Glucose 6-Phosphate Produced by Gluconeogenesis and by Glucokinase Is Equally Effective in Activating Hepatic Glycogen Synthase*

Roger R. GomisDagger §||, Cristián FavreDagger §||**, Mar García-RochaDagger §, Josep M. Fernández-NovellDagger , Juan C. FerrerDagger , and Joan J. GuinovartDagger §DaggerDagger

From the Dagger  Departament de Bioquímica i Biologia Molecular and the § Institut de Recerca Biomèdica de Barcelona-Parc Científic de Barcelona, Universitat de Barcelona, Barcelona E-08028, Spain

Received for publication, December 1, 2002, and in revised form, December 27, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glucose 6-phosphate (Glc-6-P) produced in cultured hepatocytes by direct phosphorylation of glucose or by gluconeogenesis from dihydroxyacetone (DHA) was equally effective in activating glycogen synthase (GS). However, glycogen accumulation was higher in hepatocytes incubated with glucose than in those treated with DHA. This difference was attributed to decreased futile cycling through GS and glycogen phosphorylase (GP) in the glucose-treated hepatocytes, owing to the partial inactivation of GP induced by glucose. Our results indicate that the gluconeogenic pathway and the glucokinase-mediated phosphorylation of glucose deliver their common product to the same Glc-6-P pool, which is accessible to liver GS. As observed in the treatment with glucose, incubation of cultured hepatocytes with DHA caused the translocation of GS from a uniform cytoplasmic distribution to the hepatocyte periphery and a similar pattern of glycogen deposition. We hypothesize that Glc-6-P has a major role in glycogen metabolism not only by determining the activation state of GS but also by controlling its subcellular distribution in the hepatocyte.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glucose 6-phosphate (Glc-6-P)1 is a key metabolite in hepatic carbohydrate metabolism. It is produced from glucose and ATP, by the action of glucokinase (GK), from three carbon-atom precursors through gluconeogenesis and as a result of glycogen breakdown. In turn, Glc-6-P is a substrate for glycolysis, for the pentose-phosphate pathway and for the production of glucose, via glucose 6-phosphatase (G6Pase)-catalyzed hydrolysis, or glycogen, through a pathway that involves the successive conversion of Glc-6-P into glucose 1-phosphate (Glc-1-P) and UDP-glucose (UDP-Glc), the substrate of glycogen synthase (GS).

GS and GK are the key enzymes in the control of hepatic glycogen synthesis from glucose (1). The activity of the former is tightly regulated by phosphorylation and by allosteric effectors (2), mainly Glc-6-P. This metabolite is an allosteric activator of GS, and more importantly, it also promotes the permanent, covalent activation of GS by dephosphorylation (3). Furthermore, upon incubation with glucose, GS is redistributed in the hepatocyte, which may be an additional mechanism of control (4). In the absence of glucose hepatic GS is uniformly distributed throughout the cytoplasm, but it concentrates at the hepatocyte periphery when it is present (5, 6). This affects the pattern of hepatic glycogen deposition. In hepatocytes with low initial glycogen content, the polysaccharide is synthesized first near the plasma membrane and then, as the synthesis progresses, in more internal locations (6, 7).

Glucose also affects the subcellular distribution of GK, both in vitro (8-10) and in vivo (11, 12). In the absence of substrate, GK concentrates in the nucleus, where it remains bound to its regulatory protein, and it translocates to the cytoplasm when the levels of glucose or fructose increase.

Hepatic GS, but not muscle GS, differentiates between Glc-6-P produced by GK or hexokinase I (HK I). Only Glc-6-P produced from glucose by GK can induce the dephosphorylation and consequent activation of liver GS and trigger the synthesis of glycogen (13, 14). These results indicate there are at least two pools of Glc-6-P inside the hepatocyte. The pool accessible to liver GS is replenished by the action of GK, whereas the Glc-6-P produced by HK I is delivered to a cellular compartment from which GS is excluded.

Here we examine, in cultured hepatocytes, biochemical and cellular aspects of the synthesis of glycogen from dihydroxyacetone (DHA), an efficient gluconeogenic substrate (15). We used recombinant adenovirus technology to compare the role of Glc-6-P produced by gluconeogenesis with that arising from the direct phosphorylation of glucose by GK, in the activation of hepatic GS and its translocation to the cell periphery.

