Báo cáo khoa học: Use of lithium and SB-415286 to explore the role of glycogen synthase kinase-3 in the regulation of glucose transport and glycogen synthase pdf

Use of lithium and SB-415286 to explore the role of glycogen
synthase kinase-3 in the regulation of glucose transport
and glycogen synthase
Katrina MacAulay
1
, Eric Hajduch
1
, Anne S. Blair
1
, Matthew P. Coghlan
2
*, Stephen A. Smith
2
and Harinder S. Hundal
1
1
Division of Molecular Physiology, Faculty of Life Sciences, MSI/WTB Complex, University of Dundee, UK;
2
GlaxoSmithKline,
Harlow, UK
Glycogen synthase kinase 3 (GSK3) is inactivated by insulin
and lithium and, like insulin, Li also activates glycogen
synthase (GS) via inhibition of GSK3. Li also mimics insu-
lin’s ability to stimulate glucose transport (GT), an obser-
vation that has led to the suggestion that GSK3 may
coordinate hormonal increases in GT and glycogen synthe-
sis. Here we have used Li and SB-415286, a selective GSK3
inhibitor, to establish the importance of GSK3 in the hor-
monal activation of GT in terms of its effect on GS in L6
myotubes and 3T3-L1 adipocytes. Insulin, Li and SB-415286
all induced a significant inhibition of GSK3, which was

associated with a marked dephosphorylation and activation
of GS. In L6 myotubes, SB-415286 induced a much greater
activation of GS (6.8-fold) compared to that elicited by
insulin (4.2-fold) or Li (4-fold). In adipocytes, insulin, Li and
SB-415286 all caused a comparable activation of GS despite
a substantial differentiation-linked reduction in GSK3
expression ( 85%) indicating that GSK3 remains an
important determinant of GS activation in fat cells. Whilst
Li and SB-415286 both inhibit GSK3 in muscle and fat cells,
only Li stimulated GT. This increase in GT was not sensitive
to inhibitors of PI3-kinase, MAP kinase or mTOR, but was
suppressed by the p38 MAP kinase inhibitor, SB-203580.
Consistent with this, phosphorylation of p38 MAP kinase
induced by Li correlated with its stimulatory effect on GT.
Our findings support a crucial role for GSK3 in the regula-
tion of GS, but based on the differential effects of Li and
SB-415286, it is unlikely that acute inhibition of GSK3
contributes towards the rapid stimulation of GT by insulin
in muscle and fat cells.
Keywords: adipocyte; muscle; GSK-3; insulin; p38 MAP
kinase.
One of the major physiological effects of insulin is to
promote the uptake, metabolism and storage of glucose in
adipose tissue and skeletal muscle [1]. The hormonal
regulation of these cellular processes is initiated by the
binding of insulin to its receptor and activation of the
receptor kinase, which tyrosine phosphorylates intracellular
target substrates, in particular insulin receptor substrate 1
(IRS-1) and its relatives IRS-2 and IRS-3 [2–5]. Of the
numerous IRS binding proteins, the serine/lipid kinase

phosphoinositide 3-kinase (PI3K) has been implicated
strongly as a component of the signalling cascade that
stimulates glucose transport and glycogen synthesis [6–9].
Another important component of this cascade is protein
kinase B (PKB), which lies downstream of PI3K and
whose activation is dependent upon phosphorylation of
two key amino acid residues, Thr308 and Ser473 [10,11].
3-Phosphoinositide-dependent kinase (PDK1) phosphory-
lates Thr308 [12,13], whereas phosphorylation of Ser473 is
thought to be mediated by a separate, as yet unidentified,
upstream kinase that has been tentatively called PDK2
[14]. Activated PKB has been shown to induce the
translocation of GLUT4 to the cell surface and stimulate
glucose transport in muscle and fat cells [15], whereas it
phosphorylates and inhibits glycogen synthase kinase-3
(GSK3) [14]. GSK3 is one of several kinases that
phosphorylate glycogen synthase (GS), an event that helps
to maintain the enzyme in an inactive state [16]. In order to
stimulate glycogen synthesis, insulin has to suppress
phosphorylation and simultaneously promote the dephos-
phorylation of GS via activation of glycogen-associated
protein phosphatase 1 (PP1G). The greatest decrease in
bound phosphate on GS has been shown to occur at sites
3a, 3b, 3c and 4 [17], which are target sites for GSK3.
Correspondence to H. S. Hundal, Division of Molecular Physiology,
MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, UK.
Fax: + 44 1382 345507, Tel.: + 44 1382 344969,
E-mail:
Abbreviations: GS, glycogen synthase; GSK3, glycogen synthase
kinase-3; HBS, Hepes buffered saline; HRP, horse-radish peroxidase;

