Tài liệu Báo cáo khoa học: Arsenite stimulated glucose transport in 3T3-L1 adipocytes involves both Glut4 translocation and p38 MAPK activity docx

Arsenite stimulated glucose transport in 3T3-L1 adipocytes involves
both Glut4 translocation and p38 MAPK activity
Merlijn Bazuine, D. Margriet Ouwens, Daan S. Gomes de Mesquita and J. Antonie Maassen
Department of Molecular Cell Biology, Leiden University Medical Centre, Leiden, the Netherlands
The protein-modifying agent arsenite stimulates glucose
uptake in 3T3-L1 adipocytes. In the current study we have
analysed the signalling pathways that contribute to this
response. By subcellular fractionation we observed that
arsenite, like insulin, induces translocation of the GLUT1
and GLUT4 glucose transporters from the low-density
membrane fraction to the plasma membrane. Arsenite did
not activate early steps of the insulin receptor (IR)-signalling
pathway and the response was insensitive to inhibition of
phosphatidylinositol-3¢-kinase (PI-3¢)kinasebywortman-
nin. These findings indicate that the ÔclassicalÕ
IR–IR substrate–PI-3¢ kinase pathway, that is essential for
insulin-induced GLUT4 translocation, is not activated by
arsenite. However, arsenite-treatment did induce tyrosine-
phosphorylation of c-Cbl. Furthermore, treatment of the
cells with the tyrosine kinase inhibitor, tyrphostin A25,
abolished arsenite-induced glucose uptake, suggesting that
the induction of a tyrosine kinase by arsenite is essential for
glucose uptake. Both arsenite and insulin-induced glucose
uptake were inhibited partially by the p38 MAP kinase
inhibitor, SB203580. This compound had no effect on the
magnitude of translocation of glucose transporters indica-
ting that the level of glucose transport is determined by
additional factors. Arsenite- and insulin-induced glucose
uptake responded in a remarkably similar dose-dependent
fashion to a range of pharmacological- and peptide-inhibi-
tors for atypical PKC-k, a downstream target of PI-3¢ kinase

signalling in insulin-induced glucose uptake. These data
show that in 3T3-L1 adipocytes both arsenite- and insulin-
induced signalling pathways project towards a similar cel-
lular response, namely GLUT1 and GLUT4 translocation
and glucose uptake. This response to arsenite is not func-
tionally linked to early steps of the IR–IRS–PI-3¢ kinase
pathway, but does coincide with c-Cbl phosphorylation,
basal levels of PKC-k activity and p38 MAPK activation.
Keywords:PKC-k;PKB;PI-3¢ kinase; insulin; Cbl.
Insulin induces multiple responses in target tissues such as
adipocytes and muscle through the intracellular activation
of several signal transduction pathways. These responses
include a pronounced anabolic action on protein and lipid
metabolism, an antiapoptotic response, an increase in
glucose uptake, and stimulation of glycogen synthesis
[1,2]. Insulin-stimulated glucose uptake occurs primarily
via translocation of the GLUT4 glucose transporter to the
plasma membrane [3,4]. This process is initiated by the
activation of the insulin receptor (IR) tyrosine kinase
followed by receptor autophosphorylation and tyrosine
phosphorylation of downstream effectors like insulin
receptor substrate-1 (IRS-1), IRS-2 and related proteins.
Tyrosine phosphorylated IRS proteins provide docking
sites for class I phosphatidylinositol-3¢ (PI-3¢)kinasethat
becomes activated upon binding to these proteins [5,6].
Numerous studies have shown that PI-3¢ kinase activation
provides an essential signal for the stimulation of glucose
uptake by insulin [7,8]. Downstream targets of PI-3¢ kinase
in 3T3-L1 adipocytes that have been implicated in signalling
towards GLUT4 translocation are the AGC kinase family

members PDK1, PKB and the atypical PKC-k/-f [9–11], of
which 3T3-L1 adipocytes only express the k-isoform [12].
Recent data also demonstrate the involvement of an
additional, nonPI-3¢ kinase dependent pathway involving
c-Cbl which becomes tyrosine-phosphorylated upon APS
(adapter protein with a PH and SH2 domain)-mediated
association with the activated insulin receptor [13]. Sub-
sequently, tyrosine-phosphorylated c-Cbl translocates
towards the caveolae and induces the activation of the
small GTP-binding protein, TC10 [14], ultimately signalling
towards the exocyst complex (Exo70) involved in GLUT4
translocation [15].
Apart from insulin, some other stimuli, like muscle
contraction, H
2
O
2
and hyperosmotic shock, have been
shown to stimulate GLUT4-mediated glucose uptake in
adipocytes and muscle. Most studies show that these stimuli
are not sensitive to inhibition by wortmannin, indicating
PI-3¢ kinase is not involved in glucose uptake mediated by
these agents [16–18].
Sodium arsenite is known for its atherogenic, carcino-
genic and genotoxic effects. Recently, arsenite has also
Correspondence to J. A. Maassen, Department of Molecular Cell
Biology, Leiden University Medical Centre, Wassenaarseweg 72,
PO Box 9503, 2333 AL, Leiden, the Netherlands.
Fax: + 31 71 5276437, Tel.: + 31 71 5276127,
E-mail:

Abbreviations: IR(s), insulin receptor (substrates); IBMX, 1-methyl-
3-isobutylxanthine; PI-3¢, phosphatidylinositol-3¢-kinase;
2-DOG, 2-deoxy-
D
[
14
C]glucose; BIM I, bisindolylmaleimide I;
LDM, low density microsome; PM, plasma membrane;
TPA, 12-O-tetradecanoylphorbol 13-acetate.
(Received 18 December 2002, revised 24 July 2003,
accepted 28 July 2003)
Eur. J. Biochem. 270, 3891–3903 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03771.x
been used effectively as a chemotherapeutic drug in the
treatment of acute promyelocytic leukaemia patients
[19,20]. At the protein level, arsenite exerts its biological
effects through modification of vicinal sulfhydryl groups
in specific target proteins. For instance, arsenite specific-
ally inactivates the E2 subunit of branched-chain alpha-
keto acid dehydrogenase (but not the other subunits) [21]
and activates heat shock protein 70 [22]. Arsenite is also a
potent activator of the stress kinases, JNK and p38, by
modulating the activity of an unidentified target protein
[23]. Furthermore, arsenite has been shown to induce
glucose uptake in 3T3-L1 adipocytes, baby hamster kid-
ney cells and L6 muscle cells [24–26]. As the action of
arsenite involves the modification of a limited number of
arsenite-sensitive target proteins, we hypothesized that
elucidation of the mechanism of arsenite-induced glucose
uptake may contribute to a better understanding of
insulin-induced glucose uptake.