    EXPERIMENTAL PROCEDURES
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Preparation of Recombinant Adenovirus

The AdCMV-RLGS adenovirus has been described in a previous report (1). AdCMV-GK (16) and AdCMV-G6Pase (17) were generous gifts from Dr. C. B. Newgard.

Hepatocyte Isolation and Treatment with Recombinant Adenovirus

Hepatocytes were isolated by collagenase perfusion from male Wistar rats (180-225 g) fasted for 24 h, as described previously (18). Cells were suspended in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10 mM glucose, 10% fetal bovine serum (Biological Industries, Israel), 100 nM insulin, and 100 nM dexamethasone (Sigma, St. Louis, MO) and then seeded onto plastic plates of 60-mm diameter treated with 0.1% gelatin (Sigma) at a final density of 8 × 104 cells/cm2. After cell attachment (4 h at 37 °C), the medium was replaced by DMEM without glucose, fetal bovine serum, and hormones, and hepatocytes were treated for 2 h with AdCMV-RLGS, AdCMV-GK, or AdCMV-G6Pase at a multiplicity of infection of 10 for the first adenovirus and 4 for the other two. The medium was again replaced by DMEM without glucose, fetal bovine serum, and hormones, and cells were kept at 37 °C for an additional 16-18 h, to allow for the expression of the transgen. Finally, hepatocytes were incubated in the same medium supplemented with glucose or DHA at the concentrations indicated in the figure legends. At the end of each manipulation, cell monolayers were washed in phosphate-buffered saline and frozen in liquid N2 until analysis, or fixed for immunofluorescence studies.

Metabolite Determinations

To measure glycogen content, cell monolayers were scraped into 30% KOH, the extract was then boiled for 15 min and centrifuged at 5000 × g for 15 min. Glycogen was measured in the cleared supernatants as described (19). The intracellular concentration of Glc-6-P was measured using a spectrophotometric assay (20). Glucose concentration in the incubation medium was measured as described (21).

Enzyme Activity Assays

Glucose-phosphorylating Activity and GS-- Frozen cell monolayers from 60-mm diameter plates were scraped using 100 µl of homogenization buffer, which consisted of 10 mM Tris-HCl (pH 7.0), 150 mM KF, 15 mM EDTA, 15 mM 2-mercaptoethanol, 10 µg/ml leupeptin, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride. Thawing induced cell lysis. Protein concentration was measured using a Bio-Rad assay reagent as described (22). Total GS activity was measured in homogenates in the presence of 6.6 mM Glc-6-P, as described (23). Active GS (I or a form) was measured in the absence of Glc-6-P. Glucose-phosphorylating activity was measured spectrophotometrically in the supernatant fraction of hepatocyte extracts centrifuged at 10,000 × g for 15 min, using 1 mM or 100 mM glucose at 30 °C, as described (24).

G6Pase Activity-- Frozen cells were scraped into a buffer solution containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 100 mM KCl, 600 mM sucrose, and 15 mM 2-mercaptoethanol. Homogenates were completely lysed by sonication. The activity of G6Pase was assessed spectrophotometrically in the supernatant fraction after centrifugation at 10,000 × g for 15 min at 4 °C, as in Ref. 25.

Immunofluorescence Analyses of GS and Glycogen Distribution

Hepatocytes seeded onto gelatin-coated glass coverslips (10 mm × 10 mm) were washed with PBS and fixed for 30 min in PBS containing 4% paraformaldehyde. After fixation, cells were incubated with NaBH4 (1 mg/ml) to reduce auto-fluorescence, permeabilized with 0.2% (v/v) Triton X-100 in PBS, and blocked with 3% bovine serum albumin (w/v) in PBS. Next, cells were incubated for 1 h at room temperature with the L1 anti-liver GS (6) at dilution 1/500 (v/v), washed in PBS, and treated for 30 min with T-6391 Texas Red®-X goat anti-rabbit Ig G (H+L) conjugate (Molecular Probes). Alternatively, cells were treated for 1 h at room temperature with a monoclonal IgM antibody against glycogen, a generous gift from Dr. O. Baba (26), diluted in PBS containing 3% bovine serum albumin (w/v), washed with PBS, and subjected to incubation for 30 min with a goat anti-mouse IgM secondary antibody conjugated to tetramethylrhodamine (Chemicon).

