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Endocrinology 146(1):494 –502
Copyright © 2005 by The Endocrine Society
doi: 10.1210/en.2004-1022
Spatial Reorganization of Glycogen Synthase upon
Activation in 3T3-L1 Adipocytes
Hesheng Ou, Limei Yan, Senad Osmanovic, Cynthia C. Greenberg, and Matthew J. Brady
Department of Medicine, Committee on Molecular Metabolism and Nutrition, The University of Chicago, Chicago,
Illinois 60637
The dephosphorylation of glycogen synthase is a key step in
the stimulation of glycogen synthesis by insulin. To further
investigate the hormonal regulation of glycogen synthase activity, enzymatic localization in 3T3-L1 adipocytes was determined by immunocytochemistry and confocal microscopy. In
basal cells, glycogen synthase and the protein phosphatase1-glycogen-targeting subunit, protein targeting to glycogen
(PTG), were diffusely distributed throughout the cell. Insulin
treatment had no effect on PTG distribution but resulted in a
reorganization of glycogen synthase into punctate clusters.
Glycogen synthase aggregation was restricted to discrete cellular sites, presumably where glycogen synthesis occurred.
Omission of extracellular glucose or substitution with 2-deoxy-glucose blocked the insulin-induced redistribution of glycogen synthase. Addition of the glycogenolytic agent forskolin
after insulin stimulation disrupted the clusters of glycogen
synthase protein, restoring the immunostaining pattern to
the basal state. Conversely, adenoviral-mediated overexpression of PTG resulted in the insulin-independent dephosphorylation of glycogen synthase and a redistribution of the enzyme from the cytosolic- to glycogen-containing fractions. The
effects of PTG on glycogen synthase activity were mediated by
multisite dephosphorylation, which was enhanced by insulin
and 2-deoxy-glucose, and required a functional glycogen synthase-binding domain on PTG. However, PTG overexpression
did not induce distinct glycogen synthase clustering in fixed
cells, presumably because cellular glycogen levels were increased more than 7-fold under these conditions, resulting in
a diffusion of sites where glycogen elongation occurred. Cumulatively, these data indicate that the hormonal regulation
of glycogen synthesis rates in 3T3-L1 adipocytes is mediated
in part through changes in the subcellular localization of glycogen synthase. (Endocrinology 146: 494 –502, 2005)
G
LUCOSE IS PRIMARILY stored as long, branching
polymers called glycogen. Glycogen synthesis is regulated by a wide variety of factors including hormones, innervation, activity state, and intracellular metabolites (1).
When blood glucose is elevated (i.e. after ingestion of a meal),
insulin stimulates glucose storage as glycogen primarily in
skeletal muscle but also in liver and adipose tissue. Conversely, during hypoglycemic episodes (i.e. during fasting),
liver glycogen stores are mobilized in concert with increased
gluconeogenesis to raise hepatic glucose output. Thus, the
hormonal modulation of glycogen synthesis and degradation helps maintain plasma glucose concentrations in a narrow, physiological range. However, the molecular mechanisms by which the enzymatic effectors involved in glycogen
metabolism are regulated remain poorly understood.
Insulin stimulates the storage of glucose as glycogen in
muscle and adipose tissue through the coordinate increase in
glucose uptake and modulation of glycogen-metabolizing
enzymes (1). Insulin binds to its receptor in peripheral tissues
and initiates a variety of signaling cascades to increase glucose uptake via translocation of glucose transporter 4
(GLUT4)-containing vesicles to the plasma membrane (2).
Glucose enters the cells and is phosphorylated by hexokinases to form glucose-6-phosphate (G6P). Depending on the
energy requirements of the cell, G6P can enter glycolysis to
generate ATP or be metabolized to uridine diphospho
(UDP)-glucose and stored as glycogen.
Glycogen synthase, the rate-limiting enzyme for glycogen
synthesis, catalyzes the incorporation of UDP-glucose into
glycogen chains. Glycogen synthase activity is stimulated by
insulin in liver, muscle, and adipose tissue via protein dephosphorylation, allosteric activation, and enzymatic translocation (1). Glycogen synthase is phosphorylated on up to
nine residues by a variety of kinases, resulting in its progressive inactivation (3). Insulin increases glycogen synthase
activity primarily by stimulating the dephosphorylation of
four key residues. Both activation of protein phosphatase-1
(PP1) and inactivation of glycogen synthase kinase-3 (GSK-3)
have been proposed to mediate this metabolic effect of insulin (4), although the relative contribution of each enzyme
remains unclear. Furthermore, maximal stimulation of glycogen synthase activity by insulin in adipocytes requires
increased glucose uptake and metabolism by target cells
(5–7). G6P allosterically activates glycogen synthase, overriding phosphorylation-dependent inhibition (6, 8, 9),
whereas glycogen content mediates the redistribution of glycogen synthase in hepatocytes, adipocytes, and muscle cells
(7, 10, 11). Thus, insulin potently activates glycogen synthase
through the coordinate stimulation of glucose uptake and
protein dephosphorylation.
3T3-L1 adipocytes are a widely used cellular model for the
study of hormonal regulation of glucose metabolism. Ter-
First Published Online October 14, 2004
Abbreviations: 2-DG, 2-Deoxy-glucose; FBS, fetal bovine serum; G6P,
glucose-6-phosphate; GLUT4, glucose transporter 4; GSK-3, glycogen
synthase kinase-3; PP1, type 1 protein phosphatase; PTG, protein targeting to glycogen; PTG-DE, glycogen synthase-binding mutant; UDP,
uridine diphospho.
Endocrinology is published monthly by The Endocrine Society (http://
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endocrine community.
