◽  
Alan D. Cherrington ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Glycogen Synthesis ◽  
Regulatory Protein ◽  
Rat Hepatocytes ◽  
Coupling Mechanism ◽  
Glycogen Accumulation ◽  
Quantitative Image ◽  
Tightly Coupled ◽  
Stimulation Period ◽  
Glycogen Deposition

Hepatic glucokinase (GK) is acutely regulated by binding to its nuclear-anchored regulatory protein (GKRP). Although GK release by GKRP is tightly coupled to the rate of glycogen synthesis, the nature of this association is obscure. To gain insight into this coupling mechanism under physiological stimulating conditions in primary rat hepatocytes, we analyzed the subcellular distribution of GK and GKRP with immunofluorescence, and glycogen deposition with glycogen cytochemical fluorescence, using confocal microscopyand quantitative image analysis. Following stimulation, a fraction of the GK signal translocated from the nucleus to the cytoplasm. The reduction in the nuclear to cytoplasmic ratio of GK, an index of nuclear export, correlated with a >50% increase in glycogen cytochemical fluorescence over a 60min stimulation period. Furthermore, glycogen accumulation was initially deposited in a peripheral pattern in hepatocytes similar to that of GK. These data suggest that a compartmentalization exists of both active GK and the initial sites of glycogen deposition at the hepatocyte surface.

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