IRS, insulin receptor substrate; MAPK, mitogen activated protein
kinase; a-MEM, a-minimal essential media; PDK, 3-phospho-
inositide-dependent kinase; PI3K, phosphoinositide 3-kinase;
PKB, protein kinase B; PP1G, protein phosphatase 1.
*Present address: AstraZeneca, Cardiovascular and Gastrointestinal
Research Area, Mereside, Alderley Park, Macclesfield, Cheshire,
SK10 4TG, UK.
(Received 15 May 2003, revised 17 July 2003,
accepted 31 July 2003)
Eur. J. Biochem. 270, 3829–3838 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03777.x
Thus, in addition to control by allosteric regulators the
activation status of GSK3 is likely to be a key determinant
of GS activity.
Whilst there is considerable evidence implicating GSK3
in the regulation of glycogen metabolism there are
conflicting reports in the literature as to whether the kinase
also participates in the hormonal activation of glucose
transport. Lithium (Li) is a widely used inhibitor of GSK3
and studies using this ion have shown that it can exert
insulin-like effects on both glycogen synthesis and glucose
uptake in insulin sensitive tissues [18–22]. These observa-
tions raise the possibility that GSK3 may help to coordi-
nate increases in glucose uptake and glycogen synthesis
allowing for more effective ÔchannellingÕ of glucose into
glycogen in response to insulin. However, one of the
potential difficulties in interpreting data from studies that
utilize Li as an inhibitor of GSK3 is that the ion also
affects the activity of a number of other molecules such as
casein kinase-2 and mitogen activated protein kinase
(MAPK)-2 [23] as well as enzymes involved in the

metabolism of glucose [24]. It is difficult therefore to
exclude the possibility that the observed stimulatory effects
of Li on glucose transport may be mediated by a
mechanism that is independent of GSK3. Indeed, in
3T3-L1 adipocytes the expression of a constitutively active
form of GSK3 has no significant effect on insulin
stimulated translocation of the GLUT4 glucose transporter
and glucose transport [25]. However, the value of these
findings is unclear given that the importance of GSK3 in
the regulation of glycogen metabolism in this cell type
remains poorly defined. Brady et al. have shown that
GSK3 activity is reduced substantially during differenti-
ation of 3T3-L1 adipocytes and have suggested that the
primary mechanism by which insulin stimulates GS in
mature adipocytes is through activation of PP1G rather
than inactivation of GSK3 [26].
In an attempt to establish the importance of GSK3 in the
acute regulation of glucose transport in terms of its
regulatory control of GS in muscle and fat cells we have
investigated the effects of Li and the anilinomaleimide, SB-
415286, a potent and highly selective inhibitor of GSK3
(K
i
¼ 31 n
M
) [27]. We demonstrate here that whilst expres-
sion of GSK3 declines substantially during differentiation of
3T3-L1 adipocytes, both Li and SB-415286 promote
activation of GS to a level comparable, if not greater, than
that elicited by insulin. Furthermore, whilst both Li and SB-

415286 inactivate GSK3, our data indicate that only Li
acutely stimulates glucose transport in L6 myotubes and
3T3-L1 adipocytes.
Materials and methods
Cell culture
L6 muscle cells were cultured to myotubes as described
previously [28] in a-minimal essential media (aMEM)
containing 2% (v/v) foetal bovine serum and 1% (v/v)
antimycotic/antibiotic solution (100 UÆmL
)1
penicillin,
100 lgÆmL
)1
streptomycin, 250 ngÆmL
)1
amphotericin B)
at 37 °Cwith5%CO
2
. 3T3-L1 fibroblasts (provided by
H. Green, Department of Cell Biology, Harvard Medical
School, Boston, MA, USA) were differentiated into
adipocytes as described previously [29,30]. Cells were
cultured in 10-cm dishes for lysate preparation and in
6-well plates for glucose uptake assays. Differentiated
muscle cells or adipocytes were serum starved for 5 h and
3 h, respectively, before addition of appropriate reagents
for times and at concentrations indicated in the figure
legends.
Preparation of cell lysates
L6 myotubes and 3T3-L1 adipocytes were serum starved as

described above. Plates were washed three times with 0.9%
(w/v) ice-cold saline. Two-hundred lL of lysis buffer
(50 m
M
Tris pH 7.4, 0.27
M
sucrose, 1 m
M
Na-orthovana-
date pH 10, 1 m
M
EDTA, 1 m
M
EGTA, 10 m
M
Na
b-glycerophosphate, 50 m
M
NaF, 5 m
M
Na pyrophosphate,
1% (w/v) Triton X-100, 0.1% (v/v) 2-mercaptoethanol,
0.1 l
M
microcystin-LR and protease inhibitors) was added.
Cells were scraped off the plates using a rubber policeman
and homogenized by passing through a 26-gauge hyper-
dermic needle prior to centrifugation (15000 g,4°Cfor
10min)andstoredat)20 °C.
Glucose uptake

L6 myotubes or 3T3-L1 adipocytes were serum starved as
described above and incubated with Li, wortmannin,
SB-415286, SB-203580, PD-98059, rapamycin, sucrose and
insulin at times and concentrations indicated in figure
legends. Cells were washed three times with warm Hepes-
buffered saline (HBS; 140 m
M
NaCl, 20 m
M
Hepes, 5 m
M
KCl, 2.5 m
M
MgSO
4
,1m
M
CaCl
2
, pH 7.4). Glucose
uptake was assayed by incubation of 2-deoxy-[
3
H]-
D
-glucose (1 lCiÆmL
)1
,26.2CiÆmmol
)1
)for10minas
described previously [28,31]. Nonspecific binding was deter-

