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Originally published In Press as doi:10.1074/jbc.M405660200 on August 18, 2004

J. Biol. Chem., Vol. 279, Issue 45, 46474-46482, November 5, 2004
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The Glycogenic Action of Protein Targeting to Glycogen in Hepatocytes Involves Multiple Mechanisms Including Phosphorylase Inactivation and Glycogen Synthase Translocation*

Andrew R. Green{ddagger}§, Susan Aiston{ddagger}, Cynthia C. Greenberg¶, Susan Freeman||, Simon M. Poucher||, Matthew J. Brady¶, and Loranne Agius{ddagger}**

From the {ddagger}Department of Diabetes, School of Clinical Medical Sciences, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom, Department of Medicine, University of Chicago, Chicago, Illinois 60637, and ||Cardiovascular and Gastrointestinal Discovery-AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom

Received for publication, May 20, 2004 , and in revised form, August 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of the glycogen-targeting protein PTG promotes glycogen synthase activation and glycogen storage in various cell types. In this study, we tested the contribution of phosphorylase inactivation to the glycogenic action of PTG in hepatocytes by using a selective inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a and sequential activation of glycogen synthase. Similar to CP-91194, graded expression of PTG caused a concentration-dependent inactivation of phosphorylase and activation of glycogen synthase. The latter was partially counter-acted by the expression of muscle phosphorylase and was not additive with the activation by CP-91149, indicating that it is in part secondary to the inactivation of phosphorylase. PTG expression caused greater stimulation of glycogen synthesis and translocation of glycogen synthase than CP-91149, and the translocation of synthase could not be explained by accumulation of glycogen, supporting an additional role for glycogen synthase translocation in the glycogenic action of PTG. The effects of PTG expression on glycogen synthase and glycogen synthesis were additive with the effects of glucokinase expression, confirming the complementary roles of depletion of phosphorylase a (a negative modulator) and elevated glucose 6-phosphate (a positive modulator) in potentiating the activation of glycogen synthase. PTG expression mimicked the inactivation of phosphorylase caused by high glucose and counteracted the activation caused by glucagon. The latter suggests a possible additional role for PTG on phosphorylase kinase inactivation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulation of liver glycogen metabolism by hormones and substrates is mediated by changes in the phosphorylation state of glycogen synthase and phosphorylase and by subcellular translocation of these proteins (1–3). Dephosphorylation is catalyzed by PP1C1 in association with glycogen-targeting proteins (4), of which three isoforms are expressed in liver, designated GL, PTG, and R6 (5). All isoforms have glycogensynthase phosphatase and phosphorylase phosphatase activity when assayed in vitro; however, the synthase phosphatase/phosphorylase phosphatase ratio is higher for GL-PP1 than for PTG-PP1, suggesting that GL may be the predominant synthase phosphatase (5). GL (R4) is expressed predominantly in liver and to a lesser extent in heart and human skeletal muscle (6, 7), PTG is expressed in several tissues including liver and skeletal muscle (8–10), and R6 is expressed ubiquitously (11). Both GL and PTG show adaptive changes in expression in rat liver in response to changes in insulin status (5), and the physiological role of these changes has been confirmed from studies (12, 13) in hepatocytes, which showed glycogen synthase activation and increased glycogen storage when GL and PTG were overexpressed.

A unique property of GL is its allosteric binding site for phosphorylase a, which accounts for the inhibition of glycogensynthase phosphatase activity by low concentrations of phosphorylase a (5). This mechanism enables reciprocal control of the phosphorylation state of glycogen synthase and phosphorylase (1). Accordingly, glucose or selective inhibitors of phosphorylase a that favor the tense conformation, which is a better substrate for dephosphorylation, cause depletion of phosphorylase a (conversion to phosphorylase b) and sequential activation of glycogen synthase, whereas ligands that favor the phosphorylated state cause inactivation of synthase (14–16).