For actin staining, hepatocytes were treated for 30 min with fluorescein-conjugated phalloidin (Molecular Probes). Coverslips were finally air-dried and mounted on glass microscope slides using Immuno Floure mounting medium (ICN Biomedicals, Inc.). Fluorescence images were obtained with a Leica TCS 4D (Leica Lasertechnik, Heidelberg, Germany) confocal scanning laser microscope adapted to an inverted Leitz DMIRBE microscope and a 63× (numerical aperture 1.4, oil) Leitz Plan-Apo objective. The light source was an argon/krypton laser (75 milliwatts), and optical sections (0.1 µm) were obtained.

Electrophoresis and Immunoblotting-- Active and total liver glycogen phosphorylase (GP) protein levels were measured in hepatocyte homogenates by Western blot. Electrotransfer of proteins from the gel to the nitrocellulose was performed at 200 V (constant) and room temperature for 2 h, using a Bio-Rad miniature transfer apparatus, as described (27). The nitrocellulose blots were incubated overnight at 4 °C in blocking buffer (1% bovine serum albumin, 0.05% Tween 20 in phosphate-buffered saline). Blots were then incubated for 1 h with antibodies against phosphorylated (active) or total GP. The antibody against active GP was raised in chicken using the peptide 9EKRRQI[pSer]IRGI19, which is located near the N terminus of rat liver GP and contains the Ser residue that is phosphorylated in the active enzyme. The antibody against total GP was obtained by immunizing rabbits with the peptide 829EPSDLKISLSKESSNGVNANGK850, which constitutes the C-terminal end of the protein. Membranes were then washed and incubated for 1 h with a secondary anti-chicken/rabbit antibody conjugated to horseradish peroxidase. Immunoreactive bands were visualized using an ECL kit (Amersham Biosciences), following the manufacturer's instructions.

Statistical Analysis-- Data are expressed as the mean ± S.E. Statistical significance was determined by unpaired two-tailed Student's t test. Statistical significance was assumed at p < 0.05.

    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of DHA on Glycogen Metabolism-- We evaluated glycogen deposition in hepatocytes that were synthesizing glucose from DHA by gluconeogenesis. Hepatocytes were maintained for 16 h in a medium devoid of glucose and DHA and then incubated for 2 h with increasing concentrations of DHA. These cells showed a dose-dependent increase in glycogen content (Fig. 1A), intracellular Glc-6-P concentration (Fig. 1B) and active GS levels (Fig. 1C), whereas total GS activity did not change in any of the conditions assayed (data not shown). These effects were maximal at 10 mM DHA (Fig. 1), and, therefore, this concentration of the gluconeogenic precursor was chosen to perform subsequent experiments.


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Fig. 1.   Glycogen content, Glc-6-P concentration, and active GS levels in hepatocytes incubated with DHA. After 16 h in DMEM, hepatocytes were incubated for 2 h with 1, 5, 10, or 25 mM DHA. Cells were then collected and glycogen content (A), intracellular Glc-6-P concentration (B), and active GS (C) levels were determined as described under "Experimental Procedures." Data represent the mean ± S.E. of four independent experiments.

Effect of DHA on the Subcellular Distribution of GS and Glycogen Deposition-- To examine the effect of DHA on the intracellular distribution of hepatic GS, cultured hepatocytes were incubated for 2 h in the presence of 10 mM DHA, 10 mM glucose, and 25 mM glucose or left untreated. Immunofluorescence analysis, using an antibody that specifically recognizes liver GS (6), showed that incubation with DHA produced an accumulation of the enzyme at the periphery of the hepatocytes, similar to that observed after glucose treatment. In control cells, GS was distributed throughout the cytoplasm, and, after incubation with glucose or DHA, the enzyme formed patches near the hepatocyte plasma membrane (Fig. 2A). Immunodetection of glycogen particles with a monoclonal anti-glycogen antibody (26) showed that DHA also induced the deposition of the polysaccharide at the cell periphery, as observed with the hepatocytes incubated with glucose (Fig. 2B).