mined by quantifying cell-associated radioactivity in the
presence of 10 l
M
cytochalasin B. Radioactive medium was
aspirated prior to washing adherent cells three times with
0.9% (w/v) ice-cold saline. Cells were subsequently lysed in
50 m
M
NaOH and radioactivity quantified using a Beck-
man LS 6000IC scintillation counter. Protein concentration
in cell lysates was determined using the Bradford reagent as
described previously [32].
Glycogen synthase
The activity of GS was assayed as described previously [31].
Briefly, assay buffer (67 m
M
Tris pH 7.5, 5 m
M
dithiothre-
itol, 89 m
M
UDP-glucose, 6.7 m
M
EDTA, 13 mgÆmL
)1
glycogen, 1 lCi per assay uridine diphospho-[6-
3
H]-
D
-

glucose) was added to 45 lL cell lysate in the presence
and absence of 20 m
M
glucose-6-phosphate. After a 30-min
incubation at 37 °C the reaction was terminated by spotting
the reaction mixture onto 31ETCHR Whatman filter paper
(Whatman, Maidstone, UK) and washed three times in
66% (v/v) ethanol for 20 min. Filters were finally washed in
acetone and air dried before incorporation of glucose from
uridine diphospho-[6-
3
H]-
D
-glucose into glycogen was
quantitated using a Beckman LS6000IC scintillation coun-
ter. GS activity was expressed as a ratio of the activity in the
absence of glucose-6-phosphate over that in the presence of
the allosteric activator.
3830 K. MacAulay et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Immunoblotting
Fifty lgofcelllysateproteinwassubjectedtoSDS/PAGE
on a 10% resolving gel as described previously [28].
Separated proteins were transferred onto nitrocellulose
membranes, which were subsequently blocked using NaCl/
Tris containing 0.1% (v/v) Tween 20 and 5% (w/v) milk
protein. Membranes were probed with antibodies against
the phosphorylated forms of p70S6K (1 : 1000), PKB
(1 : 1000), p42/44 MAPK (1 : 1000), p38 MAP kinase
(1 : 1000), GSK-3a/b (1 : 1000) all from New England
Biolabs or to GS phosphorylated at the GSK3 (site 3)

epitope (1 : 500) or with antibodies against native PKBa
(1 : 1000) or GSK3a and b (1 : 1000). Antibodies to PKBa,
GSK3a,GSK3b and phospho GS were a gift from the
Division of Signal Transduction and Therapy, University of
Dundee. cMyc antibodies were from Sigma. The membranes
were washed three times in NaCl/Tris/0.1% Tween 20 (v/v)
for 15 min prior to incubation with horseradish peroxidase
(HRP) anti-rabbit IgG (1 : 1000), HRP anti-mouse IgG
(1 : 1000) or HRP anti-sheep/goat IgG (1 : 500, all from
Sigma). Protein bands on nitrocellulose were visualized using
enhanced chemiluminescence by exposure to Konica Medi-
cal Film (Konica Corporation, Hohenbrunn, Germany).
GSK3 assay
L6 myotubes were deprived of serum for 4 h in a-MEM and
washed twice with warm HBS. Cells were incubated
subsequently at 37 °CinHBS/25m
MD
-glucose for 1 h.
During the last hour insulin and or wortmannin were added
at times and concentrations indicated in the figure legends
prior to cell lysis. Myotubes were extracted from 10-cm
dishes using ice-cold lysis buffer. GSK3a,GSKb or myc-
tagged GSK3
S9A
were immunoprecipitated from 100 lg cell
lysate and incubated with or without 25 mUÆmL
)1
PP2A
1
prior to assay using phospho-GS peptide-1 as substrate [33].

Cell transfection
L6 cells were transfected with pSG5 vector, which encodes
resistance to G418 sulphate. cDNA encoding myc-tagged
GSK-3b in which serine 9 was mutated to an alanine was
subcloned into the pSG5 vector. Phosphorylation of the
serine 9 site on GSK-3b is considered important for its
inactivation by insulin and thus mutation of this site to an
alanine (S9A) renders the kinase constitutively active. Con-
trol cells were transfected with the empty vector lacking the
GSK-3b
S9A
cDNA. L6 cells transfected with GSK3
S9A
were
cultured as described earlier, but with the addition of
0.8 mgÆmL
)1
G418 sulphate to the media at all stages to
select for transformed cells. Transfected cells were used for
analysis of glucose uptake and GSK3 activity as described
earlier.
Statistical analyses
For multiple comparisons statistical analysis was performed
using one-way analysis of variance (
ANOVA
) followed by a
Newman–Keuls post-test. Data analysis was performed
using
GRAPHPAD PRISM
software and considered statistically

