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J. Biol. Chem., Vol. 277, Issue 26, 23246-23252, June 28, 2002
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,From the Department of Biochemistry and Molecular Biology and Barcelona Science Park, Universitat de Barcelona, E-08028 Barcelona, Spain
Received for publication, November 26, 2001, and in revised form, March 5, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Using adenovirus-mediated gene transfer into
FTO-2B cells, a rat hepatoma cell line, we have overexpressed
hexokinase I (HK I), glucokinase (GK), liver glycogen synthase (LGS),
muscle glycogen synthase (MGS), and combinations of each of the two
glucose-phosphorylating enzymes with each one of the GS isoforms.
FTO-2B cells do not synthesize glycogen even when incubated with high
doses of glucose. Adenovirus-induced overexpression of HK I and/or LGS,
two enzymes endogenously expressed by these cells, did not produce a
significant increase in the levels of active GS and the total glycogen
content. In contrast, GK overexpression led to the
glucose-dependent activation of endogenous or overexpressed
LGS and to the accumulation of glycogen. Similarly overexpressed MGS
was efficiently activated by the glucose-6-phosphate (Glc-6-P) produced
by either endogenous or overexpressed HK I and by overexpressed GK.
These results indicate the existence of at least two pools of Glc-6-P
in the cell, one of them is accessible to both isoforms of GS and is
replenished by the action of GK, whereas LGS is excluded from the
cellular compartment where the Glc-6-P produced by HK I is directed.
These findings are interpreted in terms of the metabolic role that the two pairs of enzymes, HK I-MGS in the muscle and GK-LGS in the hepatocyte, perform in their respective tissues.
Glycogen synthase (GS)1
catalyzes the incorporation of glucose residues to the non-reducing end
of a growing glycogen molecule via The two main isoforms of mammalian GS are designated as muscle and
liver. Most tissues express the former, whereas the latter appears to
be tissue-specific (2). Although the two forms have 70% identical
amino acid sequence, the N and C termini, which contain the
phosphorylation sites that regulate the activity of the enzyme, show a
lower degree of homology (3). Moreover the two isoforms have dissimilar
intracellular distribution both in the absence and in the presence of
glucose (4, 5), suggesting that there are significant differences in
the molecular mechanisms involved in the control of glycogen synthesis
in muscle and in liver.
Using adenovirus-mediated gene transfer in cultured hepatocytes, we
have previously shown that overexpression of glucokinase (GK,
hexokinase IV or hexokinase D), the isoenzyme of the mammalian hexokinase group characteristic of hepatocytes and pancreatic The FTO-2B rat hepatoma cell line has a highly differentiated hepatic
phenotype (10, 11) and expresses genes characteristic of the liver,
such as albumin and insulin-like growth factor II receptor.
However, FTO-2B cells do not express GK and instead express high levels
of HK I (12). In this hepatoma cell line, anaerobic glycolysis is
impaired, and there is no glycogen deposition even in the presence of
high concentrations of glucose (12). The retrovirus-mediated expression
of GK in FTO-2B cells stimulates glucose uptake and utilization and
restores the ability of these cells to synthesize glycogen. It
has been suggested that the increase in the levels of Glc-6-P in the
GK-expressing cells was responsible for the glycogen deposition
(12).
In this study, we have taken advantage of the characteristics of the
FTO-2B hepatoma cell line to show that LGS, but not the muscle isoform,
is dependent on GK as the source of Glc-6-P for activation. The latter
isoform, MGS, is activated and able to synthesize glycogen in response
to glucose independently of the type of hexokinase present. This
observation underlines the differences between the liver and muscle
glycogen metabolism.
Preparation of Recombinant Adenovirus--
AdCMV-GK (7),
AdCMV-HK I (7), and AdCMV-LGS (8) were described previously. AdCMV-GFP
and AdCMV-MGS were constructed following the procedure described by
Becker et al. (13) using the cDNA of the green
fluorescent protein (GFP) and that of the human muscle glycogen
synthase (14), respectively. In the metabolic impact studies, FTO-2B
cells infected with AdCMV-GFP were used as control cells.
