From the 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
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ABSTRACT |
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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.
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.
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.
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.
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).
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).
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.
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).
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.
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.
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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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.
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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.
Glucose-phosphorylating activity, G6Pase, and total GS activity in GS-,
GK-, and G6Pase-overexpressing hepatocytes
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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.
View larger version (51K):
[in a new window]
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).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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).
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
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ABBREVIATIONS |
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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.
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REFERENCES |
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