significant at P values < 0.05.
Results and discussion
Effects of insulin, Li and SB-415286 on GSK3 activity
As an initial starting point for our studies we investigated
the effects of insulin, Li and the maleimide, SB-415286, on
GSK3 activity from L6 myotubes. Insulin caused a
significant inactivation (by up to 40%) of both GSK3
isoforms, which was blocked by prior treatment of cells with
the PI3-kinase inhibitor wortmannin (Fig. 1A). Because
both Li and SB-415286 inhibit GSK3 by competitively
blocking Mg and ATP binding, respectively [27,34], it was
not possible to directly determine the effect of these
inhibitors on cellular GSK3 activity in vivo. However, both
Li (50 m
M
) and SB-415286 (50 l
M
) induced a substantial
suppression of immunoprecipitated GSK3 activity when
they were included in the in vitro kinase assay by 73% and
97%, respectively. Identical results were also obtained with
SB-216763 (data not shown), a structurally unrelated
maleimide, which, like SB-415286, also exhibits selectively
Fig. 1. Effects of insulin and wortmannin on GSK3 a and b activity in L6
myotubes and relative abundance of GSK3 isoforms in L6 and 3T3-L1-
adipocytes. (A) L6 myotubes were pretreated for 10 min with either
100 n
M
insulin alone or with 100 n
M

wortmannin for 15 min before
exposing cells to insulin. Following these incubations cells were lysed
and GSK3 a or b immunoprecipitated for analysis of kinase activity.
GSK3 activity was expressed as a re-activation ratio (i.e. GSK3 activity
measured without PP2A
1
treatment divided by GSK3 activity after
PP2A
1
treatment). Values are the mean ± SEM for three experiments
carried out in duplicate. The asterisk signifies a statistically significant
change from the untreated sample (P < 0.01). (B) Lysates (50 lg
protein) from L6 myoblasts, L6 myotubes, 3T3-L1 fibroblasts and
3T3-L1 adipocytes were immunoblotted using antibodies against
GSK3a and b.
Ó FEBS 2003 GSK-3 and glucose metabolism (Eur. J. Biochem. 270) 3831
for GSK3 [27]. An attempt was made to assess the effects of
insulin on GSK3 activity in 3T3-L1 adipocytes, but proved
technically difficult as kinase activity in immunoprecipitates
from unstimulated fat cells was found to be extremely low.
To establish why this may be so we immunoblotted lysates
from 3T3-L1 preadipocytes, fully differentiated 3T3-L1
adipocytes as well as L6 myoblasts and myotubes with
antibodies against GSK3a and b. Fig. 1B shows that whilst
preadipocytes express both GSK3 isoforms, the abundance
of the b isoform declines by  85%, whereas that of the a
isoform is virtually undetectable in differentiated adipocytes.
In contrast, such a loss was not observed during differenti-
ation of L6 muscle cells, which, if anything, showed a
marginal increase in GSK3 abundance during differenti-

ation. Our inability to detect GSK3 activity in differentiated
adipocytes is at odds with the study of Orena et al. [21] in
which the authors reported the presence of significant GSK3
activity. The reasons for this discrepancy are unclear, but
the assay protocol used in the present study relied upon
measuring kinase activity in GSK3 immunoprecipitates,
whereas that of Orena et al. utilized whole cell extracts to
monitor phosphorylation of a primed GSK3 peptide
substrate [21]. It is conceivable that this technical difference
may give rise to the apparent discrepancy between the two
studies. Nevertheless, it should be stressed that the marked
decline in GSK3 expression that we observe in 3T3-L1
adipocytes is fully consistent with previous data showing that
GSK3 activity diminishes substantially during adipogenesis
of 3T3-L1 adipocytes [26] thereby helping to explain the low
immunoprecipitable activity that we observe in our hands.
Effects of insulin, Li and SB-415286 on signalling
elements implicated in the regulation of GSK3 and GS
To further understand the effects of Li and SB-415286 on
cell signalling events we assessed their effects and that of
insulin on p70S6K, PKB, p42/p44 MAP kinases, GSK3 and
GS. PKB is considered to be the upstream inactivator of
GSK3 in vivo [35], but evidence also exists showing that the
latter can be targeted by p70S6K and the classical MAP
kinase pathway in response to nutrients and certain growth
factors [33,36,37]. Using phospho-specific antibodies to
screen for the phosphorylation and hence activation status
of these signalling molecules we observed that, unlike
insulin, neither Li or SB-415286 had any detectable effect on
the phosphorylation of PKBSer

473
, p70S6K, p42/p44 MAP
kinases or GSK3 in L6 myotubes or 3T3-L1 adipocytes
(Fig. 2). It is noteworthy that in L6 myotubes insulin
induces phosphorylation of both GSK3 a and b,whereasin
3T3-L1 adipocytes we observed only a single phospho-band
that correlates with that of GSK3b. The lack of an
equivalent GSK3a phospho-signal is consistent with the
virtual absence of this isoform in our 3T3-L1 adipocytes
(Fig. 1B). As indicated earlier, SB-415286 potently inhibits
GSK3 by an ATP competitive mechanism [27]. Conse-
quently, this compound did not affect insulin’s ability to
induce phosphorylation of GSK3 or that of PKB, p70S6K
and the p42/p44 MAP kinases in response to insulin
(Fig. 2). In unstimulated cells, GS is phosphorylated on site
3 by GSK3 and indeed a phospho-antibody directed against
site 3 confirmed that this was the case in muscle and fat cells
(Fig. 2). The adipocyte data suggests that despite the
substantial reduction in GSK3 activity and expression that
occurs during differentiation of 3T3-L1 cells ([26] and
Fig. 1B), sufficient GSK3 activity remains in these cells to
induce phosphorylation of GS on site 3. GS phosphoryla-
tion fell significantly upon treating both muscle and fat cells
with insulin, and was undetectable following incubation of
either cell type with Li or SB-415286 (Fig. 2). As site 3
phosphorylation can be taken as a downstream read out of
GSK3 activity the observation that both Li and SB-415286
induce a complete abolition of GS phosphorylation on this
site reflects that both agents cause a far greater inhibition of
GSK3 than that elicited by insulin.