In this study, we observed that arsenite displays insulin-
like effects on GLUT4-mediated glucose transport in
adipocytes. To explore the underlying mechanism we
analysed the signalling pathways that are activated by
arsenite in comparison to insulin and that contribute to
stimulation of glucose uptake.
Experimental procedures
Materials
Dulbecco’s modified Eagle’s medium (DMEM) was pur-
chased from Life Technologies, Inc.; foetal bovine serum
was from Brunschwig, Amsterdam; bovine insulin,
1-methyl-3-isobutylxanthine (IBMX), dexamethasone,
12-O-tetradecanoylphorbol 13-acetate (TPA) and 2-deoxy-
glucose were obtained from Sigma. 2-deoxy-
D
-[
14
C]glucose
was purchased from NEN-Dupont. Tyrphostin A25,
SB203580, chelerythrine chloride, bisindolylmaleimide I,
Go
¨
6976, Ro 31-8220 and Ro 32-0432 were from Calbio-
chem. LY-294002, microcystin LR and wortmannin were
obtained from Alexis. Myristoylated pseudosubstrate pep-
tide inhibitors for PKC-a/-b and PKC-k/-f were purchased
from Biomol. For an overview of the characteristics of
the pharmacological inhibitors applied in this study, see
Table 1.
Antibodies

Polyclonal antisera recognizing IRS-1, and the regulatory
subunit of PI-3¢ kinase were described previously [27].
Table 1. Characteristics of pharmacological inhibitors applied in this study.
Pharmacological inhibitor Concentration applied Described target IC
50
Reference
Bisindolyl-maleimide I 5 l
M
PKC-a, bI, bII, c, d, e 10–100 n
M
Calbiochem
5-hydroxytryptamine3 receptor 7 n
M
[49]
Glycogen synthase kinase 3b 170 n
M
[50]
K(ACh)channels 100 n
M
[51]
MAPK activated protein-kinase 1b 50 n
M
[52]
p70 S6 kinase 100 n
M
[52]
Mitogen and stress activated kinase-1 <1 l
M
[53]
AMP-activated protein kinase 1 l

M
[53]
Phosphorylase kinase 1 l
M
[53]
Chelerythrine Chloride 10 l
M
PKC 660 n
M
Calbiochem
a
K(ACh) channels 490 n
M
[51]
Go
¨
6976 100 n
M
Trk A and B 10–100 n
M
[54]
Conventional PKC-a 2.3 n
M
[55]
Conventional PKC-bI 6.2 n
M
[55]
PKC-l (PRK) 20 n
M
[56]

LY 294002 10 l
M
Phosphatidylinositol 3¢-kinase 1.4 l
M
[57,58]
Casein kinase 2 6.9 l
M
[53]
Ro31–8220 0–20 l
M
Conventional and novel PKC 10–100 n
M
[11,59,60]
Atypical PKC-k 5 l
M
This study
Atypical PKC-f 1 l
M
in vitro
4 l
M
in vivo
[11]
Glycogen synthase kinase 3b 38 n
M
2.8 n
M
[53]
[50]
MAPK activated protein-kinase 1b 3 n

M
[52]
p70 S6 kinase 15 n
M
[52]
Mitogen and stress activated protein kinase 8 n
M
[53]
Ro32–0432 10 l
M
Conventional PKC-a 9n
M
[61]
Conventional PKC-bI28n
M
[61]
Novel PKC-e 108 n
M
[61]
SB203580 10 l
M
p38 MAP kinase (a and b) 0.6 l
M
[62,63]
Tyrphostin A25 25 l
M
Tyrosine kinases EGF 3 l
M
[64,65]
Wortmannin 100 n

M
Phosphatidylinositol 3¢-kinase 2–5 n
M
[66,67]
a
Disputed by Davies et al. [53].
3892 M. Bazuine et al. (Eur. J. Biochem. 270) Ó FEBS 2003
A polyclonal antiserum recognizing IRS-2 is described in
a recent paper from our group [28]. HRP-conjugated
mouse monoclonal anti-(phosphotyrosine Ig) pY-20,
mouse monoclonal pY-20, rabbit polyclonal antibody
against PKC-k/-f (C-20) and polyclonal against Cbl
(C-15), goat polyclonal antibodies recognizing the cata-
lytic (p110a) subunit of PI-3¢ kinase (C-17) and GLUT4
(C-20) were obtained from Santa Cruz Biotechnology,
Inc. Rabbit polyclonal antibody recognizing IRb-chain
and mouse monoclonal integrin b1, Cbl and PKC-k were
purchased from Transduction Laboratories. The phospho-
specific antibodies recognizing PKC-k (T403), caveolin-1
(Y14), PKB (T308 and S473), MAPKAP-K2 (T334), p38
(T180/Y182) and ERK-1/)2 (T202/Y204) were obtained
from Cell Signalling Technology. Sheep polyclonal anti-
body recognizing PKB was purchased from Upstate
Biotechnology. The rabbit polyclonal antibodies against
GLUT-1 and -4 (used in the PM-lawn assay) have been
described [29]. The appropriate HRP- and FITC-
conjugated secondary antibodies were obtained from
Promega.
Cell culture
3T3-L1 Fibroblasts, obtained from American Type Cul-