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Ou et al. • Regulation of Glycogen Synthase Activity
minal differentiation of 3T3-L1 cells into lipid-containing
adipocytes results in a dramatic increase in insulin-stimulated glucose uptake and storage as glycogen due to a potent
increase in GLUT4 translocation and glycogen synthase activation (12, 13). Previously, we reported that insulin and the
glycogenolytic agent isoproterenol exerted opposing effects
on glycogen synthase localization in subcellular fractions
from 3T3-L1 adipocytes (7, 14), which suggests a potential
connection between glycogen synthase activation and intracellular localization in the regulation of glycogen synthesis.
In the present study, we have further characterized the regulation of glycogen synthase localization and activation state
by insulin, glucose metabolites, and the PP1-glycogentargeting subunit, protein targeting to glycogen (PTG). The
results suggest that spatial reorganization of glycogen synthase is involved in the hormonal regulation of glycogen
metabolism.
Materials and Methods
Materials
Cell culture reagents and calf serum were supplied by Mediatech, Inc.
(Herndon, VA), and fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT.) All other chemicals were from Sigma (St. Louis, MO).
UDP-[U-3H]glucose (60 Ci/mmol) was supplied by American Radiolabeled Chemicals (St. Louis, MO). ECL reagent was purchased from
Amersham Pharmacia Biotech (Uppsala, Sweden), and GF/A filters
were supplied by Whatman (Maidstone, UK). Anti-PTG antibody was
generated and affinity purified as described (15). Commercial sources of
antibodies were as follows: antiglycogen synthase, Chemicon (Temecula, CA); anti-PP1, Santa Cruz Biotechnology (Santa Cruz, CA);
anti-phospho-glycogen synthase (Ser 640) and anti-phospho-GSK-3␤
(Ser 9), Cell Signaling Technology (Beverly, MA); anti-phospho-acetylconenzyme A carboxylase (Ser 79), Upstate Biotechnology (Charlottesville, VA); horseradish peroxidase-conjugated goat antirabbit and goat
antimouse IgG, Bio-Rad (Hercules, CA); and Alexa Fluor 488- and Alexa
Fluor 633-labeled secondary antibodies (goat antirabbit and goat antimouse, respectively), Molecular Probes (Eugene, OR).
Cell culture
3T3-L1 cells were cultured and differentiated as described (7) and
infected within 7 d after completion of the differentiation protocol (15).
For immunofluorescence experiments, 3T3-L1 adipocytes were detached by the addition of 0.25% trypsin and replated at an approximate
density of 50% onto 15 ⫻ 15-mm no.1 glass coverslips (Carolina Biological Supply, Burlington, NC) in 12-well dishes. Cells were incubated
for 24 h to allow cell attachment.
Preparation of cellular lysates
Cells were washed twice with DMEM supplemented with 25 mm
HEPES (pH 7.4), 0.5% FBS, and 5 mm glucose and incubated for 2.5 h
in the same medium. The cells were then treated as indicated in the
figure legends and washed three times with PBS on ice. For glycogen
synthase activity assays and immunoblotting experiments, cells were
scraped into homogenization buffer (50 mm HEPES, pH 7.4; 150 mm
NaCl; 10 mm NaF; 10 mm EDTA; 10% glycerol; 0.5% Triton X-100; and
protease inhibitors added just before use). Lysates were centrifuged for
10 min at 10,000 ⫻ g at 4 C, and supernatants were transferred to new
tubes.
Cellular fractionation
After washing three times with ice-cold PBS, 3T3-L1 adipocytes in
six-well plates were scraped into homogenization buffer lacking detergent and lysed by sonication (20% output, 10 sec). All subsequent steps
were performed at 4 C. The samples were centrifuged at 1000 ⫻ g for
5 min to pellet nuclei. The supernatants were centrifuged at 10,000 ⫻ g
Endocrinology, January 2005, 146(1):494 –502
495
for 15 min. The pellet was saved, and supernatants were then centrifuged at 100,000 ⫻ g for 30 min. The high-speed pellet fraction was
termed glycogen-enriched pellet, whereas the final supernatants were
saved as the cytosolic fraction. Both pellet fractions were resuspended
in detergent-free homogenization buffer using a 23-gauge needle.
Immunofluorescence confocal microscopy
3T3-L1 adipocytes plated on glass coverslips were washed two times
with ice-cold PBS and then fixed and permeabilized by the addition of
ice-cold methanol and acetone (1:1 ratio, vol/vol) for 10 min at ⫺20 C.
After fixation, the cells were washed three times with PBS containing
0.5% Tween 20 and incubated for 30 min at room temperature in a
blocking solution (5% nonfat dry milk in PBS). Cells were treated by the
addition of primary antibodies in a 1:50 dilution for 1 h at room temperature, followed by incubation in labeled secondary antibodies under
the same conditions. After washing cells three times with ice-cold PBS,
the coverslips were mounted onto labeled slides using nail polish.
Confocal microscopy was performed with an Olympus Fluoview 200
Laser Scanning system equipped with a HeNe/Argon laser (Olympus,
Tokyo, Japan). Excitation of double-label Alexa 488/Alexa 633 samples
was performed with an Argon laser (488 nm) using a 500-nm short pass,
dichroic mirror and a HeNe laser (633 nm) using a double dichroic
488/633-nm mirror, respectively. The band pass for Alexa 488 and Alexa
633 emission spectra was set at 500 –575 nm and 600 –700 nm, respectively, with a prism spectrophotometer. The laser rheostat and laser
power were both set at 20% for imaging Alexa 488, and both parameters
were set at 30% to image Alexa 633. Photomultiplier gain adjustments
were set to 50% of maximum level. Optical sections through the z-axis
(0.6-␮m thick; step size, 1.0 ␮m) were collected using a stage galvanometer, and flattened maximum projections of six-section image stacks
were generated.
Cellular ATP determination
3T3-L1 adipocytes were serum starved for 2.5 h and then washed two
times with glucose-free DMEM. Cells were then incubated for 15 min,
with or without 100 nm insulin, in DMEM plus 0.5% FBS containing 5
mm of either glucose or 2-deoxy-glucose (2-DG). Cells were washed
three times with ice-cold PBS, collected in ATP assay buffer (100 mm Tris,
pH 7.75; and 4 mm EDTA), and snap-frozen in a dry ice/ethanol bath.