Using a selective phosphorylase inhibitor (CP-91149) that causes depletion of phosphorylase a and sequential activation of glycogen synthase (17), it has been shown that phosphorylase a has a very high negative flux control coefficient on glycogen synthesis in hepatocytes, indicating that a small depletion of phosphorylase a causes a large fractional stimulation of glycogen synthesis (15). In this study we used CP-91149 to test the hypothesis that inactivation of phosphorylase has an important contributory role to the glycogenic action of PTG in hepatocytes. The results show that the glycogenic action of PTG in hepatocytes involves multiple mechanisms, including inactivation of phosphorylase and translocation of glycogen synthase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Adenoviruses expressing glucokinase (18) and muscle glycogen phosphorylase (19) were gifts from Drs. C. Newgard and A. M. Gomez-Foix, CP-91149 (17) was a gift from Pfizer, and KT-5720 was from Calbiochem or Tocris Cookson, Ltd. (Avonmouth, UK).

Hepatocyte Isolation and Treatment with Adenoviruses—Hepatocytes were isolated by collagenase perfusion (20) of the livers of male Wistar rats (170–240 g) (B & K, Hull, UK). Hepatocytes were suspended in MEM containing 6% (v/v) newborn calf serum and plated in multiwell plates (20). After cell attachment (~2 h) the medium was replaced with serum-free MEM containing the adenovirus followed by a 3-h incubation. The medium was then supplemented with serum-free MEM containing 5 mM glucose and 10 nM dexamethasone, and cells were cultured for 16 h.

Preparation of Recombinant Adenoviruses—PTG was subcloned into the tetracycline-responsive Adeno-X DNA vector (21). The PTG-adenovirus and the regulatory adenovirus (Adeno-X Tet-Off) were diluted in MEM containing 0.5 µg/ml poly-L-lysine hydrobromide. Hepatocyte monolayers were incubated with the PTG-adenovirus (75–1800 pfu/cell). Where indicated, cells were cultured in medium supplemented with 1 µg/ml doxycycline to inhibit PTG expression (21). To confirm transfection efficiency, cells were treated with an adenovirus encoding {beta}-galactosidase and stained (22).

Metabolic Incubations—Incubations for the determination of glycogen synthesis were performed for 3 h in fresh MEM containing 10 mM glucose, [U-14C]glucose (2 µCi/ml), and other additions as indicated. Incubation was terminated by washing with 150 mM NaCl, and cells were extracted in 0.1 M NaOH. The incorporation of radiolabel into glycogen was determined (20). Cell glycogen content was determined enzymatically (20). Incubation sequences for glycogen synthase and phosphorylase a were performed with 10 mM glucose without radiolabel, and cells were extracted as described previously (23). Active and total glycogen synthase were assayed without or with glucose-6-P, respectively (24), in the homogenate. Active synthase is expressed as milliunits/mg protein or as the activity ratio (active/total). Total synthase activity was not affected by CP-91149 or PTG expression but was increased by glucokinase expression (2.7 ± 0.4 to 3.4 ± 0.4 milliunits/mg). Distribution of total synthase between the 13,000 x g pellet and supernatant was determined as described previously (3) and is expressed as pellet percentage of the total. Phosphorylase a was assayed in the supernatant spectrometrically in the glycogenolytic direction and is expressed as milliunits/mg protein (15). Distribution of phosphorylase a between the pellet and supernatant was determined radiochemically (3, 23) from the incorporation of [1-14C]glucose-1-P into glycogen (25) and expressed as pellet percentage of the total. Activities were found to be 2-fold higher by the radiochemical assay compared with the spectrometric assay (3).