ture Collection (Manassas, VA, USA), were cultured and
differentiated to adipocytes as described previously [30].
Cells were routinely used 7 days after completion of the
differentiation process, with only cultures in which >95%
of cells displayed adipocyte morphology being used. Prior to
use, adipocytes were serum starved for 16 h with DMEM
supplemented with 0.5% foetal bovine serum.
Membrane isolation assay
3T3-L1 adipocytes were stimulated as indicated in the figure
legends. Subsequently cells were washed twice in ice-cold
HES buffer (20 m
M
Hepes pH 7.4, 1 m
M
EDTA and
250 m
M
sucrose) on ice and scraped in HES buffer in the
presence of protease inhibitors (complete protease inhibitor
cocktail, Boehringer Mannheim). Samples were homogen-
ized by nine times three strokes in a glass potter homo-
genizer after which low density microsome (LDM) and
plasma membrane (PM) fractions were isolated by differ-
ential centrifugation as described by Simpson et al.[31].
Equal amounts of protein as determined with BCA
protein assay reagent (Pierce) were subjected to immunoblot
analysis using various antibodies.
Plasma membrane-lawn assay
The plasma membrane-lawn assay was performed as
described previously [32]. Digital fluorescence imaging was

performed using a Leica DM-RXA epifluorescence micro-
scope (Leica, Germany) equipped with a 100-W mercury
lamp and the appropriate filters.
Assay of 2-deoxyglucose uptake
3T3-L1 adipocytes, grown in 12-well plates (Costar), were
subjected to an assay of 2-deoxy-
D
-[
14
C]glucose (0.075 lCi
per well) uptake as described previously [33].
Immunoprecipitations and Western blotting
Dishes (9 cm) of 3T3-L1 adipocytes were stimulated with
agonists. Immunoprecipitation and immunoblotting pro-
cedures were as described previously [27]. For c-Cbl
immunoprecipitation 9-cm dishes of 3T3-L1 adipocytes
were stimulated with agonists and scraped in lysis buffer
(1 m
M
Na
3
VO
4
,1m
M
EGTA, 1 m
M
EDTA, 50 m
M
Tris/HCl pH 7.4, 1% NP-40, 0.5% sodium deoxycholate,

150 m
M
NaCl, 5 m
M
NaF in the presence of protease
inhibitors). Cell lysates were tumbled for half an hour at
4 °C, cell lysate was cleared from cellular debris by spinning
at 14 000 g,for10minat4°C in a table-top centrifuge.
About 1 mg of cell lysate was subjected to immunopreci-
pitation using 5 lg of anti-Cbl mouse monoclonal 7G10
(UBI) for 1.5 h at 4 °C. Immunocomplexes were harvested
by incubating with ProtG beads for 1.5 h at 4 °C. Beads
were washed in lysis buffer and dissolved subsequently in
sample buffer. Phosphotyrosine was demonstrated by
immunoblotting using anti-pY20 followed by anti-(mouse
HRP) secondary Ig. Immunoblots were quantified using
LUMIANALYST
software on a LumiImager (Boehringer-
Mannheim).
PI-3¢ kinase activity assay
Dishes (9 cm) of 3T3-L1 adipocytes were stimulated with
agonists and immunoprecipitated using time, concentration
and antibodies as indicated in the figure legends. Cells were
lysed and IRS-1 and p85 immunoprecipitates collected on
protein A–Sepharose beads were analysed for the copreci-
pitation of in vitro PI-3¢ kinase activity using 5 lCi c-
32
P-
labelled ATP per reaction as described by Burgering et al.
[34]. Incorporated radioactivity was quantified on a

Molecular Dynamics phosphorimager.
PKC-k kinase assay
Dishes (9 cm) of 3T3-L1 adipocytes were stimulated with
100 n
M
of insulin for 10 min or 0.5 m
M
arsenite for 30 min.
Cells were lysed in an NP-40 based lysis buffer (see above) in
the presence of 1 l
M
microcystin LR and immunopreci-
pitated with 5 lg mouse monoclonal PKC-k for 1.5 h at
4 °C. Subsequently, Prot-G beads were added and com-
plexes were harvested after another 1.5 h. The precipitate
was washed three times with lysis buffer and two times with
kinase assay buffer (100 m
M
Hepes pH 7.4, 10 m
M
MgCl
2
,
1m
M
dithiothreitol). PtdSer (4 lg per sample) was dried
under N
2(g)
and dissolved in 25 lL kinase buffer per sample.
Subsequently, PtdSer was waterbath-sonicated three times

for 5 min and 25 lL sample kinase buffer, ATP (40 l
M
,
final concentration), 5 lCi c-
32
P-labelled ATP per reaction,
dithiothreitol (1.5 m
M
), PKI (1 m
M
), the indicated concen-
trations of Ro 31-8220, and PKCe-substrate (40 l
M
)were
added. As a control, 10 l
M
of a peptide identical to the
PKC-k pseudosubstrate domain was added to determine
the specificity of the assay. Kinase reactions were allowed
to proceed for 10 min at 37 °C under gentle agitation.
Twenty microlitres of each reaction was spotted on p81
paper and washed three times for 5 min with 0.85% (v/v)
phosphoric acid, and once for 5 min with acetone. P81
papers were air dried and analysed in a scintillation counter.
Ó FEBS 2003 Arsenite induced glucose-uptake (Eur. J. Biochem. 270) 3893
Statistical analyses
Data were analysed with an independent-samples t-test
using
SPSS
10.0. Curves represent fits to data by nonlinear

regression analysis using
GRAPHPAD PRISM
2.01.
Results
Arsenite induces 2-DOG uptake, GLUT1 and GLUT4
translocation in 3T3-L1 adipocytes
Incubation of 3T3-L1 adipocytes with arsenite stimulated
the uptake of hexose in a time- and dose-dependent manner.
Stimulation of 2-deoxy-
D
[
14
C]glucose (2-DOG) uptake was
maximal at a 30-min preincubation period with 0.5 m
M
arsenite (Fig. 1A,B). On average, maximal stimulation of
glucose uptake was approximately sevenfold for insulin and
threefold for arsenite. When insulin was added to adipocytes
during the final 15 minutes of a 30-min incubation with
arsenite, no significant additive response was seen on
arsenite-induced glucose uptake (Fig. 1C). Under these
conditions, insulin did induce tyrosine phosphorylation of
IRb, IRS-1 and IRS-2, indicating that arsenite does not
interfere with the insulin-induced activation of this pathway
(data not shown).
Insulin-stimulated glucose transport predominantly
involves GLUT4 translocation from an intracellular
microsomal compartment to the plasma membrane of
adipocytes along with some induction of GLUT1 trans-
location. To determine whether arsenite stimulates

