The Role of Glycogen Synthase Kinase 3
in Insulin-stimulated
Glucose Metabolism*
Scott A.
Summers
,
Aimee W.
Kao§,
Aimee D.
Kohn¶,
Gillian
S.
Backus
,
Richard A.
Roth¶,
Jeffrey E.
Pessin§, and
Morris
J.
Birnbaum
From the
Howard Hughes Medical Institute, University
of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, the § Department of Physiology and Biophysics, University of
Iowa College of Medicine, Iowa City, Iowa 52242, and the
¶ Department of Molecular Pharmacology, Stanford University School
of Medicine, Stanford, California 94305
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ABSTRACT |
To characterize the contribution of glycogen
synthase kinase 3
(GSK3
) inactivation to insulin-stimulated
glucose metabolism, wild-type (WT-GSK), catalytically inactive
(KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3
were expressed
in insulin-responsive 3T3-L1 adipocytes using adenovirus technology.
WT-GSK, but not KM-GSK, reduced basal and insulin-stimulated glycogen
synthase activity without affecting the -fold stimulation of the enzyme by insulin. S9A-GSK similarly decreased cellular glycogen synthase activity, but also partially blocked insulin stimulation of the enzyme.
S9A-GSK expression also markedly inhibited insulin stimulation of
IRS-1-associated phosphatidylinositol 3-kinase activity, but only
weakly inhibited insulin-stimulated Akt/PKB phosphorylation and glucose
uptake, with no effect on GLUT4 translocation. To further evaluate the
role of GSK3
in insulin signaling, the GSK3
inhibitor lithium was
used to mimic the consequences of insulin-stimulated GSK3
inactivation. Although lithium stimulated the incorporation of glucose
into glycogen and glycogen synthase enzyme activity, the inhibitor was
without effect on GLUT4 translocation and pp70 S6 kinase. Lithium
stimulation of glycogen synthesis was insensitive to wortmannin, which
is consistent with its acting directly on GSK3
downstream of
phosphatidylinositol 3-kinase. These data support the hypothesis that
GSK3
contributes to insulin regulation of glycogen synthesis, but is
not responsible for the increase in glucose transport.
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INTRODUCTION |
The peptide hormone insulin elicits a broad array of metabolic
responses. In muscle and adipose tissue, insulin promotes the storage
of sugar by coordinately accelerating both the rate of glucose entry
into the cell and glycogen synthase activity. The former is mediated by
the insulin-dependent redistribution of glucose transport
proteins (GLUT4) from intracellular compartments to the plasma membrane
(1), and the latter by multisite dephosphorylation of the enzyme
coupled with activation by the soluble metabolite glucose 6-phosphate
(2). Although much progress has been made characterizing the molecular
signaling events emanating from the insulin receptor and progressing to
these targets, the complete pathway has not yet been elucidated. The
general purpose of this study was to evaluate the relative contribution
of the serine/threonine kinase
GSK3
1 to insulin
regulation of these molecular events.
GSK3
was originally identified based on its kinase activity toward
an in vitro substrate, glycogen synthase (3). Subsequent studies have described roles for the enzyme in many different cellular
processes. For example, the potent GSK3
inhibitor lithium was used
to identify roles for the enzyme in spore and stalk development in
Dictyostelium (4, 5) and in expansion of dorsal mesoderm in
Xenopus (4, 5). A critical role for GSK3
in pattern formation has been confirmed through investigation of the
Drosophila ortholog shaggy/zeste-white 3 (6). Moreover,
overexpression of GSK3
in cultured cells led to the proposal of a
role for the enzyme in the initiation of apoptosis (7). Two types of
studies support the view that GSK3
could contribute to
insulin-stimulated anabolic metabolism. First, insulin inhibits GSK3
kinase activity in multiple insulin-responsive tissues (8-11), and a
novel signaling pathway has been postulated whereby GSK3
inactivation follows the sequential activation of PI3K and Akt/PKB (12,
13). Second, GSK3
inhibits basal glycogen synthase activity when
transfected into transformed cells (14). Since the effects of GSK3
on glycogen metabolism have only been evaluated in vitro or
in non-insulin-responsive tissues, the relative contribution of GSK3
to regulation of these metabolic events by insulin remains unclear.