Immunoblotting—For determination of phosphorylase and PTG protein, cell lysates were fractionated by SDS-PAGE, and protein was transferred to nitrocellulose. Phosphorylase was determined with a mouse IgG (clone 3G1) (Research Diagnostics, Flanders, NJ) (3). PTG was determined as described previously (21) followed by peroxidase-conjugated anti-IgG and visualization with an ECL kit (Amersham Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PTG Overexpression Stimulates Glycogen Synthesis and Inactivates Phosphorylase—Cells transduced with graded titers (75–1800 pfu/cell) of PTG-adenovirus showed increased expression of PTG protein, with a greater distribution in the 13,000 x g pellet compared with the supernatant (Fig. 1). Using a {beta}-galactosidase adenovirus, we confirmed that transfection efficiency was >70% at viral titers >300 pfu/cell. In the rest of this study we used viral titers from 75 to 1800 pfu/cell or of 300 and 1800 pfu/cell, representing intermediate and high expression, respectively.



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  		FIG. 1.
PTG immunoreactivity in cells treated with PTG-adenovirus. Hepatocytes were treated with varying titers (pfu/cell x 100) of PTG-adenovirus and then cultured for 18 h without or with 1 µg/ml doxycycline (+DX). PTG was determined by immunoblotting in the homogenate (H), supernatant (SN), and particulate (P) fractions. Representative results of four (–DX) or two (+DX) experiments are shown.

 
The highest PTG overexpression increased glycogen synthesis 9-fold and active glycogen synthase 3-fold and decreased phosphorylase a by 85% (Fig. 2, A, C, and E). These effects are qualitatively similar to the effects of the phosphorylase inhibitor CP-91149 (Fig. 2, B, D, and F), which causes inactivation of phosphorylase, sequential activation of glycogen synthase, and stimulation of glycogen synthesis (15, 17). There was an inverse correlation between the activity of active glycogen synthase and phosphorylase a in cells incubated with CP-91149 or overexpressing PTG (Fig 2G). However, activation of synthase by CP-91149 reached saturation at lower activities of phosphorylase a, suggesting an additional role for PTG in activating synthase independently of dephosphorylation of phosphorylase a when the latter is below a threshold. Stimulation of glycogen synthesis by PTG expression was greater than with CP-91149, also indicating involvement of additional mechanisms (Fig. 2H). We confirmed that doxycycline counteracts the stimulation of glycogen synthesis by the PTG-adenovirus but not the stimulation by insulin, indicating that the metabolic changes in cells treated with PTG-adenovirus are not caused by nonspecific effects of the adenovirus.



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  		FIG. 2.
Comparison of PTG expression and inactivation of phosphorylase with CP-91149. A, C, and E, hepatocytes were treated with varying titers of PTG-adenovirus and cultured for 16 h. They were then incubated for 3 h in medium containing 10 mM glucose. B, D, and F, hepatocytes not treated with adenovirus were incubated for 3 h with 10 mM glucose CP-91149. A and B, glycogen synthesis; C and D, active glycogen synthase; E and F, phosphorylase a activity; G, active glycogen synthase versus phosphorylase a. H, glycogen synthesis versus phosphorylase a is shown for cells expressing PTG (filled circles) or incubated with CP-91149 (open circles). Means ± S.E. are shown for seven experiments.

 
Effects of PTG Are Additive with Glucokinase Expression but Not with Phosphorylase Inactivation—To further test the role of phosphorylase inactivation in the glycogenic action of PTG, we determined the combined effects of PTG expression (300 and 1800 pfu/cell) and either incubation with CP-91149 or glucokinase expression (6–8-fold), because glucokinase and CP-91149 activate glycogen synthase by increasing glucose-6-P (26) or by depleting phosphorylase a (15), respectively. CP-91149 activated glycogen synthase and stimulated glycogen synthesis in non-transduced cells at the lower level but not at the higher level of PTG expression, whereas glucokinase expression had an additive effect with PTG expression on glycogen synthase activation and glycogen synthesis stimulation (Fig. 3). Inactivation of phosphorylase a by PTG expression was additive with the effects of CP-91149 and glucokinase expression (Fig. 3C). The lack of further activation of glycogen synthase in the combined presence of CP-91149 and PTG expression (Fig. 3, B compared with C) can be explained by inhibition of synthase phosphatase when phosphorylase a is above a certain threshold. Conversely, the additive effects of glucokinase and PTG on glycogen synthase can be explained by the role of glucose-6-P (27) in potentiating synthase phosphatase activity by PTG or GL.