ATP levels were then measured using an ATP Bioluminescence Assay
Kit CLS II (Roche Diagnostics, Basel, Switzerland) according to the
manufacturer’s instructions.
Other methods
Glycogen synthase activity assays and immunoblotting were performed as previously reported (16). Statistical analysis was performed
using the Student’s t test.
Results
Hormonal regulation of glycogen synthase localization in
3T3-L1 adipocytes
We previously reported that insulin treatment resulted in
the redistribution of glycogen synthase in cellular fractions
prepared from 3T3-L1 adipocytes (7, 14). To further investigate the spatial organization of glycogen metabolism in
these cells, glycogen synthase localization was determined
by immunocytochemistry and confocal microscopy. Fully
differentiated 3T3-L1 adipocytes were replated onto cover
slips and allowed to recover for 24 h. After a 2.5-h serum
starvation, cells were treated for 10 min in the absence or
presence of 100 nm insulin. Cells were then fixed, and the
coverslips were incubated with the indicated antibodies, followed by fluorescently labeled secondary antibodies. As expected, GLUT4 was primarily located in the perinuclear region in basal cells and translocated to the plasma membrane
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Endocrinology, January 2005, 146(1):494 –502
after insulin treatment (data not shown). In control cells, both
glycogen synthase and the PP1-glycogen targeting subunit
PTG were diffusely distributed within cytoplasm and absent
from the nucleus (lipid droplets and nuclei appear as dark
areas). Insulin had no effect on PTG distribution (Fig. 1A) or
PP1 localization (data not shown). In contrast, insulin stimulation resulted in a reorganization of glycogen synthase into
bright, punctate clusters (Fig. 1), confirming that the hormone induces enzymatic redistribution in 3T3-L1 adipocytes. Because glycogen synthase translocation in these cells
was previously linked to enzymatic activation (14), we presume that these glycogen synthase aggregates formed where
de novo glycogen synthesis was occurring. Exposure of basal
cells to the glycogenolytic agent forskolin had no effect on
glycogen synthase distribution (data not shown). However,
forskolin addition to insulin-pretreated cells reversed glycogen synthase clustering (Fig. 1B), further linking the discrete regulation of glycogen synthesis and degradation and
the intracellular localization of glycogen synthase.
FIG. 1. Hormonal regulation of glycogen synthase (GS) distribution
in 3T3-L1 adipocytes. A, Fully differentiated 3T3-L1 adipocytes were
trypsinized and replated on glass coverslips. The next day, cells were
serum starved for 2.5 h in DMEM containing 5 mM glucose and 0.5%
FBS. Cells were then treated without (basal) or with 100 nM insulin
for 10 min. After fixation, coverslips were incubated simultaneously
with monoclonal anti-GS and polyclonal anti-PTG antibodies and
then with secondary antibodies coupled to Alexa Fluor 633 (antimouse; red) or 488 (antirabbit; green). Cells were then analyzed by
confocal microscopy. B, 3T3-L1 adipocytes plated on glass coverslips
were preincubated for 2.5 h in DMEM containing 5 mM glucose and
0.5% FBS. Replicate wells were treated without (basal) or with 100
nM insulin for 10 min. Half of the insulin-treated cells were washed
two times with PBS (37 C) and then stimulated for 30 min with 10
␮g/ml of forskolin. After fixation, GS localization was analyzed by
confocal microscopy.
Ou et al. • Regulation of Glycogen Synthase Activity
Requirement for extracellular glucose in the insulin-induced
redistribution of glycogen synthase
Insulin-stimulated glucose uptake plays a critical role in
the activation of glycogen synthase in a variety of cell types
(reviewed in Ref. 1). To investigate whether the insulininduced glycogen synthase redistribution in 3T3-L1 adipocytes required increased glucose utilization, extracellular
glucose conditions were varied. 3T3-L1 adipocytes were
seeded on coverslips and serum starved for 2.5 h the next
day. Cells were preincubated in medium containing 0 or 5
mm glucose for 15 min before the insulin stimulation. Cells
were then fixed and analyzed by confocal microscopy. In the
presence of 5 mm glucose, insulin treatment resulted in the
formation of glycogen synthase clusters (Fig. 2). However,
this insulin effect was completely abolished upon withdrawal of extracellular glucose, suggesting a requirement for
a glucose metabolite. Next, cells were preincubated for 15
min in media containing 5 mm 2-DG and then treated with
100 nm insulin. 2-DG is not metabolized after hexokinasemediated phosphorylation, resulting in intracellular accumulation of 2-DG-6-phosphate. Consequently, the substitution of extracellular glucose with 2-DG potentiated glycogen
synthase activation by insulin in 3T3-L1 adipocytes (7). Interestingly, insulin did not induce a reorganization of glycogen synthase in cells incubated in 2-DG, indicating that
enzymatic activation was not in itself sufficient to promote
glycogen synthase translocation. However, acute insulin
treatment consistently increased glycogen synthase immunostaining in 3T3-L1 adipocytes incubated in 2-DG-containing media (Fig. 2). The reason for this result is not clear, but
it may reflect enhanced recognition of dephosphorylated
glycogen synthase by the antibody because total enzyme
levels were unchanged in activity assays.
Next, the levels of extracellular glucose were varied, and
glycogen synthase translocation from the cytosolic fraction
was measured by in vitro enzymatic assay and immunoblotting (7). Replicate 12-well plates of 3T3-L1 adipocytes were
serum starved for 2.5 h, washed twice in glucose-free DMEM,
and then incubated in media containing 0 –5 mm glucose in
FIG. 2. Insulin-induced glycogen synthase (GS) redistribution requires increased glucose uptake and metabolism. 3T3-L1 adipocytes
were seeded onto coverslips, serum starved for 2.5 h, and then washed
two times with PBS (37 C). Cells were then preincubated for 15 min
in DMEM plus 0.5% FBS with the indicated glucose addition. After
a 10-min treatment in the absence or presence of 100 nM insulin, cells
were fixed and GS immunostaining was analyzed using confocal microscopy.