FTO-2B Culture Conditions and Treatment with Recombinant
Adenovirus--
FTO-2B rat hepatoma cells were cultured in 60-mm
plates. Cells were kept in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 5 mM glucose and 5% fetal bovine serum.
Cells were treated for 2 h with adenovirus at a multiplicity of
infection of 5 except for AdCMV-HK I, which was used at a multiplicity
of infection of 10. Media were replaced with DMEM containing 10 mM glucose, and cells were incubated for 24 h at
37 °C. Media were then replaced by DMEM, and another incubation of
12-14 h was carried out. Cells were then incubated in DMEM at several
concentrations of glucose as detailed in the text and figure legends.
At the end of each manipulation, cell monolayers were washed in
phosphate-buffered saline and frozen in liquid N2 until analysis.
Metabolite Determinations--
For the measurement of glycogen
content, cell monolayers were scraped into 30% KOH, and the extract
was then boiled for 15 min and centrifuged at 5,000 × g for 15 min. Glycogen was measured in the cleared
supernatants as described previously (15). The intracellular
concentration of Glc-6-P was measured by a spectrophotometric assay
(16).
Enzyme Activity Assays--
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 plus
sonication caused cell bursting. Protein concentration was measured
following Bradford (17) using a Bio-Rad assay reagent. Glucose
phosphorylating activity was measured spectrophotometrically in the
supernatant fraction of FTO-2B cell extracts centrifuged at 10,000 × g for 15 min using 1 or 100 mM glucose
at 30 °C as described previously (18). GS activity was measured in
homogenates in the absence or presence of 6.6 mM Glc-6-P as
described previously (19). The activity measured in the absence of
Glc-6-P represents the active form of the enzyme (I or a
form), whereas that measured in the presence of 6.6 mM
Glc-6-P represents total GS activity.
Electrophoresis and Immunoblotting--
HK I, GK, LGS, and MGS
protein levels were measured by Western blot. Electrotransfer of
proteins from the gel to the nitrocellulose was performed at 200 V
(constant) at room temperature for 2 h using a Bio-Rad miniature
transfer apparatus as described previously (20). The nitrocellulose
blot was incubated overnight at 4 °C in blocking buffer (1% bovine
serum albumin, 0.05% Tween 20 in phosphate-buffered saline). The blot
was then incubated for 1 h at room temperature with a rabbit
antibody against rat LGS (8), human MGS (14), or rat GK (21) or a mouse
antibody against rat HK I (Chemicon), washed, and then incubated for
1 h with a secondary anti-rabbit (Amersham Biosciences) or
anti-mouse (Dako, Glostrup, Denmark) 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. Analysis of statistical differences was performed using the
unpaired two-tailed Student's t test, and statistical
significance was assumed at p < 0.05.
Characterization of FTO-2B Cells--
The FTO-2B cells show the
most consistent hepatocyte-like phenotype among the hepatoma cell lines
(10). However, these cells expressed undetectable levels of GK, both
measured as protein by Western blot (Fig.
1B, lane i) and by
enzymatic activity (Table I), and instead expressed high levels of HK I
(Fig. 1A, lane i, and Table I). These two
observations are in agreement with previous results obtained with this
cell line (11, 12). Unique among hepatic tumor cell lines, FTO-2B
expressed the liver isoform of GS (Fig. 1C, lane
i) even at higher levels than cultured hepatocytes (Table
I) and did not express detectable amounts
of MGS (Fig. 1D, lane i).
Adenovirus-mediated Overexpression of HK I, GK, LGS, or
MGS--
Protein overexpression in FTO-2B cells was achieved very
efficiently using recombinant adenoviruses. When AdCMV-GFP was used as
control at a multiplicity of infection of 5, over 95% of the adenovirus-treated cells expressed GFP as detected by fluorescence microscopy (not shown).