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  		FIG. 3.
Combined effects of PTG expression and inactivation of phosphorylase or glucokinase. Hepatocytes were either untreated (open bars) or treated with PTG-adenovirus at titers of 300 (hatched bars) or 1800 (filled bars) pfu/cell. Where indicated, cells were also treated with glucokinase adenovirus before the 16-h culture (GK) or were incubated with 10 µM CP-91149 (CP) during the final 3-h incubation with 10 mM glucose for determination of glycogen synthesis (A), glycogen synthase (B), or phosphorylase-a (C). Con, control. a, p < 0.05; aa, p < 0.01, effects of CP-91149 or glucokinase expression; b, p < 0.05; bb, p < 0.01, effect of PTG. Means ± S.E. for n = 6–8.

 
Effects of PTG Expression Are In Part Counteracted by Muscle Phosphorylase—To further test the role of phosphorylase in the glycogenic effect of PTG, we determined the effects of PTG expression in combination with either expression of the muscle isoform of phosphorylase (MGP) or with short term incubation with glucagon (Fig. 4). Expression of MGP was associated with small changes in the activity of phosphorylase a (Fig. 4C), despite a large increase in total phosphorylase as confirmed by immunoblotting (2–4-fold, results not shown), indicating that the expressed protein is predominantly in the dephosphorylated state as shown previously (15). MGP expression partially counteracted the activation of glycogen synthase and stimulation of glycogen synthesis by the lower level of PTG expression, whereas glucagon suppressed the activation of glycogen synthase and stimulation of glycogen synthesis by the higher PTG expression. The latter effect could not be explained by increased phosphorylase a because high PTG expression counter-acted the activation of phosphorylase by glucagon.



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  		FIG. 4.
Partial counteraction by MGP or glucagon of the glycogenic action of PTG. Hepatocytes were either untreated (open bars) or treated with PTG-adenovirus at titers of 300 (hatched bars) or 1800 (filled bars) pfu/cell. Where indicated, cells were additionally treated with adenovirus for expression of MGP before the 16-h culture or were incubated with 1 µM glucagon (GLN) during the final 3-h incubation with 10 mM glucose for determination of glycogen synthesis (A), glycogen synthase (B), or phosphorylase-a (C). Con, control. a, p < 0.05; aa, p < 0.01 effects of MGP expression or glucagon; b, p < 0.05; bb, p < 0.01 effect of PTG. Means ± S.E. for n = 6–8.

 
PTG Overexpression Causes Translocation of Glycogen Synthase to the Pellet—The greater stimulation of glycogen synthesis by PTG expression compared with CP-91149 (Fig. 2) suggests that inactivation of phosphorylase and activation of synthase cannot fully explain the glycogenic action of PTG. We therefore tested the effects of PTG expression on the compartmentation of glycogen synthase, using a similar approach as used previously (26, 28) to study glucose-6-P-mediated translocation. Expression of PTG caused a 5-fold increase in the distribution of total glycogen synthase in the pellet (Fig. 5A), whereas glucokinase expression and CP-91149 caused a 4-fold and 3-fold increase, respectively, in agreement with previous studies (26, 29). Translocation by PTG expression was slightly higher (30–50%) in cells treated with CP-91149 or overexpressing glucokinase (Fig. 5A). For cells expressing PTG and/or MGP without or with incubation with CP-91149 there was a correlation between glycogen synthesis and the distribution of glycogen synthase. However, stimulation by glucokinase expression and inhibition by glucagon could not be explained by changes in glycogen synthase distribution (Fig. 5B).