Moreover, since Akt/PKB and PI3K have been implicated in insulin
regulation of numerous other metabolic processes (15-18), the relative
contribution of GSK3
to these events warrants evaluation.
The studies described herein evaluated the contribution of GSK3
to
insulin-stimulated metabolism in 3T3-L1 adipocytes. These cells are a
well recognized system for investigating insulin-stimulated glucose
metabolism, as they respond to physiological doses of insulin with
increases in glucose uptake, GLUT4 translocation, glycogen synthesis,
and lipogenesis. Adenovirus-mediated overexpression of GSK3
mutants
in these cells, as well as treatment with the GSK3
inhibitor
lithium, was used to disrupt coupling of insulin regulation of the
enzyme. Data support a model whereby GSK3
inhibition contributes to
insulin regulation of glycogen synthesis, but not GLUT4 translocation.
These results further indicate that GSK3
can inhibit
insulin-stimulated IRS-1-associated PI3K activity, but has little
effect on downstream signaling events.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Crystalline porcine insulin was a gift of Lilly.
Polyclonal anti-IRS-1 antibodies were a gift from Bayer (West Haven,
CT). Polyclonal anti-pp70 S6 kinase antibodies were generously provided by Dr. Margaret Chou (University of Pennsylvania, Philadelphia, PA).
Rabbit phospho-specific anti-S6 antibodies were raised against a
peptide resembling the major phosphorylation site in ribosomal subunit
protein S6 (CRRLSpSpLRASpTSKSpEESpQK) by Rockland Inc. (Gilbertsville,
PA). Polyclonal rabbit anti-hemagglutinin antibodies were from Berkeley
Antibody Co. (Berkeley, CA). Antibodies against GSK3
were purchased
from Transduction Labs (Lexington, KY) or Quality Controlled
Biochemicals (Hopkintown, MA). An antibody that recognizes the
phosphorylated serine 9 regulatory site on human GSK3
and less well
the mouse enzyme was also from Quality Controlled Biochemicals.
GSK3
Constructs, Adenovirus Infection, and Cell
Culture--
cDNAs encoding human wild-type GSK3
(WT-GSK) and
catalytically inactive GSK3
(KM-GSK) were generously provided by
Peter Klein (University of Pennsylvania). Additionally, the serine 9 regulatory site on WT-GSK was mutated to alanine (S9A-GSK) by polymerase chain reaction. The sequence of the latter was confirmed by
DNA sequencing. The relative activities of all three constructs are
described elsewhere (14). These constructs were subcloned into the
vector pACCMV.pLpA and transferred to recombinant adenovirus by
homologous recombination (19). A multiplicity of infection of 100 was
used to infect 3T3-L1 adipocytes in Dulbecco's modified Eagle's
medium containing 1% calf serum, with the virus being left on the
cells for at least 16 h prior to its removal. Experiments were
conducted 48 h after the initial addition of the virus. This method results in infection of >90% of the cells on the dish as assessed by expression of
-galactosidase.
3T3-L1 fibroblasts were differentiated into adipocytes 1 day
post-confluence in Dulbecco's modified Eagle's/H-21 medium
supplemented with 10% fetal bovine serum (Sigma), 1 µg/ml
dexamethasone, and 112 µg/ml isobutylmethylxanthine. After 3 days,
cells were maintained in Dulbecco's modified Eagle's/H-21 medium
supplemented with 10% fetal bovine serum.
Glycogen Synthase Assay--
3T3-L1 adipocytes differentiated in
6-well culture dishes were serum-deprived for 2 h in Leibovitz
L-15 buffer supplemented with 0.2% bovine serum albumin. Cells were
then incubated for 15 min with insulin and washed in glycogen synthase
extraction buffer (100 mM NaF, 10 mM EDTA, 1 mM benzamidine, and 50 mM Tris-HCl (pH 7.8)).
Adipocytes were disrupted using a Teflon/glass homogenizer, and the
homogenate was centrifuged briefly at 2000 × g. The
supernatant without the fat cake was assayed for enzyme activity as
described previously (20), and glycogen synthase activity is the
activity measured in the presence of low (0.1 mM) glucose
6-phosphate divided by the activity in the presence of high (10 mM) glucose 6-phosphate.
Incorporation of Glucose into Glycogen--
The incorporation of
glucose into a glycogen pellet was determined as described previously
(21).