GLUT1 and GLUT4 translocation, we fractionated
adipocytes into membrane (PM) and microsomal vesicle
(LDM) fractions. Equal amounts of protein ( 10 lg)
were subjected to immunoblot analysis (Fig. 2A,C).
Plasma membrane fractions were identified using antibod-
ies against the IR b-chain and integrin-b1 (data not
shown). As can be seen in Fig. 2A, when probing the
fractions with an antibody against GLUT4, both insulin-
and arsenite-treatment resulted in a shift of GLUT4 from
the microsomal fractions towards the plasma membrane
fractions. Figure 2C shows that insulin induces some
translocation of GLUT1 towards the plasma membrane,
as did arsenite, albeit at lower levels than insulin. The
amounts of GLUT1 and GLUT4 in each fraction were
quantified and expressed as a relative amount of total
GLUT protein in these fractions. Arsenite significantly
increases the amount of GLUT4 in the PM (Fig. 2B),
albeit at a lower level than insulin. With respect to
GLUT1, although a consistent increase in the amount of
GLUT1 translocating towards the plasma membrane was
observed this did not reach significant levels compared to
basal levels of GLUT1 in the PM (Fig. 2D). Furthermore,
arsenite did not change the total amount of GLUT4 or
GLUT1 in 3T3-L1 adipocytes (data not shown). It should
be noted that both GLUT1 and GLUT4 are heavily
glycosylated and show heterogeneous mobility.
An alternative method to investigate GLUT protein
translocation is the plasma-lawn assay. In this analysis,
sonicated cells are probed with an antibody recognizing
GLUT1 or GLUT4 and subjected to immunofluorescnce

microscopy. As can be seen in Fig. 2E, both GLUT1 and
GLUT4 are present in PM-lawns at higher quantities
after arsenite treatment as compared to the unstimulated
situation.
Combined, these data indicate that arsenite-stimulated
glucose uptake involves translocation of the GLUT1 and
the insulin-responsive GLUT4 glucose transporter.
The effect of arsenite on early events in insulin
receptor signalling
To elucidate the signalling-pathways that contribute to
arsenite-induced GLUT4 translocation, we examined
Fig. 1. Arsenite induces glucose uptake in 3T3-L1 adipocytes in a dose-
and time-dependent manner. (A) 3T3-L1 adipocytes were stimulated
with the indicated concentrations of arsenite for 30 min and assayed
for 2-deoxy-
D
[
14
C]glucose (2-DOG) uptake. (B) 3T3-L1 adipocytes
were stimulated with 0.5 m
M
arsenite for the indicated times and
assayed for 2-DOG uptake. (C) 3T3-L1 adipocytes were stimulated as
indicated with 100 n
M
insulin for 15 min (ins), 0.5 m
M
arsenite for
30 min (as), or 0.5 m
M

arsenite for 30 min combined with 100 n
M
insulin added after 15 min (as/ins) and assayed for 2-DOG uptake.
Incorporated radioactivity was determined by liquid scintillation
counting. Values are mean ± SEM of at least four determinations;
*P < 0.05 compared to basal and P <0.05 for as/ins compared
to ins.
3894 M. Bazuine et al. (Eur. J. Biochem. 270) Ó FEBS 2003
whether arsenite activates intermediates of the insulin
signalling-pathway. Following stimulation of 3T3-L1 adi-
pocytes with either insulin or arsenite, IR, IRS-1 and IRS-2
were immunoprecipitated and assessed for tyrosine phos-
phorylation by Western blotting. As shown in Fig. 3A–C,
no significant increase in tyrosine phosphorylation of either
IRb, IRS-1 or IRS-2 could be detected in response to
arsenite. Under these conditions, stimulation with insulin
led to a pronounced tyrosine phosphorylation of these
proteins. In agreement with the lack of IRS-tyrosine
phosphorylation in response to arsenite, no association of
the p85 or p110a subunits of PI-3¢ kinase with the IRS
proteins was detected, again in contrast to the situation seen
after insulin stimulation (Fig. 3B). Treatment with arsenite
did not lead to phosphorylation of PKB on either Ser473 or
Thr308, nor of phosphorylation of PKC-k on T403. This
observation agrees with the absence of PI-3¢ kinase activa-
tion by arsenite in vivo (Fig. 3D). Consistent with these
observations, no increase of in vitro PI-3¢ kinase activity
was observed in IRS-1 immunoprecipitates after arsenite
treatment (Fig. 4A), nor did we find any arsenite-induced
stimulation of in vitro PI-3¢ kinase activity in immuno-

precipitates of PI-3¢ kinase (Fig. 4B).
To investigate the possibility that basal PI-3¢ kinase
activity in combination with arsenite-induced signals is
needed to stimulate glucose uptake, we examined the effect
of the PI-3¢ kinase inhibitor, wortmannin, on arsenite-
induced glucose uptake. Using concentrations that fully
inhibited insulin-induced glucose uptake, arsenite-induced
glucose uptake was unaffected by wortmannin (Fig. 4C).
Similar data were obtained using LY-294002 (data not
shown). These observations suggest that arsenite-induced
glucose uptake occurs without the need for PI-3¢ kinase
activity, a situation that is in marked contrast to insulin
induced glucose uptake. Another early target of insulin
action, the activation of ERK-1, -2 was also not activated
appreciably in response to arsenite-treatment either
(Fig. 3D).
The effect of arsenite on Cbl and caveolin-1 tyrosine
phosphorylation
A recently described PI-3¢ kinase independent pathway
involved in insulin-induced glucose uptake in 3T3-L1
adipocytes involves Tyr phosphorylation of c-Cbl and
caveolin-1 mediated by the IR. Remarkably, we found
Fig. 2. Arsenite-treatment induces GLUT1 and GLUT4 translocation to the plasma membrane in 3T3-L1 adipocytes. 3T3-L1 adipocytes were mock-
treated(basal),stimulatedfor15minwith100n
M
insulin (insulin/ins) or for 30 min with 0.5 m
M
arsenite (arsenite/as). (A) Adipocytes were
fractionated and equal amounts of protein from both microsomal (LDM) and plasma membranes (PM) were analysed by immunoblot with anti-
GLUT4 Igs. (B) GLUT4 levels in each fraction subjected to immunoblot analysis as in A were quantified using a LumiImager and expressed as a