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Fig. 2.   Subcellular distribution of GS and pattern of glycogen deposition in hepatocytes incubated with glucose or DHA. Confocal microscopy images of cultured hepatocytes treated for 2 h with the indicated concentrations of glucose or DHA. Control cells were incubated in DMEM without glucose and DHA. At the end of the incubations, hepatocytes were processed for immunofluorescence analysis with the L1 anti-GS antibody (A) or with an anti-glycogen antibody (B) as indicated under "Experimental Procedures." In B, actin was also stained, with fluorescein isothiocyanate-conjugated phalloidin, to delineate the contour of the cells. The scale bar indicates 10 µm.

Effect of GS Overexpression on Hepatocytes Incubated with DHA or Glucose-- In control uninfected hepatocytes, a 2-h treatment with 10 mM DHA induced the deposition of 16.1 ± 0.6 µg of glycogen/106 cells. This value was 40% lower than that measured for hepatocytes incubated with 25 mM glucose (28.1 ± 0.6 µg of glycogen/106 cells) (Fig. 3A), even though the levels of Glc-6-P and active GS were somewhat higher in the former condition (Fig. 3, B and C).


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Fig. 3.   Effects of GS, GK, and G6Pase overexpression on glycogen content, Glc-6-P concentration, and active GS levels in hepatocytes incubated with glucose or DHA. Hepatocytes were treated with AdCMV-RLGS (hatched bars), AdCMV-GK (gray bars), AdCMV-G6Pase (black bars), or left untreated (open bars). After 16 h in DMEM, hepatocytes were incubated for 2 h with 10 mM DHA or with 5 or 25 mM glucose and glycogen content (A), Glc-6-P concentration (B), and active GS (C) were measured as described under "Experimental Procedures." Data represent the mean ± S.E. of four independent experiments. *, p < 0.05 and **, p < 0.01 versus the respective uninfected control.

When total GS was overexpressed 4-fold by treatment of cultured hepatocytes with the AdCMV-RLGS adenovirus (Table I), the ability of these cells to accumulate glycogen increased substantially. However, the differences in glycogen content with the respective uninfected controls were higher in the glucose-treated hepatocytes than in those incubated with 10 mM DHA. Thus, GS-overexpressing hepatocytes treated with 5 or 25 mM glucose accumulated ~160% more glycogen than their corresponding controls and reached a level of 73 ± 10 µg/106 cells at 25 mM glucose (Fig. 3A). In contrast, AdCMV-RLGS-infected hepatocytes incubated with 10 mM DHA deposited 28 ± 3 µg of glycogen/106 cells, which is only 70% higher than that observed for their uninfected counterparts, and represents 40% of the glycogen accumulated by GS-overexpressing hepatocytes treated with 25 mM glucose. This large difference was observed despite the fact that intracellular Glc-6-P concentration (Fig. 3B) and active GS levels (Fig. 3C) were similar in AdCMV-RLGS-infected hepatocytes incubated with 25 mM glucose or with 10 mM DHA. On the other hand, the increase in glycogen content observed in GS-overexpressing hepatocytes incubated with DHA was accompanied by a 13% decrease in the glucose output, which changed from 215 ± 5 to 188 ± 2 µg glucose/106 cells in uninfected and GS-overexpressing cells, respectively.

                              
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Table I
Glucose-phosphorylating activity, G6Pase, and total GS activity in GS-, GK-, and G6Pase-overexpressing hepatocytes
Cultured rat hepatocytes were treated with AdCMV-GK or AdCMV-G6Pase at a moi of 4, with AdCMV-RLGS at a multiplicity of infection of 10, or were left untreated. Cells were incubated for 16 h with DMEM without glucose, treated for 2 h with 25 mM glucose, and finally collected for measurement of GK and HK, G6Pase and total GS activities as described under "Experimental Procedures." Activities are expressed in milliunits/106 cells and as a percentage of control values. Data represent the mean ± S.E. of four independent experiments.