Infection of FTO-2B cells with AdCMV-HK I produced a 5-fold increase in
HK I activity and protein content (Fig. 1A, lanes i and ii, and Table I). When the AdCMV-GK was used, GK
reached levels similar to those found in cultured hepatocytes, measured both as protein by densitometric analysis (Fig. 1B,
lane ii) or activity (Table I). AdCMV-LGS-infected cells
showed a 3-fold increase in total GS activity (Table I). This increase
was consistent with protein content analyzed by Western blot (Fig.
1C, lanes i and ii) with an antibody
specific for rat LGS. Similarly MGS was overexpressed by infection of
FTO-2B cells with AdCMV-MGS and also resulted in a 3-fold increase in
total GS activity (Table I). The presence of the muscle isoform was
also detected by Western blot using antibodies specific for MGS (Fig.
1D, lane ii). The multiplicity of infection used
was 5 in all cases except with the AdCMV-HK I adenovirus, which was
used at a multiplicity of infection of 10 to obtain intracellular
levels of Glc-6-P similar to those obtained with the AdCMV-GK infection
(see below).
In addition, FTO-2B cells were simultaneously co-infected with
AdCMV-LGS plus AdCMV-HK I or AdCMV-GK and with AdCMV-MGS plus AdCMV-HK
I or AdCMV-GK. In the double infection experiments, HK I, GK, and total
GS activities were comparable to those obtained when the viruses were
used alone (Table I). Likewise the levels of protein measured by
Western blot were also similar to those attained in the single
infections (Fig. 1, lanes iii and iv).
Metabolic Impact of the Overexpression of HK I or GK--
Despite
containing 3-fold more total LGS than cultured hepatocytes
(Table I), FTO-2B cells synthesized negligible amounts of glycogen in
response to increasing doses of glucose (Fig.
2A). Intracellular Glc-6-P
levels increased sharply when the cells were incubated with 5 mM glucose but remained constant at ~2 nmol/mg of protein
when higher concentrations of the sugar were used (Fig. 3A).
Nevertheless this accumulation of Glc-6-P induced neither the
activation of LGS (Fig. 4) nor the deposition of glycogen.
Adenovirus-mediated overexpression of HK I in FTO-2B cells led to a
marked increase in Glc-6-P levels, which at 25 mM glucose were about ~3-fold higher than in control cells (Fig.
3A). Nevertheless HK I
overexpression did not produce a significant increase in glycogen
deposition (Fig. 2A), and the amount of active LGS was not
significantly different from that found in control cells infected with
the AdCMV-GFP adenovirus (Fig. 4A). On the contrary, GK
overexpression had a dramatic effect on the capacity of FTO-2B cells to
synthesize glycogen. At high glucose concentrations, AdCMV-GK-infected
cells produced over 2-fold more glycogen than control cells or cells overexpressing HK I (Fig. 2A). In contrast to control cells,
in GK-expressing cells, endogenous GS was activated by Glc-6-P in a
concentration-dependent manner (Fig.
4AA). Interestingly the large
increase in the amount of active LGS in the AdCMV-GK-infected cells was
attained at similar or even lower levels of Glc-6-P than those found in
HK I-overexpressing cells (Fig. 4A). However, as indicated
above, no GS activation was observed in HK I-overexpressing cells.
Metabolic Impact of the Overexpression of Liver and Muscle Glycogen
Synthase--
Overexpression of LGS up to 3-fold did not increase
glycogen deposition even when infected FTO-2B cells were incubated with high doses of glucose (Fig. 2B). Overexpressed LGS behaved
like the endogenous enzyme in the sense that it was not activated in response to an increase in the intracellular concentration of Glc-6-P
(Fig. 4B). In contrast, when MGS was expressed to attain the
same total GS activity as with LGS, there was a marked increase in
glycogen deposition (Fig. 2C) in response to increasing
doses of glucose. Although LGS or MGS overexpression did not alter
Glc-6-P concentrations (Fig. 3, B and C), the
amount of active GS was much larger in AdCMV-MGS-infected cells and
increased linearly with Glc-6-P concentration (Fig.
4B).
Differential Metabolic Impact of Overexpression of LGS or MGS
Concomitantly with HK I or GK--
To compare the effect on glycogen
synthesis of the overexpression of LGS or MGS concomitantly with each
of the two hexokinases, we overexpressed LGS plus HK I or GK, and MGS
plus HK I or GK.