fraction of GLUT4 residing in either LDM or PM. (C) Subcellular fractions as in (A) were also subjected to immunoblot analysis using antibodies
against GLUT1. (D) GLUT1 levels in each fraction were quantified as in (B) and expressed as a fraction of GLUT1 residing in either LDM or PM.
The total amount of GLUT1 or GLUT4 in the HDM fraction did not alter during either arsenite- or insulin-treatment. Data are expressed as
mean ± SEM of at least three independent observations, *P < 0.05 compared to basal. (E) 3T3-L1 adipocytes were mock-treated (none),
stimulated for 15 min with 100 n
M
insulin (ins) or for 30 min with 0.5 m
M
arsenite (as). Adipocytes were subjected to PM-lawn analysis using
antibodies against either GLUT1 or GLUT4. Data shown are a representative example of five independent observations for each condition.
Ó FEBS 2003 Arsenite induced glucose-uptake (Eur. J. Biochem. 270) 3895
that arsenite did induce tyrosine-phosphorylation of both
c-Cbl (Fig. 5B) and caveolin-1 (Fig. 5A), in spite of the
absence of IR activation. This observation suggests that
arsenite does induce an as of yet unidentified tyrosine
kinase activity in 3T3-L1 adipocytes. To evaluate whether
this tyrosine kinase activity is required for arsenite-induced
glucose uptake, we applied the tyrosine kinase inhibitor,
tyrphostin A25. Insulin-induced IRS and caveolin-1 tyro-
sine phosphorylation are attenuated, but still present after
pretreatment with tyrphostin A25 (Fig. 5A). Insulin-
induced Cbl phosphorylation is even enhanced by tyr-
phostin (Fig. 5B). In contrast, arsenite-induced Cbl
and caveolin-1 phosphorylation are strongly inhibited
(Fig. 5A,B). In a glucose uptake assay, tyrphostin A25
attenuated insulin-induced glucose uptake, but completely
blocked arsenite-induced glucose uptake (Fig. 5C). These
data illustrate the distinct nature of the insulin- (namely
the IR) and arsenite-induced tyrosine kinases, and fur-
thermore they suggest that tyrosine kinase activity is a

requirement for arsenite-mediated induction of glucose
uptake in 3T3-L1 adipocytes.
The effect of the p38 MAPK inhibitor, SB203580,
on arsenite-induced glucose uptake
SB203580 is a pharmacological inhibitor of the MAP kinase
family member p38 and has been shown to inhibit insulin
induced glucose uptake by 3T3-L1 adipocytes and L6
muscle cells [35,36]. Arsenite-treatment induced p38-phos-
phorylation on Thr180 and Tyr182, and phosphorylation of
MAPKAP-K2 on Thr334 (a direct target site of p38 MAP
kinase activity [37]). Treatment with 10 l
M
SB203580
significantly inhibited arsenite-induced glucose uptake
as well as MAPKAP-K2 and p38-phosphorylation
(Fig. 6A,B).
In insulin-signalling, p38 MAPK has been implied in
enhancing the intrinsic activity of the GLUT4 glucose
transporter. Thus, SB203580 has been shown to reduce
insulin-induced glucose uptake without an effect on insulin-
induced GLUT4 translocation [35]. SB203580 had a similar
Fig. 4. Arsenite-induced in vitro PI-3¢ kinase activity. 3T3-L1 adipo-
cytes were stimulated as indicated with 100 n
M
insulin for 5 min or
0.5 m
M
arsenite for 30 min. Cell lysates were incubated for 3 h with
polyclonal antiserum against IRS-1 (A), or against the 85-kDa regu-
latory subunit of PI-3¢ kinase (B). Immunoprecipitates were washed

and subsequently subjected to an in vitro PI-3¢ kinase assay. Copre-
cipitating PI-3¢ kinase activity was determined on a phosphorimager as
the relative stimulation of [c-
32
P]ATP incorporation into phosphati-
dylinositol standardized against untreated cells. Data are expressed as
the mean ± SEM of three observations, statistically significant com-
pared to basal (*P < 0.05). (C) 3T3-L1 adipocytes were pretreated for
15 min with 100 n
M
wortmannin. Subsequently, adipocytes were
mock-treated (basal) or stimulated as indicated with 100 n
M
insulin for
15 min or with 0.5 m
M
arsenite for 30 min in the continued presence of
the pharmacological inhibitor and assayed for 2-DOG uptake. Data
are expressed as mean ± SEM of at least six observations. Statistically
significant data when compared to the samples not treated with
wortmannin are indicated (*P <0.05).
Fig. 3. The effect of arsenite on the activation status of early steps in
insulin-responsive signal-transduction pathways. 3T3-L1 adipocytes
were stimulated as indicated with 100 n
M
insulin for 5 min or 0.5 m
M
arsenite for 30 min. Cell lysates were immunoprecipitated with anti-IR
(A), anti-IRS-1 (B), anti-IRS-2 (C) followed by immunoblot analysis
with anti-phosphotyrosine (ap-Tyr), anti-PI-3¢ kinase regulatory sub-

unit (ap85) or anti-PI-3¢ kinase catalytic subunit (ap110a)asindicated.
Equal loading was confirmed using the respective antibodies used for
immunoprecipitation. (D) Total cell lysate of adipocytes (10 lg) sti-
mulated as described above was analysed by immunoblot using
phosphospecific antibodies against T202/Y204 of ERK-1/2 (apERK),
T308 and S473 of PKB (apThr308 and apSer473), T403 of PKC-k
(apPKCk)orPKB(aPKB) as indicated.
3896 M. Bazuine et al. (Eur. J. Biochem. 270) Ó FEBS 2003
effect on arsenite-induced glucose uptake (Fig. 6; compare
A with C,D) i.e. a reduction in glucose uptake without a
reduction in GLUT4 translocation.
The effect of pharmacological inhibitors
of PKC-isoforms on arsenite-induced glucose uptake
Atypical PKC isoforms (PKC lambda and zeta) have been
implicated in insulin induced glucose uptake in adipocytes
[11,12]. We compared the effect of a number of pharmaco-
logical inhibitors for various PKC isoforms on arsenite- and
insulin-induced glucose uptake in 3T3-L1 adipocytes. Ro 31-
8220 inhibited insulin- and arsenite-induced glucose uptake
with similar dose–response relations. More precisely, an IC
50
value of approximately 5 l
M
was found for the inhibition
of insulin- and arsenite-induced glucose (Fig. 7A). When
PKC-k was purified by immunoprecipitation and subjected
to an in vitro kinase assay, a similar dose dependency for the
inhibition of PKC-k (an IC
50
of 5 l