Ou et al. • Regulation of Glycogen Synthase Activity
the presence of 100 nm insulin. Additionally, some wells
were incubated in media containing 5 mm pyruvate in place
of glucose. After 15 min, lysates from triplicate wells were
pooled and subjected to differential centrifugation to obtain
the cytosolic fraction. Glycogen synthase activity was measured in the presence of 10 mm G6P to determine total enzymatic levels. Additionally, lysates were analyzed in parallel by antiglycogen synthase immunoblotting. As shown in
Fig. 3, the insulin induced redistribution of glycogen synthase from the cytosolic fraction in the presence of glucose,
with a half-maximal effect occurring between 1–2 mm
glucose. However, removal of extracellular glucose or substitution with pyruvate completely blocked this effect of
insulin. These results confirm a requirement for glucose
uptake in the translocation of glycogen synthase in 3T3-L1
adipocytes.
2-DG potentiates the activation of glycogen synthase by PTG
Previous studies have shown that the PP1-glycogen-targeting subunit PTG plays an important role in regulation of
glucose metabolism by virtue of its ability to bind to glycogen, PP1, and glycogen synthase (17, 18). Adenoviral-mediated overexpression of PTG in 3T3-L1 adipocytes resulted in
a marked increase in glycogen synthesis rates and glycogen
levels (15). To examine the effects of PTG overexpression on
glycogen synthase distribution, 3T3-L1 adipocytes were infected with a recombinant adenovirus encoding PTG. After
a 48-h recovery, glycogen synthase activity and localization
were determined in replicate wells of cells. As previously
reported (15), PTG overexpression markedly increased both
the basal and insulin-stimulated glycogen synthase activity
ratio without changing total glycogen synthase activity (data
not shown). In 3T3-L1 adipocytes, insulin-stimulated glyco-
FIG. 3. Cytosolic glycogen synthase (GS) translocation is dependent
on glucose uptake. 3T3-L1 adipocytes were serum starved for 2.5 h
and then washed twice with glucose-free DMEM. Fresh medium was
added with varying amounts of glucose or 5 mM pyruvate in the
absence (condition 1) and presence (conditions 2– 8) of 100 nM insulin.
After 15 min, cells were lysed in a detergent-free homogenization
buffer and subjected to sequential centrifugation at 1,000 ⫻ g and
100,000 ⫻ g. The supernatant from the ultracentrifugation spin was
assayed for total GS activity in the presence of 10 mM G6P. Inset,
Anti-GS immunoblot from the cytosolic lysates used in the GS activity
assay. Conditions: 1, 5 mM glucose; 2, 0 mM glucose; 3, 1 mM glucose;
4, 2 mM glucose; 5, 3 mM glucose; 6, 4 mM glucose; 7, 5 mM glucose;
and 8, 5 mM pyruvate. *, P ⬍ 0.05; **, P ⬍ 0.01 vs. basal (condition
1). Results are the average of three independent experiments performed in triplicate.
Endocrinology, January 2005, 146(1):494 –502
497
gen synthase activity returned to basal levels in 1–2 h in the
continued presence of insulin (14, 19). However, adenoviralmediated overexpression of PTG resulted in a chronic dephosphorylation of glycogen synthase that was maintained
for up to 4 d (Fig. 4A), resulting in a 12-fold increase in
cellular glycogen levels (data not shown). These data indicated that PTG overexpression overrode the cellular feedback mechanisms that inactivate glycogen synthase activity.
The molecular regulation of glycogen synthase activity by
the PTG-PP1 complex was further investigated. 3T3-L1 adipocytes were infected with PTG adenovirus, recovered for
2 d, and then stimulated with insulin for 15 min. Additionally, extracellular glucose was substituted with 5 mm 2-DG
in half of the wells. Phosphorylation of the key regulatory site
3A on glycogen synthase was analyzed by immunoblotting.
Insulin treatment in the presence of 5 mm glucose significantly promoted the dephosphorylation of site 3A, whereas
the effects of insulin were enhanced in the presence of 2-DG
(Fig. 4B). These data agree with previous work demonstrating that 2-DG potentiates the dephosphorylation of glycogen
synthase in adipocytes (5, 7). Interestingly, PTG overexpression alone markedly reduced site 3A phosphorylation to
levels below insulin-treated control cells (Fig. 4B). Insulin
treatment caused a further reduction in site 3A phosphorylation, although the majority of site 3A dephosphorylation
occurred in the absence of insulin in PTG-overexpressing
cells.
Because dephosphorylation of site 3A is critical for glycogen synthase activation (20), the effects of 2-DG and PTG
overexpression on glycogen synthase activity were determined. After infection and recovery, cells were stimulated in
the absence or presence of 100 nm insulin for 15 min, in media
containing 5 mm glucose or 2-DG. As previously reported,
inclusion of 2-DG enhanced basal and insulin-stimulated
glycogen synthase activity (Fig. 4C), which is in agreement
with the anti-phospho-glycogen synthase immunoblotting
data (Fig. 4B). Interestingly, 2-DG also markedly increased
glycogen synthase activation by insulin in PTG-overexpressing cells (Fig. 4C). Because site 3A was largely dephosphorylated upon PTG overexpression (Fig. 4B), these results
suggest that insulin and 2-DG promote glycogen synthase
activation in these cells via the dephosphorylation of other
unidentified sites on glycogen synthase. However, it is not
possible at this point to completely rule out the further increase in site 3A dephosphorylation as mediating the additional effect on glycogen synthase activation.