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  		FIG. 5.
Effects of PTG expression on translocation of glycogen synthase to the pellet. Conditions were as in Figs. 3 and 4. Total glycogen synthase activity (+ glucose-6-P) was determined in the supernatant and pellet fractions. A, activity in the pellet is expressed as a percentage of the supernatant plus pellet. Con, control; CP, CP-91149; GK, glucokinase adenovirus; GLN, glucagon. a, p < 0.05; aa, p < 0.01 relative to control; b, p < 0.05; bb, p < 0.01 effect of PTG. B, glycogen synthesis is compared with glycogen synthase distribution. (open circles), control; (filled circles), CP-91149; (filled squares), glucokinase adenovirus; (open squares), glucagon. Means ± S.E. for n = 5.

 
PTG Overexpression Causes Translocation of Phosphorylase a to the Pellet—Because glucose-6-P causes translocation of phosphorylase (determined enzymatically or by immunoblotting (3, 23)) as well as glycogen synthase from the supernatant to the pellet (26, 28), we tested the combined effects of PTG and glucokinase expression on the distribution of phosphorylase. There was an additive effect of glucokinase and PTG on translocation of phosphorylase a (Fig. 6A), which correlated with the translocation of glycogen synthase (Fig 6B). Glucagon slightly lowered the distribution of phosphorylase a in the pellet. This effect was further investigated in time course experiments (see below). It is noteworthy that translocation of glycogen synthase to the pellet correlated with the relative distribution of phosphorylase a and not with the absolute activity of phosphorylase a in the pellet. This correlation is similar to previous findings (3) on glucose-6-P-induced translocation in which translocation of phosphorylase a was associated with translocation of immunoreactive protein.



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  		FIG. 6.
Effects of PTG overexpression on translocation of phosphorylase a to the pellet. Experimental conditions were as in Fig. 5. Phosphorylase a activity was determined in the supernatant and pellet fractions. A, activity in the pellet is expressed as a percentage of the supernatant and pellet fractions. open bars, untreated hepatocytes; hatched bars, hepatocytes treated with PTG-adenovirus at titers of 300 pfu/cell; filled bars, hepatocytes treated with PTG-adenovirus at titers of 1800 pfu/cell. Con, control; GK, glucokinase adenovirus; Gln, glucagon. a, p < 0.05, relative to control; b, p < 0.05; bb, p < 0.01, effect of PTG. B, distribution of phosphorylase a is compared with the distribution of glycogen synthase. open circle, control; filled square, glucokinase adenovirus; open square, glucagon. Means ± S.E. are shown for five experiments.

 
Enzyme Translocation during PTG Expression Is Not Secondary to Glycogen Accumulation—Cells treated with adenovirus for expression of PTG show progressive glycogen accumulation (12, 13). To test whether the translocation of glycogen synthase and phosphorylase to the pellet is secondary to glycogen accumulation, hepatocytes treated with PTG-adenovirus were cultured with glucagon (2 µM) plus dibutyryl cAMP (50 µM) to suppress glycogen deposition (Fig. 7A). When the distribution of glycogen synthase and phosphorylase a in the pellet were plotted against the glycogen content, the data for incubations without or with glucagon plus dibutyryl cAMP were not superimposed (Fig. 7, B and C), showing that enzyme translocation in cells expressing PTG cannot be fully explained by glycogen accumulation. As in the other experiments there was a strong correlation (p < 0.0001) between synthase and phosphorylase a distribution in the pellet (Fig 7D).



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  		FIG. 7.
Enzyme translocation in cells expressing PTG is not caused by glycogen accumulation. Hepatocytes were treated with varying titers of PTG-adenovirus and cultured for 16 h without (open circles) or with (closed circles) 2 µM glucagon and 50 µM dibutyryl cAMP to suppress glycogen accumulation. A, glycogen content is shown. B, total glycogen synthase is shown (+ glucose-6-P) assayed in the supernatant and pellet and expressed as pellet percentage of the supernatant and pellet versus respective glycogen content. C, phosphorylase a is shown expressed as pellet percentage of the supernatant and pellet versus respective glycogen content. D, phosphorylase a distribution versus glycogen synthase distribution is shown. Means ± S.E. for n = 4.