Preparation of Total Cell Extracts--
Cells were serum-starved
for 24 h in Dulbecco's modified Eagle's medium containing 0.5%
bovine serum albumin and 10 mM HEPES (pH 7.5). Two hours
prior to solubilization, cells were washed twice in phosphate-buffered
saline and incubated at 37 °C in Leibovitz L-15 buffer containing
0.5% bovine serum albumin. Cells were washed again in
phosphate-buffered saline and then incubated for 1 h at 37 °C
in KRP buffer (136 mM NaCl, 4.7 mM KCl, 10 mM NaPO4 (pH 7.4), 0.9 mM
MgSO4, and 0.9 mM CaCl2) containing
0.2% bovine serum albumin. For experiments using lithium, the
indicated concentration of LiCl replaced an equal concentration of
NaCl. Cells were solubilized in 66 mM Tris-HCl (pH 7.5) and
2% SDS additionally containing 1 mM vanadate, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 2 µg/ml aprotinin. DNA was sheared by sonication, and insoluble material was pelleted for 10 min in a microcentrifuge.
Glucose Uptake and GLUT4 Translocation Assays--
Methods for
measuring glucose uptake rates and plasma membrane GLUT4 levels (using
the plasma membrane "sheet" assay) have been described (16). For
experiments involving lithium, cells were stepped down in KRP buffer
with the indicated concentration of lithium replacing an equal
concentration of NaCl.
PI3K Assays--
PI3K assays were performed using methods
previously described (22).
 |
RESULTS |
Insulin inhibits GSK3
kinase activity in rat skeletal muscle
and epididymal fat, mouse 3T3-L1 adipocytes, and human cultured myoblasts (8-11). Recent studies further indicate that Akt/PKB is
required for insulin inactivation of GSK3
in NIH-3T3 cells overexpressing insulin receptors (12), and in vitro
experiments suggest that this is the result of Akt-catalyzed
phosphorylation of serine 9 (13). To investigate whether insulin and
Akt/PKB stimulate phosphorylation of this regulatory residue in intact 3T3-L1 adipocytes, GSK3
was immunoprecipitated from cell lysates using a monoclonal anti-GSK3
antibody. Following transfer to nitrocellulose membranes, GSK3
phosphorylation was detected using a
phospho-specific antibody against the serine 9 portion of GSK3
. Insulin rapidly stimulated GSK3
phosphorylation in 3T3-L1 adipocytes infected with an empty vector (Fig.
1A). Moreover, stable
expression of a constitutively active form of Akt/PKB (Myr-akt,
described in Ref. 16) stimulated GSK3
phosphorylation in the absence of insulin (Fig. 1A). Immunoblotting with the monoclonal
anti-GSK3
antibody confirmed that equal amounts of GSK3
were
precipitated under all conditions (Fig. 1B). Mock
lanes denote immunoprecipitations done in the absence of cell
lysates to visualize the IgG bands.

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Fig. 1.
Effect of insulin on GSK3
phosphorylation. 3T3-L1 adipocytes infected with empty
vector or Myr-akt were treated with (+) or without ( ) insulin (100 nM, 15 min) prior to solubilization. Insoluble material was
precipitated by centrifugation, and endogenous GSK3 was
immunoprecipitated (IP) from the supernatants using
monoclonal antibodies against GSK3 . The mock sample represents the
immunoprecipitation done in the absence of cell lysates. Proteins were
eluted in Laemmli solubilization buffer, resolved by SDS-PAGE,
transferred to nitrocellulose, and immunoblotted. Antibodies used for
Western blotting (WB) included the monoclonal antibody to
GSK3 ( GSK) (A) or a polyclonal antibody
raised against a peptide resembling the phosphorylated serine 9 site on
GSK3 ( P-GSK) (B). Blots were then probed
with horseradish peroxidase-conjugated secondary antibodies and
detected by enhanced chemiluminescence. IgG bands are shown by
arrows. Data are representative of four independent
experiments.