Next, the potential role of glycogen synthase association
with PTG in these effects was determined. 3T3-L1 adipocytes
were infected with an adenovirus encoding full-length PTG,
containing two alanine substitutions in the glycogen synthase-binding mutant (PTG-DE). Mutation of the aspartic
and glutamic acid residues 225 and 228 to alanine in PTG
completely blocked glycogen synthase binding to PTG and
the stimulation of glycogen accumulation in CHO-IR cells
(18) and 3T3-L1 adipocytes (data not shown). Although
PTG-DE was overexpressed at comparable levels to PTG
(Fig. 4C, inset), the mutant PTG molecule had no effect on
glycogen synthase activation under any condition tested.
Cumulatively, these results suggest that elevation of intracellular 2-DG-6-phosphate levels by insulin promotes the
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Endocrinology, January 2005, 146(1):494 –502
Ou et al. • Regulation of Glycogen Synthase Activity
FIG. 4. Overexpression of PTG increases glycogen synthase (GS) activity. A, Replicate wells of 3T3-L1 adipocytes were mock infected (M) or
infected with PTG adenovirus (P). Half of the wells received 1 ␮g/ml doxycycline (D) during the recovery period to suppress exogenous protein
expression (15). Fresh medium was added after 48 h, and cells were collected 4 d after infection. Cell lysates were prepared and analyzed by
anti-GS immunoblotting. PTG overexpression resulted in an increase in the electrophoretic mobility of GS, indicative of dephosphorylation and
activation. B, Cells were infected as indicated, and 2 d later, they were incubated in the absence and presence of 100 nM insulin for 15 min in
media containing 5 mM glucose or 2-DG. Lysates were prepared and analyzed by anti-phospho-GS (pGS) immunoblotting (Ser 640). C, Cells
were mock infected or infected with adenoviral constructs encoding wild-type PTG or a full-length PTG construct containing a double alanine
substitution in the GS-binding domain (PTG-DE). Cells were allowed to recover for 2 d, and then they were treated for 15 min in the presence
or absence of 100 nM insulin in media containing 5 mM glucose or 5 mM 2-DG. GS activity ratios were then determined in vitro; the activity
in mock-infected basal cells was set at 100%, which corresponds to an activity ratio of 0.03– 0.07. *, P ⬍ 0.05; **, P ⬍ 0.01 vs. identical condition
in the mock-infected cells. Inset, Anti-PTG immunoblots from mock- (M), PTG- (P), and PTG-DE- (DE) infected cells. All immunoblotting results
are representative of two to four independent experiments, whereas GS activity measurements are the average of two to five independent
experiments.
association of glycogen synthase of PTG, resulting in its
subsequent dephosphorylation and activation in PTG-overexpressing cells.
Effects of 2-DG and PTG on cellular ATP levels
Both incubation of cells with 2-DG or adenoviral-mediated
overexpression of PTG increased glycogen synthase activity
in 3T3-L1 adipocytes. Presumably, both effects occurred by
increasing PP1 activity against glycogen synthase, but theoretically, either manipulation could affect cellular ATP concentration and, thus, indirectly result in glycogen synthase
dephosphorylation. 2-DG is phosphorylated by hexokinases
but does not enter glycolysis to replenish ATP levels. Additionally, there is a marked increase in glucose storage as
glycogen upon PTG overexpression, which potentially could
be diverting glucose flux into the glycolytic pathway. To
further investigate these possibilities, the effects of these
experimental conditions on cellular ATP levels were determined. First, serum-starved 3T3-L1 adipocytes were incubated for 15 min with or without 100 nm insulin and in the
presence of 5 mm glucose or 2-DG in the extracellular media.
Surprisingly, acute inclusion of 2-DG in the absence of insulin markedly reduced ATP levels (Fig. 5A). Furthermore,
addition of insulin, which stimulates 2-DG uptake and phosphorylation, caused a greater than 90% drop in ATP levels in
15 min. These results are in contrast to previous work with
primary rat adipocytes (5), in which 10 mm 2-DG in the
presence of insulin had no effect on ATP levels within 5 min
and 1 mm 2-DG had no similar effect on ATP levels for up
to 1 h. These differences may reflect a higher rate of glucose
uptake and phosphorylation in the cultured 3T3-L1 adipocytes vs. primary cells, resulting in a faster depletion of ATP
stores. The effects of the drop in ATP levels were mirrored
by an inhibition of insulin-stimulated GSK-3␤ phosphorylation and a concomitant increase in phospho-acetyl-coenzyme A carboxylase levels (Fig. 5B), which is reflective of
cellular energy depletion and AMP kinase activation (21).
The addition of 5 or 10 mm pyruvate to the 2-DG-containing
media did not reverse these effects (Fig. 5B) or the drop in
ATP levels (data not shown), indicating that pyruvate entry
into the glycolytic pathway was not sufficient to preserve
ATP levels under these conditions of enhanced 2-DG
phosphorylation.
In parallel, the effects of PTG overexpression on ATP
stores were examined. Cells were incubated for 2 d in the
absence and presence of PTG adenovirus. Additionally,
half of the wells were treated with 1 ␮g/ml doxycycline
during the recovery period, which completely suppresses
exogenous protein expression (15). PTG overexpression
resulted in a trend toward increased ATP levels, although
the differences obtained were not statistically significant
(Fig. 5C). Thus, the presumed effects of 2-DG on glycogen
synthase dephosphorylation in 3T3-L1 adipocytes may involve both increased PP1 activity and a decrease in cellular
ATP levels. In contrast, glycogen synthase dephosphorylation upon PTG overexpression appeared to be mediated
Ou et al. • Regulation of Glycogen Synthase Activity
FIG. 5. Effects of 2-DG or PTG overexpression on cellular ATP levels.