 
PTG Expression Counteracts the Acute and Sustained Activation of Phosphorylase by Glucagon—The inactivation of phosphorylase caused by PTG expression could be caused by either an increase in phosphorylase phosphatase or by inactivation of phosphorylase kinase. To test the latter possibility, we compared the effects of PTG expression with incubation with the phosphorylase inhibitor (CP-91149) or a potent inhibitor (KT-5720) of phosphorylase kinase (30) on the time course of activation of phosphorylase by glucagon. In non-transduced cells, glucagon caused rapid activation of phosphorylase that peaked within 2 min in the homogenate and pellet but reached a maximum in the supernatant after 30 min (Fig. 8A). The activity in the pellet, expressed as a percentage of the total (Fig. 8B), showed a biphasic effect with a maximum at 2 min, suggesting that activation by phosphorylase kinase occurs at least in part in the particulate fraction followed by translocation to the soluble fraction. The phosphorylase kinase inhibitor caused a delay in peak activation, whereas the phosphorylase inhibitor caused both a delay in peak activation and suppression of maximum activation. PTG expression caused a greater suppression of the glucagon-induced activation than either of the inhibitors (Fig. 8C).



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  		FIG. 8.
Effects of PTG expression and inhibitors of phosphorylase and phosphorylase kinase on activation of phosphorylase by glucagon. A, phosphorylase a in homogenate (open circles), supernatant (upper U shape), and pellet (lower U shape) after addition of 1 µM glucagon in non-transduced hepatocytes. B and C, hepatocytes were either non-transduced (open symbols) or transduced (filled circles) with 1800 pfu/cell PTG-adenovirus. They were incubated with 1 µM glucagon for the time intervals shown, without (open circles) or with 10 µM CP-91149 (open triangle) or 20 µM KT-5720 (open squares). Phosphorylase a was determined radiochemically in the homogenate, supernatant, and pellet fractions and is expressed either as milliunits/mg protein (A and C) or as the activity in the pellet as a percentage of the total. *, p < 0.05; **, p < 0.005 relative to control. Means ± S.E. for n = 4.

 
Inactivation of Phosphorylase a by Glucose and PTG Is Not Additive—Because glucose is the main substrate regulator of phosphorylase in liver, we tested whether glucose (25 mM) has additive effects with PTG expression on phosphorylase a activity in the homogenate and supernatant fractions (Fig. 9). Glucose (25 mM versus 5 mM) decreased the activity of phosphorylase a by 46% in the supernatant and by 29% in the homogenate in untreated cells. The decrease in phosphorylase a activity in the homogenate is a measure of dephosphorylation, whereas the decrease in the supernatant is the combined effect of dephosphorylation and translocation. The effects of glucose and PTG were not additive suggesting that PTG mimics the effect of glucose.



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  		FIG. 9.
Inactivation of phosphorylase by glucose is not additive with the effects of PTG. Hepatocytes were treated with the PTG-adenovirus, cultured for 16 h, and incubated for 1 h with 5 mM (open circles), 10 mM (filled circles), or 25 mM (filled squares) glucose. Phosphorylase a was determined in the homogenate (A) and supernatant (B). Means ± S.E. for n = 4. *, p < 0.05; **, p < 0.01 relative to 5 mM glucose.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
PTG expression activates glycogen synthase and stimulates glycogen storage in various cell types (8, 12, 13, 21). Because phosphorylase a has major roles in the control of synthase phosphatase (1) and the rate of glycogen synthesis in hepatocytes (15, 29), we tested the role of inactivation of phosphorylase in the glycogenic action of PTG in hepatocytes. We show that, in addition to a direct role as a synthase phosphatase (8), there are two key mechanisms by which PTG promotes glycogen storage: depletion of phosphorylase a and translocation of glycogen synthase.