In FTO-2B cells overexpressing LGS plus GK, glycogen synthesis was
strongly enhanced (Fig. 2B) compared with control cells or
cells overexpressing LGS alone. In contrast, when LGS was overexpressed concomitantly with HK I glycogen deposition in response to increasing doses of glucose remained negligible (Fig. 2B). Furthermore
LGS was very efficiently activated by Glc-6-P in a
dose-dependent manner when GK was present but only
experienced a minor activation when the source of Glc-6-P was
overexpressed HK I (Fig. 4C).
When MGS was co-expressed with GK or HK I, glycogen synthesis was
strongly enhanced compared with the cells overexpressing MGS alone. In
both cases, glycogen accumulation increased in response to glucose
(Fig. 2C), and the levels of active GS (Fig. 4D)
increased with Glc-6-P in a dose-dependent manner
independently of the hexokinase activity present in the cells. In
contrast to what occurs with LGS, both hexokinases, HK I and GK,
appeared to be equally effective in mediating the activation of MGS in
response to glucose. The differences observed in the amount of glycogen
and in the levels of active GS between the cells overexpressing MGS and
HK I and those expressing MGS and GK can be attributed to the
simultaneous activation of the endogenous LGS in the latter case.
In liver and muscle, GS activation occurs through the
dephosphorylation of the enzyme, which is probably produced by protein phosphatases of type I (2, 23) and is enhanced by Glc-6-P. It is
generally accepted that this metabolite binds to GS and triggers a
conformational change that renders the enzyme a better substrate for
protein phosphatases (1). Previous studies by our group, using cultured
hepatocytes, have shown that Glc-6-P arising from the catalytic action
of GK is much more effective in mediating the activation of LGS than
the same metabolite produced by HK I (6, 7). In the present work we
have taken advantage of the biochemical features of the FTO-2B cells to
show that LGS but not MGS differentiates between Glc-6-P produced by GK
or by HK I.
Using specific antibodies, here we show that FTO-2B cells express high
levels of LGS and do not express measurable levels of MGS. However,
these cells do not express GK, the main glucose-phosphorylating enzyme
in hepatocytes, and instead express HK I, a ubiquitous hexokinase.
Because of these characteristics, this system is ideally suited to
study the dependence of GS activation on the source of Glc-6-P.
It has already been reported that FTO-2B cells do not accumulate
glycogen when incubated with glucose but that the overexpression of GK
restores this ability (12). It was hypothesized that the large
increment in the levels of Glc-6-P derived from the expression of GK
was solely responsible for this. However, here we show that the source
of this metabolite is also crucial. In the first set of experiments,
the adenovirus-mediated overexpression of GK led to the activation of
the endogenous LGS present in FTO-2B cells and to the consequent
accumulation of glycogen. In contrast, the overexpression of HK I, such
that similar levels of Glc-6-P were attained, had no effect on the
amount of active GS and glycogen content.
The observation that endogenous LGS was activated in response to
increasing concentrations of glucose when GK was present but not when
the source of Glc-6-P was HK I indicates the existence of at least two
pools of Glc-6-P inside the cell. One of these pools is
replenished by the action of GK and is accessible to LGS, while
the pool of Glc-6-P produced by HK I is localized to a cellular
compartment from which LGS is excluded.
The second set of experiments, in which we overexpressed LGS and MGS,
corroborated this conclusion. The overexpression of LGS did not have
any effect on the levels of glycogen. Glc-6-P produced by the
endogenous HK I of the FTO-2B cells did not facilitate the
dephosphorylation and activation of overexpressed LGS. However, exogenous MGS was effectively activated in response to glucose, and
MGS-overexpressing cells synthesized a large amount of glycogen. This
finding indicates that, in contrast to LGS, MGS has access to the
compartment where the Glc-6-P produced by HK I is directed.
The third set of experiments, consisting of double infections with each
of the two GS isoforms combined with each one of the two
glucose-phosphorylating enzymes, further confirms the previous conclusions and allows us to make one additional conclusion. LGS was only activated by glucose when GK was present, but Glc-6-P produced
by GK or HK I was equally effective in inducing the activation of MGS.