Effect of GK Overexpression on Hepatocytes Incubated with DHA or Glucose-- A 3-fold overexpression of GK (Table I) caused an increase of about 120% in the intracellular Glc-6-P concentration of cultured hepatocytes treated with 5 (0.45 ± 0.10 nmol of Glc-6-P/106 cells) or 25 mM glucose (1.54 ± 0.16 nmol of Glc-6-P/106 cells), when compared with their uninfected controls (0.21 ± 0.05 and 0.66 ± 0.07 nmol Glc-6-P/106 cells, respectively) (Fig. 3B). As a result, the levels of active GS (1.74 ± 0.07 and 2.10 ± 0.16 milliunits/106 cells, respectively) (Fig. 3C) and glycogen content (31.7 ± 3.4 and 67.2 ± 6.9 µg/106 cells, respectively) (Fig. 3A) of AdCMV-GK-infected hepatocytes treated with 5 or 25 mM glucose also rose significantly and in a glucose-concentration dependent manner, as described previously (1).

In contrast, GK overexpression did not affect the Glc-6-P concentration of hepatocytes treated with 10 mM DHA (0.80 ± 0.05 nmol of Glc-6-P/106 cells in uninfected hepatocytes versus 0.86 ± 0.14 nmol of Glc-6-P/106 cells in AdCMV-GK-treated hepatocytes) (Fig. 3B). Accordingly, the levels of active GS (1.47 ± 0.06 and 1.43 ± 0.16 milliunits/106 cells, respectively) (Fig. 3C) and glycogen content (16.0 ± 0.9 and 19.3 ± 1.5 µg/106 cells, respectively) (Fig. 3A) remained unchanged compared with their respective controls.

Effect of G6Pase Overexpression on Hepatocytes Incubated with DHA or Glucose-- The ability of hepatocytes to accumulate glycogen was drastically reduced when G6Pase was overexpressed 3-fold in these cells (Table I). Cultured hepatocytes treated with AdCMV-G6Pase and incubated with 10 mM DHA and 5 or 25 mM glucose showed a decrease of 75, 35, and 50% in glycogen content, respectively, compared with their uninfected controls (Fig. 3A). The larger drop in glycogen accumulation in hepatocytes incubated with 10 mM DHA was consistent with the larger effect of the overexpression of G6Pase on Glc-6-P concentration and active GS levels in these cells, which were respectively 25 and 45% of those determined for the uninfected controls (Fig. 3, B and C). The concentration of Glc-6-P and active GS attained in G6Pase-overexpressing hepatocytes incubated with 25 mM glucose were, respectively, 50 and 75% of the corresponding values in uninfected hepatocytes (Fig. 3, B and C). Conversely, the glucose output in G6Pase-overexpressing hepatocytes incubated with 10 mM DHA (253 ± 3 µg of glucose/106) was almost 20% higher than in the uninfected cells (215 ± 5 µg of glucose/106 cells).

Correlation of Glycogen Content and Endogenous Active GS with Glc-6-P Concentration in Hepatocytes Incubated with DHA or Glucose-- We analyzed the relative effectiveness of Glc-6-P, produced by direct phosphorylation of glucose or by gluconeogenesis from DHA, in activating GS and promoting glycogen deposition in cultured hepatocytes. The values of glycogen content (Fig. 4A) and active GS (Fig. 4B), which were determined in previous experiments, were plotted against the corresponding Glc-6-P concentration values. The points derived from hepatocytes infected with the AdCMV-RLGS adenovirus were not included in the plots, because in these experiments the total amount of GS was artificially augmented and, therefore, they are not comparable with the rest of the series, as we previously pointed out (1).


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Fig. 4.   Correlation of glycogen content and endogenous active GS versus Glc-6-P concentration in hepatocytes incubated with glucose or DHA. The mean values of glycogen content (A) or endogenous active GS (B) shown in Figs. 1 and 3 were plotted against the corresponding mean values of Glc-6-P concentration. Open symbols correspond to hepatocytes incubated with glucose, and closed symbols to correspond to hepatocytes incubated with DHA. Cells treated with AdCMV-GK (triangles), AdCMV-G6Pase (squares), or left untreated (diamonds). Regression coefficients are indicted in the plots. The slopes of the two lines in A are 44 and 19 µg of glycogen/nmol Glc-6-P for hepatocytes incubated with glucose and DHA, respectively.