The role of PTG as a phosphorylase phosphatase was evident from the progressive decline in phosphorylase a with PTG expression and from the counteraction of the activation of phosphorylase by glucagon. To test the contribution of depletion of phosphorylase a to the activation of glycogen synthase and stimulation of glycogen synthesis, we used a selective inhibitor of phosphorylase (CP-91149) that causes activation of glycogen synthase by depleting phosphorylase a (17). There was an apparently similar inverse correlation between activation of glycogen synthase and depletion of phosphorylase a in response to either CP-91149 or PTG expression; however, in the former but not the latter case, synthase activation approached saturation. This suggests that activation of synthase by PTG involves two components: an indirect mechanism via depletion of phosphorylase a and a direct mechanism as a synthase phosphatase that is apparent when depletion of phosphorylase a approaches saturation. The lack of additive effects of PTG expression and CP-91149 on activation of synthase indicates that PTG shares a common mechanism with CP-91149 and supports the role of phosphorylase a depletion as a contributing mechanism to the activation of synthase. The partial counter-action by muscle phosphorylase expression on synthase activation by PTG is further evidence supporting the role of phosphorylase. It is noteworthy that, although phosphorylase a is a more potent inhibitor of synthase phosphatase than phosphorylase b (1), the latter also has inhibitory activity (31). The effect of MGP may be caused by binding to GL or the result of competition with glycogen synthase for PTG (32).

PTG expression was more effective in counteracting the activation of phosphorylase by glucagon than either the phosphorylase inactivator or the inhibitor of phosphorylase kinase (KT-5720). The latter compound was identified as a cell permeable inhibitor of protein kinase A (33) and was subsequently shown to be a potent inhibitor (IC50 11 nM versus 3.3 µM) of phosphorylase kinase (30). At 20 µM, KT-5720 caused a delay in the activation of phosphorylase by glucagon but did not affect the sustained activation. The greater efficacy of PTG expression in counteracting activation of phosphorylase by glucagon than either the phosphorylase kinase inhibitor or CP-91149 could be the result of a dual role of PTG in promoting dephosphorylation of both phosphorylase and phosphorylase kinase.

The additive effects of PTG and glucokinase expression on synthase activation can be explained by the complementary roles of glucose-6-P, derived from glucokinase (a positive modulator) and depletion of phosphorylase a (a negative modulator) on synthase phosphatase activity by substrate-directed mechanisms (Fig. 10). The additive activation of synthase by glucokinase and PTG expression can also be explained by the direct effect of PTG as a synthase phosphatase (8) and may be caused by the elevated glucose-6-P resulting from glucokinase expression (27) potentiating synthase phosphatase activity.



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  		FIG. 10.
Model showing effects of PTG expression and glucose-6-P on enzyme translocation and dephosphorylation. PTG expression causes translocation of glycogen synthase (GS) and phosphorylase a (Phos-a) from the soluble to the pellet fraction and inactivation of phosphorylase a (conversion to phosphorylase b (Phos-b)) in the pellet. Phosphorylase a is an inhibitor of synthase phosphatase associated with GL-PP1. Dephosphorylation of phosphorylase a by PTG-PP1 allows deinhibition of synthase phosphatase in association with GL and conversion of inactive glycogen synthase (GSB) to active glycogen synthase (GSA). Glucose-6-P, like PTG, causes translocation of glycogen synthase and phosphorylase a to the pellet and dephosphorylation of phosphorylase a. It promotes dephosphorylation of glycogen synthase by a substrate-directed mechanism, explaining the synergistic activation of glycogen synthase by PTG and glucokinase expression. Glucagon-induced phosphorylation of phosphorylase b by phosphorylase kinase (PhK) occurs in the pellet fraction.