Regulation of GS activity
To establish the importance of GSK3 inhibition on GS
activity we monitored the effects of insulin, Li, SB-415286
and wortmannin (a PI3K inhibitor) on the incorporation of
Fig. 2. Representative immunoblots showing
the effects of insulin, SB-415286 and lithium on
the phosphorylation status of key signalling
molecules. (A) L6 myotubes and (B) 3T3-L1
adipocytes were pretreated for 60 min with
50 l
M
SB-415286 or 50 m
M
lithium prior to a
10-min incubation of cells with 100 n
M
insulin.
Lysates (50 lg protein) were immunoblotted
using phospho-specific antibodies against
p70S6K, PKB, p42/44 MAP kinases,
GSK3a/b and GS. Equal loading of cell lysate
protein was determined by probing with an
antibody to native PKBa. The blots are
representative from up to four separate
experiments.
3832 K. MacAulay et al. (Eur. J. Biochem. 270) Ó FEBS 2003
labelled UDP-glucose into glycogen in the absence and
presence of glucose-6-phosphate (the allosteric activator of
GS). Insulin stimulated GS activity in both L6 myotubes
and 3T3-L1 adipocytes by 4.2- and 2.5-fold, respectively

(Fig. 3A). This stimulation was reduced significantly in both
cell lines by wortmannin, suggesting that activation of PI3K
precedes that of GS. This proposition is consistent with the
observation that the inhibition of GSK3 (mediated by PKB)
and the activation of PP1 in response to insulin are both
PI3K-dependent processes in L6 muscle cells and 3T3-L1
adipocytes [10,26]. GS was also activated by Li and SB-
415286 in both cell types, but, unlike insulin, activation of
the enzyme in response to these stimuli was not sensitive to
wortmannin (Fig. 3). This finding is compatible with the
suggestion that Li and SB-415286 target GSK3 directly and
that inhibition of the kinase by these agents does not rely
upon activation of upstream signalling molecules, such as
PI3K or PKB (Fig. 2). Collectively, these findings suggest
strongly that targeted inactivation of GSK3, using either Li
or SB-415286, is sufficient to induce activation of GS to a
level similar or greater than that by insulin. Since the activity
of GS depends on the relative activities of GS kinases and
PP1G, inhibiting GSK3 (one of the principal GS kinases)
will shift the balance towards dephosphorylation and
activation of GS. The notion that GSK3 is critical for
glycogen metabolism is strengthened further by our finding
that despite the significant decline in GSK3 abundance
during differentiation of 3T3-L1 adipocytes, selective inhi-
bition of this kinase, using SB-415286, appears to mimic the
hormonal activation of GS in this cell type. Consequently,
whilst PP1G is likely to play a significant role in the
hormonal activation of GS in 3T3-L1 adipocytes, the
importance of GSK3 in the insulin-mediated regulation of
this enzyme in fat cells should not be readily discounted [26].

Moreover, it is also noteworthy that an analysis of the GS
activity ratio in 3T3-L1 preadipocytes reveals that in
unstimulated cells, basal GS activity was  80% lower than
that measured in differentiated adipocytes. This lower GS
activity is fully concordant with the much higher level of
GSK3 expression that prevails in preadipocytes.
Is GSK3 a regulator of glucose transport
in insulin-responsive cells?
The potential involvement of GSK3 in the regulation of
glucose transport remains unclear at present. Two recent
studies have suggested that acute inhibition of GSK3 using
Li or long-term suppression of the kinase using inhibitors
that exhibit selectivity towards GSK3, enhance glucose
uptake in muscle and fat cells [21,38]. In contrast, another
study expressing a constitutively active form of GSK3
reported no significant changes in insulin-stimulated glucose
uptake or GLUT4 translocation, although a slight reduc-
tion in basal glucose uptake was noted [25]. In an attempt to
clarify this matter we investigated the effects of both Li and
SB-415286 on basal and insulin-stimulated glucose uptake
in L6 myotubes and 3T3-L1 adipocytes. Fig. 4 shows that
insulin enhances glucose uptake in both muscle and fat cells
by 2- and 3.4-fold, respectively. When both cell types were
exposed to Li, at a concentration that inhibits GSK3,
glucose uptake was stimulated by  1.8 fold (L6 myotubes)
and 2.6 fold (3T3-L1 adipocytes) (Fig. 4A and B). In
contrast, however, incubation of muscle and fat cells with
50 l
M
SB-415286, circumstances during which there is a