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To ascertain the role of GSK3
in insulin action, the effects of
various overexpressed GSK3
constructs on glucose uptake and glycogen
synthase enzyme activity were investigated. Using recombinant
adenovirus, the wild-type (WT-GSK) and kinase-dead (KM-GSK) forms of
GSK3
were expressed in 3T3-L1 adipocytes. In addition, a GSK3
construct with the regulatory serine residue changed to alanine
(S9A-GSK) was also expressed. A previous study characterized the
relative activity of all three constructs, including their effects on
basal glycogen synthase activity, in non-insulin-responsive 293T cells.
To evaluate the levels of expression, whole cell lysates were resolved
by SDS-PAGE, transferred to nitrocellulose, and analyzed by
immunoblotting. All three constructs were overexpressed roughly
3-5-fold (Fig. 2A) 48 h
after infection with adenovirus. Moreover, immunofluorescent detection
using an antibody against the hemagglutinin epitope revealed that
80-90% of the cells expressed S9A-GSK (Fig. 2B) 48 h
after infection. This is comparable to the percentage of cells that
expressed a
-galactosidase gene when a similar infection protocol
was used with a control virus.

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Fig. 2.
Adenovirus-mediated GSK3
overexpression. A, 3T3-L1 adipocytes were
infected with recombinant adenoviruses encoding WT-GSK, KM-GSK, or
S9A-GSK. Forty-eight hours after infection, cells were treated with or
without insulin (100 nM, 15 min) and solubilized. Total
cell extracts were harvested, resolved by SDS-PAGE, transferred to
nitrocellulose, and immunoblotted. Monoclonal antibodies against
full-length GSK3 ( -GSK) from Quality Controlled
Biochemicals (QCB) or Transduction Labs (TL) were
used. Data are representative of three independent experiments.
B, 3T3-L1 adipocytes were infected with recombinant
adenoviruses encoding S9A-GSK. Forty-eight hours after infection, cells
were fixed and analyzed by immunofluorescence using Hoechst or
anti-hemagglutinin ( -HA) antibodies.
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As shown in Fig. 3, overexpression of
WT-GSK, but not KM-GSK, decreased glycogen synthase enzyme activity in
3T3-L1 adipocytes, with reductions apparent under both basal and
insulin-stimulated conditions. Nonetheless, insulin still stimulated
glycogen synthase activity ~2-2.5-fold over the new, lowered basal
value, which was comparable to the stimulation induced in uninfected or
KM-GSK-infected cells. Overexpression of the uninhibitable S9A-GSK
construct similarly reduced basal and insulin-stimulated glycogen
synthase activity. More important, however, S9A-GSK also reduced the
-fold stimulation of glycogen synthase activity by insulin (Fig. 3).
This finding suggests a role for GSK3
phosphorylation in insulin
regulation of glycogen synthase.

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Fig. 3.
Effect of GSK3
overexpression on glycogen synthase activity. 3T3-L1
adipocytes were infected with adenovirus producing KM-GSK, WT-GSK, or
S9A-GSK 48 h prior to stimulation with insulin (100 nM) for 10 min. Following stimulation, glycogen synthase
assays were performed as described under "Experimental Procedures."
Data are presented as low Glu-6-P (G6P)/high Glu-6-P ratios
and means ± S.E. of three independent experiments. *, the value
was statistically different from the control (+insulin) value at
p 0.05; **, the value was statistically different
from the WT-GSK (+insulin) value at p 0.05.
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Akt/PKB has been implicated as a mediator of other metabolic responses
to insulin, including glucose uptake and GLUT4 translocation (15-18).
Since GSK3
is one of only a few identified substrates for Akt, we
also sought evidence of the role of GSK3
in these insulin-regulated
events. Insulin stimulated the uptake of non-metabolizable 2-deoxyglucose significantly in uninfected 3T3-L1 adipocytes. Overexpression of S9A-GSK slightly inhibited rates of 2-deoxyglucose uptake in both the presence and absence of insulin (Fig.
4). However, the dose-response curves for
both infected and uninfected cells were nearly identical. The major
effect of insulin on glucose uptake is due to the translocation of the
GLUT4 isoform from intracellular storage sites to the plasma membrane
(1). The commonly utilized GLUT4 sheet assay was used to measure plasma
membrane levels of GLUT4 (16). Briefly, 3T3-L1 adipocytes
differentiated on coverslips were sonified, liberating cellular
structures from the coverslip, but leaving an intact sheet containing
the plasma membrane with its cytosolic face exposed. Probing the sheet
with antibodies against the carboxyl terminus of GLUT4 reflects the
total amount of GLUT4 on the membrane. Expression of KM-GSK, WT-GSK,
and S9A-GSK had no effect on plasma membrane GLUT4 levels (Fig.