M
) was observed
(Fig. 7B). Co-incubation with 50 l
M
of a peptide resembling
the PKC-k pseudosubstrate domain reduced
32
P incorpor-
ation by 90%, demonstrating the specificity of the assay. In
support of the data presented in Fig. 3D, arsenite did not
induce PKC-k activation over basal levels. Hence, arsenite
appears to require basal levels of PKC-k in conjunction with
other signals to induce glucose uptake.
In addition the PKC-inhibitors, chelerythrine chloride,
Ro 32-0432, bisindolylmaleimide I (BIM I) and Go
¨
6976
were studied at concentrations well above the IC
50
values
for their respective conventional and novel PKC target
proteins (Table 1). All inhibitors reduced TPA-induced
ERK phosphorylation in 3T3-L1 adipocytes, demonstra-
ting their functional interference with PKC (Fig. 7C). Their
effects on arsenite- and insulin-induced glucose uptake were
minimal (Fig. 7D) (although BIM I had a small but
significant inhibitory effect), demonstrating that neither
conventional nor novel PKC isoforms are involved in
arsenite- or insulin-induced glucose uptake.
The effect of myristoylated PKC-k/-f and PKC-a/-b

pseudosubstrate peptides on arsenite-induced
glucose uptake
To substantiate the observations made using Ro 31-8220 we
investigated the effect of myristoylated peptide-inhibitors
for PKC [11] on insulin- and arsenite-induced glucose
uptake. As can be seen in Fig. 8B, a myristoylated peptide
with a sequence similar to the pseudosubstrate domain of
the atypical PKCs was capable of inhibiting insulin- as well
as arsenite-induced glucose uptake. A peptide resembling
the pseudosubstrate domain of conventional PKC-a/-b had
no significant inhibitory effect on either insulin- or arsenite
induced glucose uptake (Fig. 8B), whereas it did block
TPA induced ERK phosphorylation demonstrating its
functionality (Fig. 8A). These observations corroborate
the observations made with Ro 31-8220.
Discussion
Insulin-induced glucose uptake by adipocytes is determined
by multiple factors, including: the translocation of glucose
transporters from intracellular sites to the plasma mem-
brane, expression levels of individual members of the
glucose transporter family and by modulating the intrinsic
activity (or, degree of occlusion) of glucose transporters
(Fig. 9).
Fig. 5. The involvement of tyrosine kinase activity in arsenite-induced
glucose uptake. 3T3-L1 adipocytes were pretreated with for 15 min
with 25 l
M
tyrphostin A25 as indicated. Subsequently, adipocytes
were mock-treated (-), stimulated for 5 min with 100 n
M

insulin (INS)
or for 30 min with 0.5 m
M
arsenite (As) in the continued presence of
the pharmacological inhibitor. (A) Total cell lysate (10 lg) was sub-
jected to immunoblot analysis using antibodies against phosphoY14 of
caveolin-1 (ap-Cav1), phosphotyrosine (ap-Tyr) (shown are the IRS-
bands at 180 kDa) and IRS-1 (aIRS) for equal loading. (B) 3T3-L1
adipocytes treated as described above were immunoprecipitated using
antibodies against c-Cbl followed by immunoblot analysis using
antibodies against phosphotyrosine (ap-Tyr) or Cbl (ac-Cbl). (C) 3T3-
L1 adipocytes were pretreated for 15 min with 25 l
M
tyrphostin A25.
Subsequently, adipocytes were mock-treated (basal), stimulated for
15 min with 100 n
M
insulin or for 30 min with 0.5 m
M
arsenite in the
continued presence of the pharmacological inhibitor and assayed for
2-DOG uptake. Data are expressed as mean ± SEM of at least six
observations. Statistically significant (*P < 0.05) when compared to
the samples not treated with tyrphostin. Statistically significant
(P < 0.05) when compared to the basal (or arsenite) samples treated
with tyrphostin A25.
Ó FEBS 2003 Arsenite induced glucose-uptake (Eur. J. Biochem. 270) 3897
Arsenite is a protein modifying agent known to react with
sulfhydryl groups in a discrete number of proteins. As a
result these proteins are modified in their function and this

can couple back to altered activity of signal transduction
pathways.
In this report we demonstrate that arsenite displays
insulin-mimicking effects in 3T3-L1 adipocytes: thus, arse-
nite stimulates 2-DOG uptake and induces translocation of
the insulin-responsive GLUT4 glucose transporter from the
low-density microsomal fraction towards the plasma mem-
brane. Comparable to other stress-inducing agents [38,39]
arsenite acutely blocks insulin-induced glucose uptake
(Fig. 1C), however, and in contrast to oxidative and
osmotic stress, arsenite did not interfere with early events
in insulin-induced signalling. A normal level of phosphory-
lation of IRS-1,2 and PKB was observed by insulin-
induction after incubation with arsenite (data not shown).
Whereas PI-3¢ kinase activity is pivotal for insulin-
induced GLUT4 translocation, arsenite increases the uptake
of 2-DOG without the need for PI-3¢ kinase activity as
judged from the absence of an effect of arsenite on
PI-3¢ kinase activation and the lack of inhibition by either
wortmannin or LY 294002. Furthermore, arsenite does not
activate other signalling steps normally activated in response
to insulin, such as IR tyrosine kinase, IRS-1 and IRS-2
tyrosine phosphorylation, phosphorylation of PKC-k on
T403 or phosphorylation of PKB on either Ser473 or
Thr308. These observations suggest that the target of
arsenite action resides downstream of PI-3¢ kinase or in a
separate pathway.
API-3¢ kinase-independent pathway in insulin-induced
GLUT4 translocation has recently been identified and
involves tyrosine phosphorylation on several residues of