Simultaneous overexpression of LGS and HK I had no net effect on
glycogen deposition, while the co-expression of LGS and GK led to a
much larger accumulation of glycogen than in FTO-2B cells singly
infected with the AdCMV-GS or AdCMV-GK viruses. In contrast, glycogen
accumulation was enhanced in FTO-2B cells overexpressing MGS when the
glucose phosphorylating capacity of the cells was also increased
regardless of whether the source of Glc-6-P was HK I or GK.
On the basis of the different effectiveness in inducing the activation
of LGS by the Glc-6-P produced by GK or by HK I, the channeling of this
sugar phosphate from GK to GS was hypothesized. However, in the light
of the new data presented here, an alternative hypothesis emerges:
compartmentation of Glc-6-P. GK delivers its product into a cellular
compartment, which is accessible by several enzymes that use Glc-6-P,
while the compartment where the Glc-6-P produced by HK I is directed
has a more restricted access. In this study we show that overexpression
of GK in FTO-2B cells enhances glycogen deposition irrespectively of
the GS isoform present. Glycolysis, which is an alternative metabolic
fate of Glc-6-P, is also greatly stimulated in GK-overexpressing FTO-2B
cells (12). In cultured hepatocytes (6, 7, 24) and in cultured human muscle cells (25), GK overexpression also stimulates both processes, glycogen deposition and glycolysis. On the contrary, Glc-6-P produced by HK I is not sensed by LGS and therefore is not used for the synthesis of glycogen even when the intracellular levels of this metabolite are substantially increased.
The four enzymes overexpressed in this study have been shown to change
their intracellular distribution in response to glucose. In the absence
of the sugar, GK is localized to the nucleus of the hepatocyte but
moves into the cytosol when the levels of glucose increase (21, 26).
MGS is also concentrated in the nucleus at low glucose and translocates
to the cytosol, where it adopts a particulate pattern, at high glucose
concentrations (5, 14). In contrast, LGS presents a cytosolic
distribution in the absence of glucose and concentrates at the
periphery of the hepatocyte when the concentration of the hexose
increases (27). Finally HK I has been shown to reversibly bind to the
outer mitochondrial membrane through a hydrophobic N-terminal sequence
(28). This association, which is at least in part controlled by the
intracellular levels of Glc-6-P, plays a role in the regulation of HK I
activity in vivo. Low levels of Glc-6-P favor the
association with mitochondria and stimulate HK I activity, while high
levels of this metabolite have the opposite effect (29).
HK I, which accounts for more than 75% of total hexokinase activity
present in skeletal muscle (30), and MGS are, respectively, at the
beginning and at the end of the glycogen synthetic pathway in muscle.
Therefore, the ability of HK I to mediate the glucose-induced activation of MGS has a clear physiological meaning. The high affinity of HK I for glucose implies that this sugar is readily converted into Glc-6-P upon entering the cell, and thus the pair HK
I-MGS is sensitive to the low concentrations of glucose present in the
muscle cell. This explains why the control of glycogen deposition in
muscle does not reside in the glucose phosphorylating capacity of the
cell but rather in the insulin-stimulated import of the sugar by the
GLUT-4 transporter and in the MGS (9).
However, the situation in the liver is rather different. In the
hepatocyte, extracellular glucose and intracellular glucose are in
equilibrium partly due to the high capacity of the hepatic glucose
transporter GLUT-2 (31). Although GK represents the main glucose
phosphorylating activity of the hepatocyte, HK I is also present at
considerable levels (32). When blood glucose levels are low (below ~5
mM), there is no significant flux through GK because of its
high Km for glucose (32) and to the action of its
regulatory protein, which further decreases the apparent affinity of GK
for glucose in the hepatocyte (22). Therefore, under these conditions
only HK I can phosphorylate glucose, but as shown in this study, the
Glc-6-P thus produced cannot be diverted toward the synthesis of
glycogen since this metabolite does not activate LGS. Only when blood
sugar concentration increases above a threshold level does GK
translocate to the cytosol and start to produce Glc-6-P, thus giving
the signal that triggers the synthesis of hepatic glycogen. In this
case, the control of glycogen synthesis is not exerted by glucose
transport but rather by GK and GS (8). It appears that the inability of
the Glc-6-P produced by HK I to stimulate the activation of LGS is one
way for the hepatocyte to ensure that hepatic glycogen synthesis is only engaged when needed, that is when blood glucose levels are high.