substantial inhibition of GSK3 (based on analysis of site 3
GS phosphorylation, Fig. 2) and an attendant activation of
GS (Fig. 3), did not elicit any change in basal or insulin-
stimulated glucose uptake (Fig. 4). Since both Li and SB-
415286 inhibit GSK3, but only one of these stimulates
glucose uptake the findings imply that GSK3 may not have
any significant regulatory input into the acute activation of
glucose transport by insulin in our experimental system.
These observations are, to some extent, consistent with the
recent work of Henriksen et al. [39] who reported that
whilst acute inhibition of GSK3 with Li enhanced glucose
uptake in skeletal muscle of lean Zucker rats, inhibition of
the kinase using a selective organic inhibitor (CT 98014)
had no stimulatory or insulin potentiating effect on glucose
uptake. This inhibitor also failed to stimulate glucose
transport in skeletal muscle of Zucker diabetic rats, but
Fig. 3. Effects of insulin, wortmannin, lithium and SB-415286 on GS
activity. (A) L6 myotubes and (B) 3T3-L1 adipocytes/preadipocytes
were pretreated for 5 min with 100 n
M
wortmannin prior to treatment
with 100 n
M
insulin (10 min), 50 m
M
lithium (60 min) and 50 l
M
SB-
415286 (60 min). Glycogen synthase activity was determined by
assaying incorporation of glucose from uridine diphospho-[6-

3
H]-
D
-
glucose into glycogen and expressed as a ratio of the activity in the
absence divided by that in the presence of glucose-6-phosphate. Values
are the mean ± SEM for three experiments each carried out in
duplicate.
Ó FEBS 2003 GSK-3 and glucose metabolism (Eur. J. Biochem. 270) 3833
interestingly potentiated the effects of insulin on muscle
glucose uptake in these animals. This potentiation was
associated with an increase in sarcolemmal GLUT4 content
following insulin-treatment of muscle. Precisely how inhi-
bition of GSK3 under these circumstances leads to an
increase in cell surface GLUT4 still remains poorly defined.
However, given that GSK3 activity is thought to be
enhanced in insulin-resistant muscle and the kinase has
been implicated in down-regulating insulin signalling via its
ability to serine phosphorylate IRS1 [40], it is possible that
inhibition of GSK3 potentiates insulin signalling at the level
of proteins such as IRS1. This possibility is supported by the
observations of Nikoulina et al. [38] who found that whilst
acute inhibition of GSK3 had no stimulatory effect on
glucose uptake in cultured human muscle cells, sustained
inhibition of GSK3 (over 96 h) led to an increase in both
basal and insulin-stimulated sugar uptake. This adaptive
increase in glucose uptake could not be linked to alterations
in cellular GLUT4 expression, but was associated with
changes in the abundance of both IRS1 and GSK3,
which were elevated and repressed, respectively. Whether

induction of IRS1 is sufficient to elicit the increase in
glucose uptake reported by Nikoulina et al. [38] remains
unclear at present, given that phosphorylation of PKB/Akt,
a kinase implicated in the hormonal regulation of glucose
transport [15], was unaffected by prolonged inhibition of
GSK3.
To assess whether SB-415286 may have an insulin
sensitizing effect in our muscle cell system we compared the
effects of the maleimide on the phosphorylation of PKB and
GSK3 and upon the stimulation of glucose transport in
response to a submaximal and maximal insulin concentra-
tion. Fig. 5 shows that insulin induces phosphorylation of
both PKB and GSK3, and modestly stimulates glucose
uptake at submaximal concentrations, although the
responses were clearly lower than that observed in response
Fig. 4. Effects of insulin, SB-415286 and lithium on glucose transport.
(A) L6 myotubes and (B) 3T3-L1 adipocytes were pretreated with
50 l
M
SB-415286 or 50 m
M
lithium for 60 min prior to a 30-min sti-
mulation with 100 n
M
insulin and analysis of 2-deoxyglucose uptake.
Values are the mean ± SEM for three experiments carried out in
triplicate, asterisks signify statistically significant changes from the
untreated sample (P < 0.01).
Fig. 5. Effects of SB-415286 on the phosphorylation of PKB and GSK3
and the stimulation of glucose transport induced by submaximal and

maximal insulin treatments in L6 muscle cells. L6 myotubes were pre-
incubated with 50 l
M
SB-415286 for 60 min prior to incubation with
insulin (1 n
M
or 100 n
M
) for 10 min (for phospho-blots) or for 30 min
(uptake assays). Cells were lysed and 50 lglysateproteinwas
immunoblotted using phospho-specific antibodies against PKB and
GSK3a/b. As a loading control, lysates were immunoblotted with an
antibody to native PKBa. The blots are representative from up to three
separate experiments. Alternatively cells following insulin treatment
were assayed for 2-deoxyglucose uptake. Values are the mean ± SEM
for three experiments carried out in triplicate, asterisks signify statis-
tically significant changes from the untreated sample (P < 0.01).
3834 K. MacAulay et al. (Eur. J. Biochem. 270) Ó FEBS 2003
to a maximally effective insulin dose. Pre-incubating L6 cells
with SB-415286 did not enhance the phosphorylation of
either kinase nor did it increase sugar uptake in response to a
submaximal insulin dose (Fig. 5). These findings are not
entirely out of line with work from rodent studies showing
that whilst GSK3 inhibition improves insulin responsiveness
in muscle of insulin resistant animals it had no insulin
potentiating effect in skeletal muscle of lean animals [39,41].
To assess whether chronic inhibition of GSK3 modifies
glucose uptake in muscle cells, we incubated L6 myotubes
chronically with SB-415286 prior to analysis of basal and
insulin-stimulated glucose uptake. However, it proved tech-