the proto-oncogene c-Cbl [13,40] and caveolin-1 [41] by
the activated insulin receptor. Arsenite also induces c-Cbl
and caveolin-1 tyrosine phosphorylation in 3T3-L1 adipo-
cytes, however, given that arsenite does not activate the
insulin receptor (Fig. 3A) the two processes are mechan-
istically different. The distinct nature of the insulin- and
arsenite-induced tyrosine kinase activities is illustrated by
the effects of tyrphostin A25. Whereas insulin-induced
tyrosine kinase activity was attenuated (and Cbl-tyrosine
phosphorylation levels even potentiated), all arsenite-
induced tyrosine phosphorylation was strongly reduced.
The effects of tyrphostin A25 on insulin- and arsenite-
induced glucose uptake mirrored these observations, i.e.,
insulin-induced glucose uptake was attenuated whereas
arsenite-induced glucose uptake was lost. These data also
demonstrate that a tyrosine kinase activity is apparently
required for the induction of glucose uptake by arsenite,
Fig. 6. The effect of the p38 MAP kinase inhibitor, SB203580, on arsenite-induced glucose uptake and translocation of GLUT4. 3T3-L1 adipocytes
were pretreated for 30 min with 10 l
M
SB203580 (A–D). Subsequently adipocytes were mock-treated (basal) or stimulated as indicated with
100 n
M
insulin for 15 min (insulin) or with 0.5 m
M
arsenite for 30 min (arsenite) in the continued presence of the pharmacological inhibitor and
assayed for 2-DOG uptake (A). Data are expressed as the mean value ± SEM of at least six observations. Statistically significant (*P <0.05)when
compared to samples without SB203580. (B) 3T3-L1 adipocytes were lysed and subjected to immunoblot analysis using antibodies against p38
MAPK (p38), phospho-specific antibodies against p38 (p-p38) and MAPKAP-K2 (p-MAPKAP-K2). (C) 3T3-L1 adipocytes treated as described
above were subjected to cell fractionation and the effect of SB203580 on GLUT4 translocation was determined by immunoblotting followed by

quantification in a lumni-imager as described for Fig. 2B. Samples pretreated with SB203580 are indicated with SB. Data are expressed as
the mean ± SEM of three independent experiments. (D) Representative immunoblot probed with anti-GLUT4 Igs, used to obtain the data
described in C.
3898 M. Bazuine et al. (Eur. J. Biochem. 270) Ó FEBS 2003
as is the case for insulin. The identity of the tyrosine-
kinase activity activated in response to arsenite remains to
be resolved.
The pharmacological p38 MAPK inhibitor, SB203580,
affects insulin-induced glucose transport by affecting
intrinsic GLUT4 activity [35,36]. Our results with this
inhibitor confirmed this observation. Furthermore, we
demonstrate a similar effect on arsenite-induced glucose
uptake. Pre-treatment with 10 l
M
SB203580 had no
effect on GLUT4 (or GLUT1) translocation but did
reduce arsenite-induced glucose uptake by  30%. Thus,
our data show a similar contribution of p38-MAPK
activity in combination with GLUT4 translocation in
insulin- and arsenite-induced glucose uptake at the level
of modulating the GLUT4 mediated transport activity.
Though we cannot fully exclude a similar effect of
SB203580 on GLUT1 as well, this seems unlikely given
that SB203850 had no effect whatsoever on arsenite- or
insulin-induced glucose uptake levels in 3T3-L1 preadipo-
cytes (expressing GLUT1 and no GLUT4) (data not
shown).
The term coined for the modulatory effect of SB203850
is Ôintrinsic activityÕ [35], possibly by altering the speed of
transition between ÔoutwardÕ and ÔinwardÕ conformations

of the transporter [42]. The term ÔocclusionÕ has been used
to describe a state in which the GLUT4 transporter is
fully inserted to the plasma membrane, but incapable of
binding and/or transporting glucose yet [43]. This could
be due to associating proteins blocking glucose transport,
differences in LDM-derived and PM-membrane compo-
sition and/or a conformational change in the GLUT4-
transporter that is required for its activation. If SB203580
hampers the progress through these stages, similar
consequences are expected (i.e., GLUT4 being present
in the plasma membrane, but less glucose being taken
up). Arsenite-induced glucose uptake, which demonstrates
a similar sensitivity to SB203580, may provide an
additional tool for future research addressing these
models.
Cellular stresses like hypoxia [44] and hyperosmolarity
[45] increase glucose uptake through an upregulation of the
amount of GLUT1. Arsenite in contrast, does not increase
Fig. 7. The effect of PKC-inhibitors on arsenite-induced glucose uptake. 3T3-L1 adipocytes were incubated with the indicated concentrations of
Ro 31-8220 for 30 min prior to stimulation. Subsequently, adipocytes were mock-treated (basal), stimulated with 100 n
M
insulin for 15 min
(insulin) or 0.5 m
M
arsenite for 30 min (arsenite) in the continued presence of Ro 31-8220. (A) 2-DOG uptake was assayed and data are expressed
as mean ± SEM of two independent experiments each performed in triplicate. (B) In vitro kinase assay performed in the continued presence of
Ro 31-8220. Incorporated counts (in k c.p.m.) are expressed as mean ± SEM of two independent experiments each performed in duplicate. (C,D)
3T3-L1 adipocytes were pretreated for 30 min with 0.1 l
M
Go