There was a strong correlation between the levels of active GS and Glc-6-P concentration, irrespective of the origin of this metabolite. This linear correlation held throughout the range of Glc-6-P concentrations, from the lowest values, attained by means of G6Pase overexpression, to the highest, obtained in GK-overexpressing hepatocytes (Fig. 4B).

However, when glycogen content was plotted against Glc-6-P concentration, two sets of points were clearly distinguishable (Fig. 4A). The points corresponding to hepatocytes incubated with glucose or with DHA fell in two separate straight lines with distinct slopes. Thus, the sensitivity of glycogen deposition to intracellular Glc-6-P concentration, measured as the slope of the plot, is about 2-fold higher in cultured hepatocytes incubated with glucose than in those treated with DHA (Fig. 4A).

Activation State of Glycogen Phosphorylase in Hepatocytes Incubated with DHA or Glucose-- To determine whether incubation with glucose or DHA affected the levels of total or active glycogen phosphorylase (GP), we performed Western blot analysis of hepatocyte homogenates, using two antibodies directed, respectively, against two unrelated peptides contained in the sequence of rat liver GP. The first antibody, which was raised against a peptide that consists of the 22 C-terminal amino acids of the protein, was used to provide a measure of total GP. In Western blots the immune, but not the pre-immune serum, recognized a band of the expected molecular mass in rat liver extracts but not in muscle homogenates (data not shown). Muscle expresses a different GP isoform, which does not contain in its sequence the peptide used for the immunizations of the rabbits. The second antibody was raised in chicken against an 11-amino acid peptide, which includes the phosphorylated Ser residue at position 15 of the rat liver GP, and it recognized a band of the expected molecular mass in liver extracts. This band was more intense in Western blots of homogenates from cultured hepatocytes treated with 100 µM forskolin, a drug that is known to trigger the phosphorylation and, thus the activation, of GP through an increase in the intracellular levels of cAMP (Fig. 5A). The binding of this antibody to active GP was blocked by the presence of the phosphorylated peptide used for the immunizations, but not by the analogous peptide in which the critical Ser residue (Ser-15) is not phosphorylated (Fig. 5A). These observations indicate that the phospho-specific antibody only recognizes the active phosphorylated form of liver GP.


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Fig. 5.   Western blot analysis of active and total GP in hepatocytes incubated with glucose or DHA. A, after 16 h in DMEM, hepatocytes were incubated for 2 h with 30 mM glucose or 100 µM forskolin, as indicated. The blot was performed in the presence or in the absence of the peptide used for immunization (Peptide 1: 9EKRRQI[pSer]IRGI19) or the analogous non-phosphorylated peptide (Peptide 2: 9EKRRQISIRGI19), as indicated. M designates the lane of molecular mass markers. B, after 16 h in DMEM, hepatocytes were incubated for 2 h with 10 mM DHA or with 5 or 25 mM glucose, as indicated. Cell homogenates were subjected to Western blot using an anti-rat liver GP antibody (total GP) or a chicken antibody that specifically recognizes phosphorylated GP (p-Ser GP).

Western blot analysis of cultured hepatocytes showed that the levels of total GP remained essentially unchanged in the incubations with 10 mM DHA and 5 or 25 mM glucose, compared with control cells (Fig. 5B). However, whereas the levels of active GP in control cells and those incubated for 2 h with 10 mM DHA did not significantly change, incubation with 5 or 25 mM glucose led to a marked decrease in the amount of active GP. The final concentration of glucose in the medium of the hepatocytes incubated with the gluconeogenic precursor was 0.7 ± 0.1 mM.

    DISCUSSION
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In previous reports we have shown that Glc-6-P produced by GK in cultured hepatocytes (13, 28) and in FTO2B cells (14) is more effective in promoting the covalent activation of hepatic GS and the deposition of glycogen than Glc-6-P derived from the catalytic action of HK I. This result suggested that Glc-6-P is compartmentalized in at least two pools in these cells. Liver GS is excluded from the compartment where the Glc-6-P produced by HK I is directed, while it has access to the Glc-6-P pool replenished by the GK-mediated phosphorylation of glucose. This second pool is also accessible to other enzymes and provides substrate for several metabolic routes. Thus, overexpression of GK enhances not only glycogen deposition, but also glycolysis, both in cultured hepatocytes (28) and in FTO2B cells (29). Glc-6-P from this pool can also be directed to hydrolysis by G6Pase, because overexpression of the catalytic subunit of this system reduces glycogen deposition and lactate production, while it increases hydrolysis of Glc-6-P (21).