nically difficult to extend the incubation period beyond 24 h
as the integrity and plate-adherent properties of terminally
differentiated myotubes was severely compromised. Never-
theless, we observed that sustained exposure of L6 myotubes
to 50 l
M
SB-415286 for 24 h led to a small, but significant
enhancement in basal glucose uptake (basal untreated,
32.9 ± 2.8 pmolÆmin
)1
per mg protein
)1
; basal treated
47.5 ± 4.4 pmolÆmin
)1
per mg protein
)1
,valuesare
mean ± SEM from three observations). However, irres-
pective of whether cells were exposed to SB-415286, we did
not observe any potentiation in insulin stimulated glucose
uptake (insulin treatment alone, 56.7 ± 2.2 pmolÆmin
)1
per
mg protein
)1
; insulin + SB-415286, 54.7 ± 6.1 pmolÆmin
)1
per mg protein
)1

, values are mean ± SEM from three
observations). The precise mechanism underlying the
observed increase in basal glucose uptake remains poorly
understood, but it is plausible that changes in the cellular
expression of proteins regulating this process may contribute
to this phenomena as reported by Nikoulina et al.[38].
An important question that emerges from these studies
concerns the mechanism by which Li stimulates glucose
transport in muscle and fat cells. To gain some insight into
this issue we subsequently monitored the effects of a number
of inhibitors that target PI3K, the MAP kinase pathway,
p38 MAP kinase and mTOR on Li-stimulated glucose
uptake in L6 myotubes. In line with previous work [28],
Fig. 6 shows that wortmannin (a PI3K inhibitor) suppresses
basal glucose uptake by  50% and induces a complete
inhibition of insulin-stimulated glucose transport. This
latter finding is in full agreement with the widely accepted
belief that PI3K plays a critical role in the hormonal
regulation of glucose transport [1]. However, despite the fall
in basal glucose uptake the net stimulation in glucose uptake
elicited by Li was largely unaffected by wortmannin
implying that PI3K was not involved in this regulatory
response. Similar analyses, using PD-98059 and rapamycin,
excluded the involvement of the classical MAP kinase
pathway and mTOR, respectively (Fig. 6A). However, the
acute stimulation of glucose uptake by Li was virtually
abolished in the presence of SB-203580, which inhibits p38
MAP kinase [31]. Interestingly, whilst SB-203580 blocked
Li-stimulated glucose transport it had no effect on the ion’s
Fig. 6. Effects of wortmannin, SB-203580, PD-98059 and rapamycin on insulin and lithium (Li)-stimulated glucose uptake and GS activation. (A) L6

myotubes were pretreated for 5 min with 100 n
M
wortmannin, 10 l
M
SB-203580, 10 l
M
PD-98059 or 10 l
M
rapamycin prior to cell stimulation
with 100 n
M
insulin for 30 min or 50 m
M
Li for 60 min. Inhibitors were present throughout the period of incubation with insulin and Li. At the end
of these incubation periods 2-deoxyglucose was assayed as described in Materials and methods. Values are the mean ± SEM for three experiments
each performed in triplicate, asterisks signify statistically significant changes from the untreated sample (P < 0.01), whereas the double asterisk
indicates a significant change from the wortmannin-treated sample (P < 0.01). (B) For GS activity, cells were treated with 50 m
M
Li or 10 l
M
SB-
203580 for 60 min or with 100 n
M
insulin for 10 min prior to assaying incorporation of glucose from uridine diphospho-[6-
3
H]-
D
-glucose into
glycogen and expressed as a ratio of the activity in the absence divided by that in the presence of glucose-6-phosphate. Values are the mean ± SEM
for three experiments each carried out in duplicate, single asterisks signify statistically significant changes from the untreated sample, whereas the

double asterisk signifies a significant change to the wortmannin-treatment alone (P <0.01).
Ó FEBS 2003 GSK-3 and glucose metabolism (Eur. J. Biochem. 270) 3835
ability to induce a stimulation of GS (presumably via
inhibition of GSK3) in muscle cells (Fig. 6B). This latter
finding adds further support to the argument that the
increase in glucose uptake elicited by Li is likely to be
mediated by a mechanism that is distinct from that used to
stimulate GS.
The observation that SB-203580 suppresses Li-stimulated
glucose uptake implies that Li stimulates the p38 MAP
kinase pathway. The notion that Li activates this stress
signalling pathway is not unprecedented. Li has been shown
to acutely activate p38 MAP kinase in a human intestinal
epithelial cell line, HT-29, and promote the transcription of
the interleukin-8 gene [42]. In line with previous studies,
Fig. 7A shows that Li induced the phosphorylation/activa-
tion of p38 MAP kinase in L6 muscle cells and that, like the
stimulation of glucose uptake, this was suppressed by
SB-203580, but not by wortmannin. Fig. 7B shows that
phosphorylation of p38 MAPK was induced by Li in a
dose-dependent manner with maximal phosphorylation
being induced in response to 50 m
M
Li. At this concentra-
tion, Li also maximally stimulated glucose uptake in muscle
cells (Fig. 7B). It is conceivable that the use of Li at the high
concentrations that are used typically to inhibit GSK3 may
stimulate glucose transport as a result of an increase in
extracellular osmolarity. However, the finding that equi-
valent concentrations of sucrose fail to elicit any significant