We conclude that the characteristics of the two pairs of isoenzymes
LGS/GK and MGS/HK I and the relationships that they establish are
tailored to suit specific metabolic roles of the tissues in which they
are expressed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-1,4-glycosidic bonds using
UDP-glucose as substrate. GS can be inactivated by phosphorylation at
multiple sites, but even highly phosphorylated forms of the enzyme
become active "in vitro " in the presence of high
concentrations of glucose-6-phosphate (Glc-6-P), which acts as an
allosteric activator (1). "In vivo," Glc-6-P binding to
GS also converts the enzyme into a better substrate for phosphatases
and induces its dephosphorylation and activation (1).
cells, enhances glycogen synthesis, whereas overexpression of hexokinase I (HK I) has no effect (6). This is attributed to a
different capacity of Glc-6-P produced by GK or HK I to induce the
activation of liver glycogen synthase (LGS) (7). Further studies in
cultured hepatocytes have shown that LGS shares the control of glycogen
synthesis with GK (8). In contrast, the control of glycogen deposition
from glucose in muscle is shared between glucose transport and muscle
glycogen synthase (MGS) (9).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (49K):
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Fig. 1.
HK I, GK, LGS, and MGS expression in
adenovirus-treated FTO-2B cells. A, cells were treated
with AdCMV-GFP (i), AdCMV-HK I (ii), AdCMV-LGS/HK
I (iii), or AdCMV-MGS/HK I (iv). B,
cells were treated with AdCMV-GFP (i), AdCMV-GK
(ii), AdCMV-LGS/GK (iii), or AdCMV-MGS/GK I
(iv). C, cells were treated with AdCMV-GFP
(i), AdCMV-LGS (ii), AdCMV-LGS/GK
(iii), or AdCMV-LGS/HK I (iv). D,
cells were treated with AdCMV-GFP (i), AdCMV-MGS
(ii), AdCMV-MGS/GK (iii), or AdCMV-MGS/HK I
(iv) as described under "Materials and Methods." The
multiplicity of infection was 5 for all the viruses except for AdCMV-HK
I, which was used at a multiplicity of infection of 10. After
infection, cells were incubated for 24 h with DMEM containing 10 mM glucose, the medium was then replaced by DMEM without
glucose, and incubation was continued for 12-14 h. Finally cells were
incubated for 2 h in DMEM with 25 mM glucose and
collected, and homogenates were analyzed by Western blotting using
antibodies against HK I (A), GK (B), LGS
(C), or MGS (D). The bands corresponding to each
one of the proteins analyzed ran at their appropriate respective
molecular weight.
HK I, GK, and total GS activity in FTO-2B cells overexpressing HK
I, GK, LGS, or MGS
View larger version (25K):
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Fig. 2.
Effects of HK I, GK, LGS, and MGS
overexpression on glycogen deposition in FTO-2B cells.
A, cells were treated with AdCMV-GFP (open bars),
AdCMV-HK I (hatched bars), or AdCMV-GK (filled
bars). B, cells were treated with AdCMV-GFP (open
bars), AdCMV-LGS (widely spaced hatched bars),
AdCMV-LGS plus AdCMV-HK I (closely spaced hatched bars), or
AdCMV-LGS plus AdCMV-GK (filled bars). C, cells
were treated with AdCMV-GFP (open bars), AdCMV-MGS
(widely spaced hatched bars), AdCMV-MGS plus AdCMV-HK I
(closely spaced hatched bars), or AdCMV-MGS plus AdCMV-GK
(filled bars) as described under "Materials and
Methods." After infection, cells were incubated for 24 h in DMEM
containing 10 mM glucose. The medium was replaced by DMEM
without glucose, and incubation was continued for 12-14 h. Cells were
finally incubated for 2 h at a range of glucose concentrations.