The main conclusion of this study is that the gluconeogenic pathway and the direct phosphorylation of glucose by GK deliver their common product to the same "general" Glc-6-P pool, which feeds the above mentioned metabolic processes. The activation state of GS strongly correlates with the intracellular levels of Glc-6-P in hepatocytes incubated with glucose (3, 30, 31). As shown in the present study, this positive correlation also holds when cultured hepatocytes are incubated with gluconeogenic precursors, such that Glc-6-P arising from gluconeogenesis is as effective in activating hepatic GS as Glc-6-P produced by GK (Fig. 4B). The conversion of Glc-6-P into glucose by G6Pase and later re-phosphorylation to Glc-6-P prior to triggering the covalent activation of GS can be ruled out, because, in contrast to what occurs in hepatocytes incubated with glucose, GK overexpression has negligible effects on GS activation and glycogen deposition in hepatocytes incubated with DHA (Fig. 3).

However, although liver GS activation state is equally sensitive to Glc-6-P produced by GK or by gluconeogenesis, glycogen accumulation in cultured hepatocytes is more efficient when Glc-6-P originates from direct phosphorylation of glucose by GK (Fig. 4A). Similar levels of Glc-6-P attained by treating hepatocytes with glucose or with DHA have distinct quantitative effects on glycogen deposition. An explanation for this could reside in the observation that, although incubation with DHA has the main effect of increasing intracellular Glc-6-P concentration, treatment with glucose simultaneously triggers the partial dephosphorylation and inactivation of GP (Fig. 5) (2, 32), thus causing the arrest of glycogenolysis and allowing higher rates of glycogen accumulation. This is possible, because GLUT-2, the main glucose transporter in hepatocytes, essentially maintains intra- and extracellular glucose concentration in equilibrium. Hepatocytes can synthesize glucose from gluconeogenic precursors, but after 2 h of incubation with 10 mM DHA, the medium in which the cultured cells were kept only contained 0.7 mM glucose. This concentration is too low to significantly inactivate GP.

Through the use of GP inhibitors that cause its inactivation by dephosphorylation, it has been shown (15, 33, 34) that active GP plays a role in controlling the phosphorylation state of GS. The covalent inactivation of GP relieves glycogen synthase phosphatase from the allosteric inhibition caused by active GP, thus leading to the dephosphorylation and activation of GS (2). However, in our experimental conditions, the levels of active GS strongly correlate with the intracellular Glc-6-P concentration, regardless of the GP activation state, indicating that GP inactivation is not a prerequisite for the covalent activation of GS. An identical conclusion was obtained from two previous studies in which the treatment with fructose (35) or lithium chloride (36) of hepatocytes isolated from fasted rats led to the simultaneous activation of GS and GP. These two apparently contradictory conclusions may be reconciled by assuming that the levels of active GP do have an effect in determining the GS activation state, as long as the intracellular concentration of Glc-6-P remains constant. However, an increase in the Glc-6-P concentration would override the allosteric inhibition of glycogen synthase phosphatase by active GP, thus causing a corresponding increase in the levels of active GS in cultured hepatocytes.

Our results also suggest that the lower efficiency shown by Glc-6-P produced by gluconeogenesis in stimulating glycogen deposition could be attributed to a futile cycle through GS and GP, because both enzymes co-exist as their respective active forms in hepatocytes incubated with DHA. Glycogen cycling has been shown to occur in vivo and in vitro by several techniques that include the utilization of stable or radioactive isotopes (37-40) or selective GP inhibitors (41). Meijer and coworkers (15, 40) have analyzed how the degree of glucose cycling, through GK and G6Pase, and glycogen cycling affect glycogen accumulation in isolated hepatocytes. By means of acute inhibition of the hepatic G6Pase system with S4048, a chlorogenic acid derivative that inhibits translocation of Glc-6-P across the endoplasmic-reticulum membrane, these authors have altered the partitioning of Glc-6-P produced by gluconeogenesis into glucose production and storage as glycogen. This partitioning can also be modulated by the overexpression of the enzymes involved in this metabolism. Thus, GS overexpression in cultured hepatocytes re-directs part of the Glc-6-P produced by gluconeogenesis from DHA to glycogen deposition, with the consequent reduction of glucose output. Conversely, the overexpression of the catalytic subunit of G6Pase has opposite effects.