A, 3T3-L1 adipocytes were serum starved and washed twice with
glucose-free DMEM. Cells were then incubated for 15 min in DMEM
containing 5 mM glucose (Glucose) or 5 mM 2-DG in the presence or
absence of 100 nM insulin. Cellular ATP levels were determined as
described in Materials and Methods. **, P ⬍ 0.01; ***, P ⬍ 0.001 vs.
respective condition in glucose-containing media. Results are the
average of four independent determinations, each performed in duplicate. B, Cellular lysates were prepared from 3T3-L1 adipocytes
that were incubated for 15 min with or without insulin and in the
presence of glucose, 2-DG, and pyruvate. Samples were analyzed by
anti-phospho-acetyl-coenzyme A carboxylase (pACC) and anti-phospho-GSK-3␤ (pGSK-3␤) immunoblotting. Lanes: odd, no insulin;
even, ⫹ 100 nM insulin; 1 and 2, 5 mM glucose; 3 and 4, 0 mM glucose;
5 and 6, 0 mM glucose ⫹ 5 mM pyruvate; 7 and 8, 5 mM 2-DG; 9 and
10, 5 mM 2-DG ⫹ 5 mM pyruvate; and 11 and 12, 5 mM 2-DG ⫹ 10 mM
pyruvate. Immunoblots are representative of three independent experiments. C, Replicate wells of 3T3-L1 adipocytes were infected as
indicated, with 1 ␮g/ml doxycycline (plus dox) added to half of the
wells during recovery to suppress exogenous protein expression (15).
After 48 h, cells were stimulated for 15 min with or without 100 nM
insulin, and cellular ATP levels were determined. Results are the
average of three independent experiments, each performed in duplicate.
Endocrinology, January 2005, 146(1):494 –502
499
by differential centrifugation. PP1, PTG, and glycogen synthase levels in the fractions were analyzed simultaneously by
immunoblotting. In control cells, most of the glycogen synthase protein was localized in the 100,000 ⫻ g and 10,000 ⫻
g fractions, with lesser amounts in the cytosol (Fig. 6). Amylase treatment of these fractions completely released glycogen synthase from the pellets (data not shown), indicating
that both fractions contain cellular glycogen. As previously
reported, insulin treatment of control cells reduced cytosolic
glycogen levels (7), although a corresponding increase in
glycogen synthase immunoreactivity in the pellet fractions
was not reproducibly detected due to high basal levels. In
contrast, most of the PP1 was present in the cytosolic fraction,
and its distribution was unchanged by insulin (Fig. 6).
Adenoviral-mediated overexpression of PTG significantly
elevated PP1 levels in the 100,000 ⫻ g pellet fraction and, to
a lesser extent, in the 10,000 ⫻ g pellet fraction. Interestingly,
PTG overexpression also resulted in the redistribution of
glycogen synthase from the cytosolic- to glycogen-containing fractions (Fig. 4). Thus, PTG overexpression alone appeared to mimic insulin action by increasing glycogen synthase activity (Fig. 4C) and inducing glycogen synthase
translocation (Fig. 6), resulting in a dramatic increase in
glycogen accumulation (15).
To further investigate the effects of PTG on glycogen synthase localization, cells were examined by immunostaining.
After infection of 3T3-L1 adipocytes with adenovirus and a
2-d recovery, cells were transferred onto coverslips. The next
day, fixed cells were incubated simultaneously with antiglycogen synthase and anti-PTG antibodies, followed by
fluorescently labeled antimouse and antirabbit secondary
antibodies. PTG levels and glycogen synthase distribution
were then determined by confocal microscopy. As shown in
Fig. 7, infection of 3T3-L1 adipocytes with PTG adenovirus
markedly increased PTG immunoreactivity (Fig. 7, closed
arrows). However, because an infection efficiency of roughly
75% was obtained using this viral titer, uninfected cells were
present in the same field (Fig. 7, open arrow) and served as a
by enhanced PP1 activity and was independent of changes
in ATP generation.
Overexpression of PTG results in redistribution of
glycogen synthase
Glycogen synthase localization was examined by subcellular fractionation and immunostaining. 3T3-L1 adipocytes
were infected with adenovirus encoding PTG and allowed to
recover for 48 h. Cells were serum-starved for 2 h, and half
of the cells were treated with 100 nm insulin for 15 min. Cells
were collected in detergent-free homogenization buffer and
lysed by sonication. Crude cellular fractions were prepared
FIG. 6. PTG overexpression induces the redistribution of glycogen
synthase (GS) and protein phosphatase-1 (PP1) in 3T3-L1 adipocytes.
PTG was overexpressed in 3T3-L1 adipocytes by adenoviral-mediated
gene transfer. After a 48-h recovery, cells were serum starved for
2.5 h, followed by treatment without (B) or with 100 nM insulin (I) for
15 min. Cells were collected in detergent-free homogenization buffer
and lysed by sonication. Lysates were subjected to sequential centrifugation at 1,000 ⫻ g, 10,000 ⫻ g, and 100,000 ⫻ g. The latter two
pellets were resuspended in homogenization buffer using a 23-gauge
needle. Samples were then analyzed simultaneously by anti-GS and
PP1 immunoblotting.
500
Endocrinology, January 2005, 146(1):494 –502
FIG. 7. Effects of PTG overexpression on glycogen synthase (GS) immunostaining. 3T3-L1 adipocytes were infected in the absence (⫺)
and presence (⫹) of adenovirus encoding PTG. Cells were allowed to
recover for 48 h before replating onto glass coverslips. The next day,
cells were serum starved for 2.5 h before fixation. Coverslips were
treated in Fig. 1, and GS and PTG immunostaining of the same cells
was examined by confocal microscopy. An infection efficiency of approximately 75% was obtained; infected cells are shown with a closed
arrow, and a non-PTG-overexpressing cell is shown with an open
arrow.
control. Despite the marked activation and redistribution of
glycogen synthase after PTG overexpression, there was no
detectable clustering of glycogen synthase in infected vs.
control cells (Fig. 7). This result was most likely due to the
greater than 7-fold increase in cellular glycogen levels during
the recovery period (15), which may have resulted in the
marked increase in glycogen chains available for elongation
and the diffusion of sites within the cell where glycogen
synthesis was occurring. Glycogen synthase immunostaining was more intense in the PTG-overexpressing cells, despite no change in total glycogen synthase activity or protein
levels (15). As before, this result may be due to differences
in antibody recognition of dephosphorylated vs. phosphorylated glycogen synthase.