5), demonstrating that GSK3
does not
contribute to insulin stimulation of GLUT4 translocation. Collectively,
these data indicate that GSK3
is unlikely to mediate
insulin-stimulated glucose uptake.

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Fig. 4.
Effect of GSK3
overexpression on insulin-stimulated glucose uptake. 3T3-L1
adipocytes were infected with or without adenoviral S9A-GSK 48 h
prior to stimulation with increasing concentrations of insulin for 10 min. Following stimulation, 2-deoxyglucose uptake was measured over a
period of 4 min. Data are means ± S.D. of quadruplicate samples
and are representative of four independent experiments.
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Fig. 5.
Effect of GSK3
overexpression on insulin-stimulated GLUT4 translocation.
3T3-L1 adipocytes were infected with WT-GSK, KM-GSK, or S9A-GSK
adenoviruses 48 h prior to stimulation with insulin
(Ins; 100 nM) for the times indicated. Following
stimulation, plasma membrane sheets were prepared by sonication. GLUT4
levels on fixed sheets were determined by immunofluorescence using
anti-GLUT4 antibodies. Data are representative of two independent
experiments.
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To further investigate the consequences of GSK3
phosphorylation,
experiments were conducted with the GSK3
inhibitor lithium. Lithium
specifically inhibits GSK3
in the millimolar range, but has no
effect on numerous other protein kinases, including
cAMP-dependent protein kinase, mitogen-activated protein
kinase, and casein kinase II (4). Lithium thus mimics the effects of
physiological GSK3
inhibition, indicating which of insulin's
metabolic actions can be mediated solely by its effects on GSK3
. A
2-h treatment with 50 mM lithium stimulated glycogen
synthesis 60-70% as well as insulin in control 3T3-L1 adipocytes
(Fig. 6). Lithium-stimulated glycogen
synthesis was not sensitive to wortmannin, which inhibited insulin-stimulated glycogen synthesis completely. Interestingly, lithium and insulin added simultaneously stimulated glycogen synthesis greater than either agent alone. Lithium also stimulated glycogen synthase enzyme activity (Fig. 7) and,
once again, was partially additive with insulin. Under conditions in
which lithium stimulated glycogen synthesis (Fig.
8A), it weakly stimulated
glucose uptake (Fig. 8B). To determine whether lithium could
mimic the effects of insulin on GLUT4 translocation, plasma membrane
GLUT4 levels from lithium-treated cells were also determined using the
aforementioned sheet assay. Lithium did not stimulate GLUT4
translocation in the absence of insulin (Fig. 8C), nor did
it augment insulin-stimulated glucose uptake or GLUT4 translocation
(data not shown).

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Fig. 6.
Effect of lithium on glycogen synthesis.
3T3-L1 adipocytes were serum-deprived for 2 h prior to the
addition of the radiolabeled glucose. Insulin (100 nM),
wortmannin (300 nM), and lithium (50 mM) were
added 15, 60, and 60 min prior to the addition of radiolabeled glucose,
respectively. As described under "Experimental Procedures," LiCl
replaced an equal concentration of NaCl in the buffer to prevent
osmolarity changes. Following a 60-min incubation with radiolabeled
glucose, glycogen pellets were isolated as described under
"Experimental Procedures." The data presented are means ± S.E. of four independent experiments.
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Fig. 7.
Effect of lithium on glycogen synthase
activity. Glycogen synthase experiments were conducted as
described under "Experimental Procedures" in 3T3-L1 adipocytes that
were serum-deprived for 2 h prior to homogenization. Lithium (50 mM) and insulin (100 nM) were present for the
last 60 and 15 min prior to homogenization, respectively. As described
under "Experimental Procedures," LiCl replaced an equal
concentration of NaCl in the buffer to prevent osmolarity changes.
Glycogen synthase activity was determined as described under
"Experimental Procedures," and the data presented are mean -fold
stimulations ± S.E. of three independent experiments.
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Fig. 8.