increase in glucose uptake would tend to negate this
possibility (Fig. 7B).
To further investigate whether inactivation of GSK3 has
any regulatory input into the stimulation of glucose uptake
by insulin we expressed a constitutively active Myc-tagged
form of GSK3b in L6 cells in which serine 9 was mutated to
an alanine (S9A). Immunoprecipitation and immunoblot-
ting using GSK3b or Myc antibodies confirmed the
Fig. 7. Li induces p38 MAPK phosphorylation and stimulates glucose
uptake in L6 myotubes in a dose-dependent manner. (A) L6 myotubes
were stimulated with 100 n
M
insulinfor10minorpretreatedwith
100 n
M
wortmannin or 10 l
M
SB-203580 for 5 min prior to a 60-min
incubation with 50 m
M
Li. At the end of this incubation cells were
lysed and 50 lg of lysate protein immunoblotted using antibodies
against phospho-p38 MAP kinase. The same blot was reprobed with
an antibody to native PKB to establish equal loading of protein in the
different sample lanes. (B) L6 myotubes were incubated with 20 m
M
,
50 m
M
or 100 m

M
Li or sucrose for 60 min. At the end of this incu-
bation cells were lysed and 50 lg lysate protein were resolved by SDS/
PAGE and immunoblotted with a phospho-specific antibody against
p38 MAPK. Alternatively, at the end of the 60 min incubation cells
were used for assaying glucose uptake. Values are mean ± SEM from
three experiments each performed in triplicate, asterisks signify sta-
tistically significant changes from the appropriate sucrose treatment
(P < 0.01). The immunoblots are representative from three similar
experiments.
Fig. 8. Effects of insulin on glucose uptake in L6 cells expressing a
constitutively active GSK3
S9A
. L6 cells were transfected with myc-
tagged GSK3
S9A
which was immunoprecipitated using antibodies to
either c-myc or GSK3b and the immunoprecipitate probed with the
reciprocal antibody. L6 cells transfected with the empty expression
vector (L6-EV) were used as a control. L6-EV or GSK3
S9A
expressing
cells were incubated with 100 n
M
insulin for 30 min prior to assaying
glucose uptake. The uptake values are mean ± SEM for three
experiments, each conducted in triplicate. Asterisks signify a significant
change from the respective basal value (P < 0.05).
3836 K. MacAulay et al. (Eur. J. Biochem. 270) Ó FEBS 2003
expression of GSK3

S9A
in L6 cells. Whilst insulin inacti-
vated GSK3b from L6 cells transfected with the empty
expression vector by 46 ± 5% (mean ± SEM of four
experimental observations) the hormone failed to induce
any inhibition of the kinase when immunoprecipitated from
GSK3
S9A
expressing cells (data not shown). Consistent with
this observation, insulin did not stimulate GS in cells
expressing the GSK3
S9A
mutant [activity ratios (± glucose-
6-phosphate) for GS in the absence and presence of insulin
in control cells were 0.029 ± 0.01 (basal), 0.67 ± 0.01
(insulin), and in GSK3
S9A
expressing cells were 0.06 ± 0.03
(basal), 0.05 ± 0.01 (insulin)]. Nevertheless, when
GSK3
S9A
expressing cells were stimulated with insulin and
glucose uptake assayed we observed no significant differ-
ences in sugar uptake compared with cells transfected with
the empty vector (Fig. 8). This observation is consistent
with our pharmacological data and is in line with previous
work by Summers et al. who reported that whilst expression
of a GSK3
S9A
in 3T3-L1 adipocytes reduced basal glucose

uptake slightly it failed to influence insulin’s ability to
acutely stimulate glucose uptake or GLUT4 translocation
in this cell line [25].
In summary, we have shown that suppressing GSK3
activity in L6 myotubes and 3T3-L1 adipocytes, using Li or
SB-415286, is capable of stimulating GS to a level that is
comparable to that observed in response to insulin.
However, whilst clearly important for the hormonal regu-
lation of GS, our data does not support a role for GSK3 in
the acute regulation of glucose transport based on (a) the
differential effects of Li and SB-415286 on hexose uptake
and (b) the inability of a constitutively active GSK3 to
modulate insulin-stimulated glucose uptake. Nevertheless,
given that inhibition of GSK3 (using Li or SB-415286)
appears to be sufficient for inducing activation of GS in
muscle and fat cells, and that inhibition of the kinase
potentiates insulin action in muscle of insulin-resistant rats
[39], and that prolonged GSK3 inhibition not only enhances
basal glucose uptake but elevates IRS1 expression [38]
suggests that long-term manipulation of GSK3 may be of
therapeutic value in improving glucose utilization and
sensitivity of muscle and adipose tissue to insulin.
Acknowledgements
We are grateful to our colleagues in the MRC Protein Phosphorylation
Unit and the DSTT for providing some of the reagents used in this
study. We also thank D. J. Powell for technical help and useful
discussions. This work was supported by the MRC, BBSRC, Diabetes
and Wellness Research Foundation, Diabetes UK and GlaxoSmith-
Kline. K. M. is supported by a BBSRC studentship and A. B. was
supported by a MRC-CASE studentship.

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