Discussion
Insulin potently stimulates glycogen synthesis in muscle
and adipose tissue through the synchronized increase in
glucose uptake and regulation of glycogen-metabolizing enzymes. After binding of its receptor, insulin activates several
signaling cascades that promote the release of GLUT4-containing vesicles from the perinuclear region and translocation and fusion of these vesicles at the plasma membrane (2).
In parallel, insulin promotes the dephosphorylation of glycogen synthase and phosphorylase, resulting in enzymatic
activation and inactivation, respectively (1). Activation of
PP1 by insulin plays an important role in these effects because glycogen synthase and phosphorylase are excellent
substrates for the phosphatase in vitro. Additionally, a family
of four targeting subunits have been described that bind to
PP1 and glycogen, thus targeting the phosphatase to glycogen particles (22). The redundancy of glycogen-targeting
subunits in insulin-sensitive tissues suggests an important
role for PP1 in the regulation of glycogen metabolism. However, other kinases and phosphatases are likely involved in
the regulation of glycogen synthase and phosphorylase activities, so the molecular mechanisms by which insulin controls glycogen-metabolizing enzymes remain uncertain.
In addition to regulation by covalent modification, glycogen synthase and phosphorylase activities are modulated by
Ou et al. • Regulation of Glycogen Synthase Activity
allosteric regulators and changes in intracellular localization
(reviewed in Ref. 1). Binding of G6P to glycogen synthase
increased enzymatic activity, overriding inhibition caused by
phosphorylation (3). Conversely, hepatic phosphorylase activity was suppressed by elevation of intracellular glucose or
G6P (1, 23). Interestingly, both hormones and extracellular
glucose levels have been reported to alter the subcellular
distribution of glycogen synthase (7, 10, 24). In skeletal muscle cells, insulin treatment and increased intracellular glycogen stores caused a clustering of glycogen synthase in
single muscle fibers (11). Because glucose transport in hepatocytes is not regulated by insulin, elevation of extracellular
glucose alone was sufficient to induce the translocation of
glycogen synthase from cytosolic to particulate fractions (1).
Immunocytochemistry revealed that glycogen synthase
translocated to an area proximal to the plasma membrane,
where glycogen synthesis was occurring (25). These effects
on hepatic glycogen synthase were mediated by elevations of
intracellular G6P levels because they were not blocked by
substitution of extracellular glucose with 2-DG (24). Less is
known about the regulation of glycogen phosphorylase localization, although the cellular distribution of glycogen may
also be affected by hormones and intracellular glucose metabolites (1).
In the present study, we found that acute insulin treatment
of 3T3-L1 adipocytes induced a redistribution of glycogen
synthase into bright, discrete punctate clusters within the
cell. Increased glucose uptake and metabolism was required
for this effect because withdrawal of extracellular glucose
or substitution with 2-DG blocked the change in glycogen
synthase immunostaining patterns. Thus, insulin caused a
translocation of GLUT4-containing vesicles to the plasma
membrane, resulting in enhanced glucose transport and
metabolism, and a subsequent translocation of glycogen
synthase. Interestingly, sequential treatment of 3T3-L1 adipocytes with insulin and the adenylyl cyclase activator forskolin reverted glycogen synthase immunostaining to the
basal state (Fig. 1B). This result indicated that glycogen synthase localization was subject to reciprocal effects of insulin
and elevation of intracellular cAMP. We have reported that
insulin treatment of 3T3-L1 adipocytes induced the movement of glycogen synthase from cytosolic to denser pellet
fractions using sucrose gradients or differential centrifugation techniques (7, 14). Interestingly, the fraction of glycogen
synthase that translocated on the sucrose gradients was preferentially activated by insulin (14), suggesting a potential
link between enzymatic activation and intracellular redistribution to glycogen. In agreement with reports of skeletal
muscle and liver cells (11, 25), we found, in the present study,
that glycogen synthesis is apparently restricted to discrete
sites within 3T3-L1 adipocytes. Thus, in addition to dephosphorylation and allosteric activation, insulin may regulate
glycogen synthase activity in these cells by promoting its
localization to elongating glycogen chains. However, further
work is required to substantiate this supposition.
Substitution of extracellular glucose with 2-DG enhances
the activation of glycogen synthase in 3T3-L1 adipocytes (7)
most likely through intracellular accumulation of 2-DG-6phosphate (26). Interestingly, 2-DG also markedly increases
insulin-stimulated glycogen synthase activity in PTG-over-
Ou et al. • Regulation of Glycogen Synthase Activity
expressing cells. This effect requires a functional glycogen
synthase-binding domain on PTG because overexpression of
the glycogen synthase-binding mutant PTG-DE had no effect
on glycogen synthase activation. These results suggest that
elevation of 2-DG-6-phosphate and, presumably, also G6P
might promote glycogen synthase association with PTG, resulting in its dephosphorylation and activation. Moreover,
the effect of 2-DG on glycogen synthase activation in PTGoverexpressing cells appeared to involve the dephosphorylation of sites other than 3A on glycogen synthase. Sites 1 and
2A are likely candidates because they have been implicated
in the effects of PP1 on glycogen synthase activation, and
work is currently underway to test this supposition. However, the marked and unexpected depletion of cellular ATP
levels upon acute incubation of 3T3-L1 adipocytes with 5 mm
2-DG clouds interpretation of these results. The drop in ATP
was unexpected because normally much higher concentrations of extracellular 2-DG are used to deplete ATP (27).
Additionally, previous work in rat primary adipocytes did
not find any similar changes in ATP levels upon incubation
with 2-DG (5), although the conditions used were not identical. Thus, the enhancement of glycogen synthase dephosphorylation by 2-DG, which was presumed to reflect increased PP1-mediated dephosphorylation, may also involve
reduction in ATP levels and, consequently, activity of glycogen synthase kinases in 3T3-L1 adipocytes.