Another point that we have addressed in this report is the study of the subcellular distribution of hepatic GS. We have previously shown that incubation of isolated (5) or cultured hepatocytes (6) with glucose causes the translocation of GS from the cytoplasm to the cell periphery, where initial glycogen synthesis takes place. When added to cultured hepatocytes, DHA has the same effect as glucose on the localization of GS and the pattern of glycogen deposition. We propose that Glc-6-P is the key metabolite that controls the subcellular distribution of hepatic GS, such that an increase in the intracellular concentration of Glc-6-P triggers the accumulation of GS at the hepatocyte periphery. This is based in the following reasoning.

First, the mere activation of GS induced by treatment with lithium chloride, a known inhibitor of glycogen synthase kinase-3 (42, 43), is not sufficient to cause GS translocation to the hepatocyte periphery (6). Second, the common metabolites in the biosynthetic pathway of glycogen from glucose and from gluconeogenic precursors are Glc-6-P, which is in equilibrium with Glc-1-P, and UDP-Glc, the substrate of GS and immediate precursor in glycogen synthesis. Of these three intermediates, UDP-Glc can be ruled out as the signal molecule, because its intracellular concentration appears to be buffered and remains unchanged upon wide variations of Glc-6-P. Thus, GK (1) and G6Pase (21) overexpression in cultured hepatocytes led, respectively, to a large increase or decrease in the Glc-6-P levels, which in turn translated into increased or decreased glycogen accumulation. However, the intracellular concentration of UDP-Glc did not significantly change in any of these situations. Third, Glc-6-P is involved in the control of the aggregation state of hepatic GS and its translocation from a soluble form to a form that sediments at low centrifugal forces, both in isolated hepatocytes (44) and in vivo (31).

Therefore Glc-6-P, not only constitutes the crossroad of several metabolic pathways, but it also plays a key role as a signal molecule in the regulation of hepatic glycogen metabolism. It determines the activation state of GS and controls the aggregation state and the subcellular distribution of GS in hepatocytes.

    ACKNOWLEDGEMENTS

We thank Dr. C. B. Newgard for the generous gift of the AdCMV-GK and AdCMV-G6Pase adenoviruses and Dr. Otto Baba for the monoclonal anti-glycogen antibody. We also thank Anna Adrover for excellent technical assistance and Robin Rycroft for assistance in preparing the English manuscript.

    FOOTNOTES

* This study was supported in part by Grant PB98-0992 from the Dirección General de Enseñanza Superior (Ministerio de Educación y Cultura, Spain), Grant 992310 from the Fundació La Marató de TV3, and the Juvenile Diabetes Foundation International.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Doctoral Fellowship (Formación de Profesorado Universitario) from the Spanish Government (Ministerio de Educación y Cultura).

|| Both authors contributed equally to this work.

** Recipient of a Postdoctoral Fellowship from the Argentinean Government (Consejo Nacional de Investigaciones Científicas y Técnicas).

Dagger Dagger To whom correspondence should be addressed: Institut de Recerca Biomèdica de Barcelona-Parc Científic de Barcelona, c/Josep Samitier, 4-5, Barcelona E-08028, Spain. Tel.: 34-934-037-163; Fax: 34-934-037-114; E-mail: guinovart@pcb.ub.es.

Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M212151200

    ABBREVIATIONS

The abbreviations used are: Glc-6-P, glucose 6-phosphate; DHA, dihydroxyacetone; GK, glucokinase; HK I, hexokinase I; G6Pase, glucose 6-phosphatase; Glc-1-P, glucose 1-phosphate; UDP-Glc, UDP- glucose; GS, glycogen synthase; RLGS, rat liver GS; GP, glycogen phosphorylase; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; CMV, cytomegalovirus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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