Cells were then flash frozen and assayed for glycogen content. Data
represent the mean ± S.E. for six to eight independent
experiments. Letters indicate statistical significance
(p < 0.05) for comparisons with the cells treated with
AdCMV-GFP (a), AdCMV-HK I (b), AdCMV-LGS
(c), AdCMV-LGS/HK I (d), or AdCMV-MGS
(e). Prot, protein.
View larger version (16K):
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Fig. 3.
Effects of HK I, GK, LGS, and MGS
overexpression on Glc-6-P levels in FTO-2B cells. A,
cells were treated with AdCMV-GFP (open squares), AdCMV-HK I
(open triangles), or AdCMV-GK (filled triangles).
B, cells were treated with AdCMV-GFP (open
squares), AdCMV-LGS (filled squares), AdCMV-LGS plus
AdCMV-HK I (filled triangles), or AdCMV-LGS plus AdCMV-GK
(filled circles). C, cells were treated with
AdCMV-GFP (open squares), AdCMV-MGS (filled
triangles), AdCMV-MGS plus AdCMV-HK I (filled circles),
or AdCMV-MGS plus AdCMV-GK (filled squares) and incubated as
described in the legend of Fig. 2. Cells were then collected, and
intracellular Glc-6-P concentrations were measured. Data represent the
mean ± S.E. for six to eight independent experiments.
Prot, protein.
View larger version (17K):
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Fig. 4.
Correlation of GS activation and
intracellular Glc-6-P concentration in FTO-2B cells. A,
cells were treated with AdCMV-GFP (open squares), AdCMV-HK I
(open triangles), or AdCMV-GK (filled triangles).
B, Cells were treated with AdCMV-GFP (open
squares), AdCMV-LGS (filled squares), or AdCMV-MGS
(filled circles). C, cells were treated with
AdCMV-GFP (open squares), AdCMV-LGS plus AdCMV-HK I
(filled triangles), or AdCMV-LGS plus AdCMV-GK (filled
circles). D, cells were treated with AdCMV-GFP
(open squares), AdCMV-MGS plus AdCMV-HK I (filled
circles), or AdCMV-MGS plus AdCMV-GK (filled squares)
and incubated as described in the legend of Fig. 2. Cells were then
collected, and GS activity was measured in the homogenates. Active GS
was measured in the absence of Glc-6-P as described under "Materials
and Methods." Data represent the mean ± S.E. for six to
eight independent experiments. mU, milliunits;
Prot, protein.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Susanna Baqué for valuable help in the preparation of the AdCMV-LGS, AdCMV-MGS, and AdCMV-GFP adenoviruses and Dr. C. B. Newgard for the generous gift of the AdCMV-GK and AdCMV-HK I adenoviruses. We also thank Anna Adrover for skillful technical assistance and Tanya Yates for assistance in preparing the English manuscript.
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FOOTNOTES |
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* This work was supported by Grant PB98-0992 from the Dirección General de Enseñanza Superior (Ministerio de Educación y Cultura, Spain) and by Grant 992310 from the Fundació La Marató de TV3.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).
§ Recipient of a doctoral fellowship from the Generalitat de Catalunya (Comissió Interdepartamental de Recerca i Innovació Tecnològica).
¶ To whom correspondence should be addressed: Dept. de Bioquímica i Biologia Molecular, Universitat de Barcelona, Martí i Franquès, 1, E-08028 Barcelona, Spain. Tel.: 34-93-4021206; Fax: 34-93-4021219; E-mail: guinovart@pcb.ub.es.
Published, JBC Papers in Press, March 6, 2002, DOI 10.1074/jbc.M111208200
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ABBREVIATIONS |
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The abbreviations used are: GS, glycogen synthase; LGS, liver glycogen synthase; MGS, muscle glycogen synthase; Glc-6-P, glucose 6-phosphate; GK, glucokinase; HK I, hexokinase I; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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