 
Although the lack of additive stimulation of glycogen synthesis by PTG expression and the phosphorylase inactivator confirms a role for inactivation of phosphorylase, it does not exclude additional mechanisms. We have shown that, in hepatocytes, inhibitors of glycogen synthase kinase-3 cause a large activation of glycogen synthase but have little impact on glycogen synthesis (29). The greater maximal stimulation of glycogen synthesis by PTG expression compared with the phosphorylase inhibitor indicates that in addition to synthase activation other mechanisms must be involved. Translocation of glycogen synthase to the pellet is a likely contributing mechanism as shown by the correlation between glycogen synthesis and synthase distribution in the pellet in cells expressing PTG or treated with CP-91149. This translocation is not secondary to glycogen accumulation and is best explained by a targeting role of PTG. Fig. 10 shows a tentative model for the changes in distribution (between soluble and particulate fractions) and in the activation state of glycogen synthase and phosphorylase in response to the stimuli tested in the present and previous studies (3, 29). Translocation of glycogen synthase and phosphorylase a occurs in tandem in response to elevated glucose-6-P (3, 23) or PTG expression (present study). The effect of glucose-6-P could be tentatively explained by an increased affinity of glycogen synthase for PTG-PP1 (see above). The phosphorylase inhibitor causes translocation of glycogen synthase to the particulate fraction concomitant with dephosphorylation of phosphorylase a (29). Thus phosphorylase a may have a dual role in the particulate fraction as an allosteric inhibitor of GL-PP1 synthase phosphatase and as a substrate for phosphorylase phosphatase in association with PTG or GL. The time course of activation of phosphorylase b by glucagon suggests that phosphorylation occurs in the particulate fraction and may be followed by translocation to the soluble fraction. It seems likely therefore that the particulate fraction is the site for both dephosphorylation and phosphorylation of glycogen synthase and that phosphorylase and the soluble fraction in part represent a storage pool of the enzymes. PTG may have a dual role in the dephosphorylation of phosphorylase and phosphorylase kinase and translocation of enzymes from the soluble pool to the particulate fraction.

High glucose concentration suppresses glycogenolysis not only through glucose-mediated allosteric inhibition of phosphorylase a and dephosphorylation (1) but also through glucose-6-P-mediated dephosphorylation of phosphorylase a and translocation (3, 23). Two explanations are possible for the lack of additive effects of glucose and PTG on inactivation of phosphorylase a. First, glucose is more effective at promoting dephosphorylation at high activities of phosphorylase a. Second, glucose and/or glucose-6-P acts on a similar pool of phosphorylase a as PTG expression and induces translocation and dephosphorylation by similar mechanisms.

In summary, the glycogenic action of PTG in hepatocytes is caused by multiple mechanisms. In addition to a direct effect as a synthase phosphatase (8), inactivation of phosphorylase by PTG is an upstream event in the mechanism by which PTG activates glycogen synthase and translocation of glycogen synthase contributes to the glycogenic mechanism.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a Biotechnology and Biological Science Research Council Case studentship with AstraZeneca. Back

** To whom correspondence should be addressed: Dept. of Diabetes, School of Clinical Medical Sciences, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom. Tel.: 044-191-2227033; Fax: 044-191-2220723; E-mail: Loranne.Agius{at}ncl.ac.uk.

1 The abbreviations used are: PP, protein phosphatase; glucose-6-P, glucose 6-phosphate; GL, hepatic glycogen targeting subunit; PTG, protein targeting to glycogen; MEM, minimum essential medium; pfu, plaque-forming units; MGP, muscle glycogen phosphorylase. Back


   ACKNOWLEDGMENTS
 
We thank Diabetes UK for project and equipment support, Dr. J. Treadway for CP-91149, Drs. A. Gomez-Foix and C. Newgard for adenoviruses, and Dr. L. Johnson for advice on the phosphorylase kinase inhibitor.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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