Effect of lithium on glycogen synthesis,
glucose uptake, and GLUT4 translocation. 3T3-L1 adipocytes were
serum-deprived for 2 h prior to assay of glycogen synthesis
(A), glucose uptake (B), or GLUT4 translocation
(C). Lithium was added at the indicated concentrations 60 min prior to
initiation of the assay. LiCl replaced an equal concentration of NaCl
in the buffer to prevent osmolarity changes. All results are normalized
to the response achieved with a 10-min stimulation by 100 nM insulin. The data presented are means ± S.E. of
three to five independent experiments.
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Although GSK3
had no effect on glucose uptake or GLUT4
translocation, a prior report indicated that GSK3
is a negative
regulator of IRS-1-mediated activation of PI3K in transformed cells
(23). Consequently, GSK3
was proposed as a possible contributor to insulin resistance. To evaluate whether GSK3
also inhibited
signaling in insulin-responsive 3T3-L1 adipocytes, IRS-1-associated
PI3K activity was assayed in S9A-GSK-expressing cells (Fig.
9). S9A-GSK expression markedly inhibited
insulin-stimulated activation of PI3K in anti-IRS-1 immunoprecipitates.
However, S9A-GSK did not reduce insulin-stimulated phosphorylation of
Akt, mitogen-activated protein kinase, and ribosomal S6 protein (Fig.
10). No effect was seen at submaximal
doses of insulin either (data not shown). S9A-GSK expression did not
affect the insulin-stimulated mobility shift for IRS-1 separated on
SDS-polyacrylamide gel (Fig. 10). Thus, if GSK3 is inhibiting PI3K by
direct phosphorylation of IRS-1, the kinases must be utilizing a subset
of those sites phosphorylated in response to insulin or a distinct set
of sites. Lithium, under conditions in which glycogen synthase was
activated (Fig. 8), did not affect insulin stimulation of PI3K, Akt,
mitogen-activated protein kinase, and ribosomal S6 protein (Fig.
11). These latter studies also indicate
that GSK3
is unlikely to mediate insulin and Akt stimulation of pp70
S6 kinase.

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Fig. 9.
Effect of GSK3 on
IRS-1-associated PI3K activity. 3T3-L1 adipocytes were infected
with adenoviral KM-GSK, WT-GSK, and S9A-GSK 48 h prior to
stimulation with insulin (100 nM) for 10 min. Following
solubilization of cells, IRS-1 was immunoprecipitated, and PI3K
activity was assayed as described previously (22). Data are expressed
as the percent of the maximal insulin response and represent the
mean ± range of two independent experiments.
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Fig. 10.
Effect of GSK3 on
phosphorylation of IRS-1, Akt, mitogen-activated protein kinase, and
ribosomal S6 protein. 3T3-L1 adipocytes infected with S9A-GSK or
empty vector were treated with or without insulin (100 nM)
for 15 min as indicated. Total cell extracts were harvested, resolved
by SDS-PAGE, and transferred to nitrocellulose. Immunoblotting was
conducted using antibodies against IRS-1 ( IRS1) and the
phosphorylated forms of Akt/PKB ( P-Akt),
mitogen-activated protein kinase ( P-MAPK), and ribosomal
S6 protein ( P-S6). Blots were then probed with
horseradish peroxidase-conjugated secondary antibodies and detected
using enhanced chemiluminescence. Data are representative of two
independent experiments.
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Fig. 11.
Effect of lithium on IRS-1, PI3K,
mitogen-activated protein kinase, Akt, and ribosomal S6-protein.
3T3-L1 adipocytes were treated with or without lithium (50 mM) for 60 min or insulin (100 nM) for 15 min
as indicated. As described under "Experimental Procedures," LiCl
replaced an equal concentration of NaCl in the buffer to prevent
osmolarity changes. A, total cell extracts were harvested,
resolved by SDS-PAGE, and transferred to nitrocellulose. Immunoblotting
was conducted using antibodies against IRS-1 ( -IRS1),
pp70 S6 kinase ( -p70 S6-kinase), and the phosphorylated
forms of Akt/PKB ( -P-Akt), mitogen-activated protein
kinase ( -P-MAPK), and ribosomal S6 protein
( -P-S6). Blots were then probed with horseradish
peroxidase-conjugated secondary antibodies and detected using enhanced
chemiluminescence. B, PI3K assays were done as described
previously (22), and incorporation of 32P into
phosphatidylinositol is presented. Data are representative of two
independent experiments. PI3P, phosphatidylinositol
3-phosphate.