In contrast, insulin treatment in the presence of 2-DG did
not induce the cellular redistribution of glycogen synthase in
3T3-L1 adipocytes (Fig. 2). This result is in contrast to findings in primary hepatocytes where addition of 2-DG to the
medium was sufficient to induce glycogen synthase movement to particulate fractions (24). The reason for the different
results is unknown but may reflect differential glycogen synthase isoform expression in the two cell types and/or potentially a requirement for ATP in this effect. However, activation of glycogen synthase by insulin in the presence of
2-DG was not sufficient to induce glycogen synthase movement in 3T3-L1 adipocytes, which is in agreement with previous results (7). Thus, a glucose metabolite downstream of
G6P, an ATP-dependent motor protein, and/or glycogen
accumulation is required for the intracellular redistribution
of glycogen synthase. Additionally, these results suggest that
glycogen synthase activation by insulin precedes and can be
uncoupled from its translocation to glycogen-containing
fractions in 3T3-L1 adipocytes.
Activation of PP1 plays an important role in the stimulation of glycogen synthase activity by insulin (28). We have
recently reported that adenoviral-mediated overexpression
of the PP1-glycogen-targeting subunit PTG in 3T3-L1 adipocytes was sufficient to markedly increase glycogen synthase dephosphorylation and activation (15). In this study,
we found that PTG overexpression also induced the translocation of glycogen synthase from cytosolic to particulate
fractions in these cells (Fig. 6). Thus, PTG overexpression
mimicked the effects of insulin on glycogen metabolism (dephosphorylation of the regulatory site 3A, resulting in stimulation of glycogen synthase activity, enzymatic redistribution,
and increased glucose storage as glycogen). Interestingly, these
effects occurred without changing basal facilitative glucose
transporter number at the plasma membrane (15), indicating
Endocrinology, January 2005, 146(1):494 –502
501
that PTG overexpression could pull enough glucose into the cell
through existing transporters to sustain the enhanced glycogen
synthesis rates. In contrast, PTG overexpression did not cause
the distinct clusters of glycogen synthase immunoreactivity that
were present after an acute stimulation of 3T3-L1 adipocytes
with insulin. However, this result is most likely due to the small
changes in glycogen levels after a 15-min insulin stimulation vs.
a greater than 7-fold increase in cellular glycogen upon PTG
overexpression. This increased number of glycogen chains in
PTG-overexpressing cells presumably caused a diffusion of
cellular glycogen synthesis from the discrete sites seen in control cells.
Similar to reports of skeletal muscle (reviewed in Ref. 29),
accumulation of intracellular glycogen reduced glycogen
synthase activation by insulin in 3T3-L1 adipocytes (14).
Although the mechanism of this feedback inhibition of glycogen synthase activity is unclear, it may represent the spatial separation of glycogen synthase from its insulin-sensitive
activators. Interestingly, insulin treatment of 3T3-L1 adipocytes resulted in the redistribution of glycogen synthase
away from PTG, as determined by immunostaining (Fig. 1A)
and cellular fractionation (data not shown). In insulin-pretreated cells, glycogenolytic agents restored the basal intracellular distribution of glycogen synthase (Fig. 2 and Ref. 14)
and subsequent activation by insulin (14). Overexpression of
PTG in 3T3-L1 adipocytes resulted in the persistent dephosphorylation and activation of glycogen synthase for up to 4 d
and preservation of insulin stimulation, despite a greater
than 12-fold elevation of glycogen stores. Thus, elevation of
PTG levels overrode the intrinsic negative regulation of glycogen accumulation on glycogen synthase activity, preventing the inactivation of enzymatic activity. Based on this
present study and previous work, we propose the following
model for the regulation of glycogen synthase activity by
insulin in 3T3-L1 adipocytes. Insulin treatment promotes
GLUT4 vesicle translocation to the plasma membrane, resulting in an increase in glucose uptake and metabolism.
Insulin also stimulates glycogen synthase dephosphorylation through the activation of PP1 and inactivation of glycogen synthase kinases such as GSK-3. The regulation of
glycogen synthase activity by the PTG-PP1 complex may be
mediated, in part, by elevation of G6P levels in insulinstimulated cells. The enhancement of glucose uptake and
metabolism also induces a redistribution of glycogen synthase presumably to elongating glycogen chains. Through an
underdetermined mechanism, glycogen accumulation results in the rephosphorylation and inactivation of glycogen
synthase and the inhibition of subsequent stimulation by
insulin. Subsequent breakdown of glycogen in 3T3-L1 adipocytes restores glycogen synthase distribution to the basal
state, priming the enzymes for subsequent stimulation by
insulin (7, 14). However, several key issues are still unresolved. What are the relative roles of phosphatase activation
vs. kinase inactivation in the insulin-induced dephosphorylation of glycogen synthase? What is the precise nature of the
interdependence between glycogen synthase intracellular localization and enzymatic activation? And finally, what are
the mechanisms by which increased glycogen levels feed
back to inhibit glycogen synthase activity? Further work will
be needed to resolve these important issues and determine
502
Endocrinology, January 2005, 146(1):494 –502
whether similar mechanisms regulate glycogen metabolism
in hepatocytes and myocytes.
Acknowledgments
Received August 4, 2004. Accepted October 6, 2004.
Address all correspondence and requests for reprints to: Matthew J.
Brady, MC1027, 5841 South Maryland Avenue, Chicago, Illinois 60637.
E-mail: [email protected].
This work was supported by National Institutes of Health Grant
DK64772 and a Career Development Award from the American Diabetes
Association (to M.J.B.).
Present address for H.O.: Cardiothoracic Surgery, Suite 0200, Baylor
College of Medicine, 2450 Holcombe Boulevard, Houston, Texas
77021-2024.
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