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DISCUSSION |
Insulin activates a number of pleiotropic metabolic responses that
are governed by a similarly broad array of signaling events. Definitive
assignment of particular metabolic functions to specific signaling
cascades has been a recent challenge in insulin action research.
Although insulin activates the Ras/Raf/mitogen-activated protein kinase
cascade, which contributes to its regulation of DNA synthesis, studies
from multiple laboratories have excluded these signaling molecules from
insulin regulation of glucose uptake, GLUT4 translocation, or glycogen
synthesis (21, 24-28). Similarly, insulin activates a protein
phosphatase (SHPTP2) that contributes to its regulation of mitogenesis
and GLUT1 expression, but is not required for acute stimulation of
GLUT4 translocation (29). Instead, insulin stimulation of glucose
uptake, GLUT4 translocation, and glycogen synthesis requires a
signaling cascade involving PI3K (30, 31) and possibly the downstream
effector Akt/PKB (16-18, 32). The studies described herein demonstrate
further divergence in insulin signaling downstream of PI3K and Akt.
Although GSK3
is likely to contribute to insulin and Akt regulation
of glycogen synthase, it apparently does not contribute to their activation of glucose transport, GLUT4 translocation, and pp70 S6 kinase.
Evidence supporting a role for GSK3
inactivation in insulin
regulation of glycogen synthase includes the following. 1) Insulin and
constitutively active forms of Akt/PKB stimulated phosphorylation of an
inhibitory residue on GSK3
(Fig. 1). 2) Insulin and constitutively active forms of Akt/PKB inhibit the activity of GSK3
in intact cells
(9-11, 32). 3) Expression of wild-type (but not catalytically inactive) GSK3
inhibited basal and insulin-stimulated glycogen synthase activity without affecting the -fold stimulation of the enzyme
by insulin (Fig. 3). 4) Expression of a GSK3
mutant incapable of
being phosphorylated on this inhibitory residue similarly decreased basal and insulin-stimulated glycogen synthase activity, but also partially blocked the -fold stimulation of the enzyme by insulin (Fig.
3). 5) Treatment with lithium, which mimics insulin's inhibitory effects on GSK3
, stimulated glycogen synthesis and glycogen synthase enzyme activity in the absence of increased GLUT4 translocation (Figs.
8-10). Since the phosphate groups on glycogen synthase turn over
rapidly (33), inhibition of GSK3
could serve as a physiologically relevant mechanism for regulating glycogen synthase activity.
Previous studies suggest that GSK3
inhibition is not insulin's only
mechanism for stimulating glycogen accumulation. In a prior study in a
different line of 3T3-L1 adipocytes, insulin was capable of stimulating
glycogen synthase despite the fact that GSK3
was either not
detectable or present in very low amounts (32). Similarly, another
group reported that GSK3
expression decreases during 3T3-L1
differentiation, thus switching insulin's primary mechanism for
glycogen synthase activation from GSK3
inactivation to protein
phosphatase activation (8). In contrast to these reports, GSK3
was
clearly present in the 3T3-L1 adipocytes used in this study, and its
expression was unaltered by adipogenesis (data not shown). Subtle
differences in cell lines or differentiation conditions could account
for discrepancies between these studies. Although the data presented
above strongly support a role for GSK3
inhibition in glycogen
synthesis, these studies also indicate the existence of an alternative
pathway. First, insulin still stimulated glycogen synthase partially in
cells infected with S9A-GSK, although its stimulation was significantly
compromised. Second, lithium was partially additive with insulin in
activating glycogen synthesis and glycogen synthase. The latter
observation is particularly suggestive since lithium is likely to be a
more effective inhibitor of GSK3
than insulin. Although these data could result from an effect of lithium independent of the inhibition of
GSK3
, this is unlikely in view of the failure of other investigators to identify such a site of action (4). More probable is that the
coupling of insulin stimulation of a protein phosphatase with its
inhibition of GSK3
is the mechanism for its complete regulation of
glycogen synthase.
A second role suggested by the literature for GSK3
is as a regulator
of glucose uptake. The GSK3
upstream inhibitor Akt/PKB is implicated
in insulin-stimulated glucose uptake and GLUT4 translocation (15-18),
although this has been recently challenged (34), and GSK3
inactivation is implicated in other Akt-mediated events (7). GSK3
has a remarkably broad array of in vitro substrates and
cellular functions (3, 35-38). Numerous sporadic reports over the last
30 years have attributed to lithium an "insulinomimetic" effect,
i.e. the ability to stimulate both glucose uptake and glycogen synthesis in various systems (39-44). In some of these studies, the effect of lithium on glucose uptake was smaller than that
observed on glycogen synthesis, whereas in others it was not.
Nonetheless, the results presented above do not support the existence
of a role for GSK3
in insulin-stimulated glucose uptake. Although,
in the experiments presented above, lithium did weakly stimulate
glucose uptake, it did not stimulate GLUT4 translocation. This
conclusion was further strengthened by the finding that WT-GSK and
S9A-GSK expression did not alter insulin stimulation of glucose uptake
or GLUT4 translocation.
A third role postulated for GSK3
is as a negative regulator of
IRS-1-associated PI3K and as a contributor to insulin resistance (23).
The results presented above confirm that GSK3
can inhibit insulin-stimulated PI3K activity in 3T3-L1 adipocytes, but suggest that
this has little effect on insulin stimulation of glucose metabolism:
under conditions in which S9A-GSK inhibited PI3K, it had no effect on
glucose uptake or GLUT4 translocation; neither S9A-GSK nor lithium
affected insulin stimulation of Akt/PKB or pp70 S6 kinase
phosphorylation. Several mechanisms could account for the lack of
correlation between IRS-1-associated PI3K activity and glucose uptake
rates. First, only a small amount of PI3K activity might be required
for full stimulation of glucose transport. Most studies evaluating the
necessity of PI3K have utilized methods (such as potent PI3K inhibitors
or dominant-negative antibodies) that completely eliminate PI3K
activity. A partial inhibition of PI3K activity may not significantly
affect insulin stimulation of downstream metabolic events.
Alternatively, compensatory mechanisms could contribute to the
requisite activation of PI3K. For example, in IRS-1 knockout mice,
other members of the IRS family take over the responsibilities of
IRS-1, stimulating PI3K to nearly normal levels (45). Moreover, recent
work indicates that an IRS-independent, yet PI3K-dependent
pathway contributes to insulin stimulation of GLUT4 translocation (46,
47). Such alternative mechanisms for activating PI3K could account for
the nearly normal stimulation of glucose transport and downstream
signaling events.
Although GSK3
was discovered over 15 years ago, its contribution to
insulin signaling has not been fully addressed. The recent observation
of molecular events linking activated insulin receptors to GSK3
rekindled interest in the enzyme. This identified signaling cascade,
which includes PI3K and Akt/PKB, is postulated to regulate numerous
metabolic responses to insulin (15-18). In the studies above, we
carefully evaluated both the effect of disrupting insulin inhibition of
the enzyme and the consequences of inhibiting the enzyme in the absence
of other signaling events. The studies confirm that GSK3
is likely
to contribute to insulin stimulation of glycogen synthesis, but not
glucose uptake or GLUT4 translocation. Alternative signaling events
must account for these important metabolic responses.
 |
ACKNOWLEDGEMENTS |
We thank Peter Klein, who kindly donated
cDNA encoding WT-GSK and KM-GSK; Margaret Chou, who kindly donated
anti-pp70 S6 kinase antibodies; and Cass Lutz, who provided assistance
in the typing and editing of this manuscript.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants DK39615 (to M. J. B.), DK33823 (to J. E. P.), DK34926 (to R. A. R.), and DK09375 (to S. A. S.) and by Medical Scientist Training Program Grant 5T32 GM07365 (to A. D. K.).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.
Supported by the Cox Institute. To whom correspondence should
be addressed: Howard Hughes Medical Inst., University of Pennsylvania Medical School, Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-5001; Fax: 215-573-9138; E-mail:
birnbaum{at}hhmi.upenn.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
GSK3
, glycogen
synthase kinase 3
;
PI3K, phosphatidylinositol 3-kinase;
PAGE, polyacrylamide gel electrophoresis.
 |
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