¨
6976, 5 l
M
bisindolylmaleimide I (BIM I), 10 l
M
chelerythrine chloride (Chel-
erythrine), or 10 l
M
Ro 32–0432 as indicated. Subsequently, 3T3-L1 adipocytes were stimulated with 100 n
M
TPA for 15 min and analysed for
ERK-1/2 phosphorylation (C). 2-DOG uptake was tested in a separate experiment (D). After pretreatment with the indicated pharmacological
inhibitors, adipocytes were mock-treated (basal), stimulated with 100 n
M
insulin for 15 min (insulin), or 0.5 m
M
arsenite for 30 min (arsenite) in the
continued presence of the inhibitors. 2-DOG uptake was assayed and data are expressed as mean ± SEM of at least two independent experiments
each performed in triplicate, statistically significant compared to the uninhibited samples (*P < 0.05).
Ó FEBS 2003 Arsenite induced glucose-uptake (Eur. J. Biochem. 270) 3899
the amount of GLUT1. Indeed, treatment of the adipocytes
with the protein synthesis inhibitors cycloheximide or
emetine had no effect on arsenite-induced glucose uptake
(data not shown). Furthermore, the time-course of arsenite-
induced glucose uptake, being maximal after 30 min and
declining thereafter (Fig. 1B) already seems to argue against
de novo synthesis of GLUT-transporters in mediating
arsenite-induced glucose uptake.
Although most GLUT1 is already localized in the plasma
membrane of an unstimulated adipocyte [46], some GLUT1

is known to cotranslocate with GLUT4 [47] and Fig. 2
C,D,E. Moreover, treatment of 3T3-L1 adipocytes with
TPA induced the specific translocation of GLUT1 and not
GLUT4 towards the plasma membrane of 3T3-L1 adipo-
cytes [48]. Arsenite in contrast, induces the translocation of
GLUT1 at about half the levels obtained with insulin.
Though failing to reach statistical significance (Fig. 2D) this
effect was consistently reproducible (e.g. Fig. 2C,E). Thus,
clearly, arsenite differs from other types of cellular stress in
that it projects towards a more insulin-like response (i.e.,
translocation of GLUT1 and 4) albeit at a lower level of
efficiency.
When applying multiple pharmacological- and peptide-
inhibitors for several PKC isoforms we observed a common
pattern of inhibition for insulin- and arsenite-induced
glucose uptake and similar concentration dependencies for
the various agents. Most notably, Ro 31-8220 inhibits both
insulin- and arsenite-induced glucose uptake with an IC
50
of
5 l
M
suggesting an involvement of atypical PKCs. The IC
50
of atypical PKC for BIM I is 5.8 l
M
, hence the significant
reduction in glucose uptake measured using 5 l
M
(Fig. 7D)

fits with the observations made with Ro 31-8220. Inhibitors
against conventional or novel PKCs such as Go
¨
6976,
chelerythrine chloride and Ro 32-0432 (a compound struc-
turally related to Ro 31-8220) (Table 1) remained without
effect, or acted even in a slightly potentiating manner.
Furthermore, both insulin- and arsenite induced glucose
uptake was inhibited by treatment with a myristoylated
PKC-k/f pseudosubstrate peptide, but not significantly
sensitive to treatment with a PKC-a/b pseudosubstrate. A
formal exclusion of other intracellular targets with similar
sensitivities to the inhibitors mentioned cannot, however, be
excluded.
In the case of arsenite-induced glucose uptake, the
myristoylated PKC-k/f pseudosubstrate peptide inhibited
this response by approximately 50% (Fig. 8B). Remark-
ably, when the effect of Ro 31-8220 on arsenite-induced
GLUT4 translocation was determined a similar reduction in
GLUT4 translocation was observed (data not shown). This
is in contrast to the situation in response to insulin, where
the inhibition is complete.
Another observation was that in contrast to insulin,
arsenite did not induce T-loop phosphorylation of PKC-k
(Fig. 3D), nor did we observe an increase in the amount of
incorporated radiolabelled phosphate in immunoprecipi-
tated PKC-k (data not shown). Indeed, when analysing
PKC-k activity in an in vitro kinase assay, no induction of
PKC-k activity in response to arsenite was observed
(Fig. 7B). Thus, taken together, these data suggest that

arsenite does not activate PKC-k, but does require the basal
Fig. 8. Arsenite-induced glucose uptake is inhibited by a myristoylated
PKC-k/-f, but not by PKC-a/-b pseudosubstrate peptide. 3T3-L1
adipocytes were incubated with either myristoylated PKC-a/-b
pseudosubstrate (myrPKC-a/b ps), or myristoylated PKC-k/–f
pseudosubstrate (myrPKC-k/f ps) at the indicated concentrations for
1 h prior to stimulation. (A) 3T3-L1 adipocytes were treated with
100 n
M
TPA for 15 min and subjected to immunoblotting as described
in the legend of Fig. 7C. (B) 2-DOG uptake was tested in a separate
experiment. 3T3-L1 adipocytes were mock-treated (basal), stimulated
for 15 min with 100 n
M
insulin (insulin) or for 30 min with 0.5 m
M
arsenite (arsenite). Data are expressed as mean ± SEM of at least two
independent experiments each performed in triplicate, statistically
significant compared to the uninhibited samples (*P < 0.05).
Fig. 9. A model highlighting the insulin- and arsenite-induced pathways
to glucose uptake in 3T3-L1 adipocytes. Our data suggest some com-
mon steps in both arsenite- and insulin-induced glucose uptake: acti-
vation of p38 MAPK and tyrosine-phosphorylation of c-Cbl. In
contrast to insulin, arsenite does not activate PI-3¢ kinase (and con-
sequently does not activate PKC-k). However, the data suggests that
basal levels of PKC-k activity are needed for arsenite-induced glucose
uptake, as is indicated by the dashed arrow.
3900 M. Bazuine et al. (Eur. J. Biochem. 270) Ó FEBS 2003
activity of this enzyme (in conjunction with other signals)
to induce GLUT-4 translocation in 3T3-L1 adipocytes. Of

note, the basal levels of PKC-k activity are already quite
high, with only a 1.2 to 2-fold induction in response to
insulin [9,11,12] (Fig. 7B). These data are consistent with a
common step in both insulin- and arsenite-induced glucose
transport downstream of PI-3¢ kinase involving the activity
of atypical PKC-isoforms, however, additional factors
contribute to the level of arsenite-induced GLUT4 trans-
location and the magnitude of glucose uptake.
In summary, our data suggest a model as depicted in
Fig. 9, in which arsenite and insulin activate distinct
signalling pathways that converge at several steps (e.g.
c-Cbl tyrosine phosphorylation, PKC-k activity and p38
MAPK activation) upstream of GLUT4 translocation and
glucose uptake in 3T3-L1 adipocytes.
Acknowledgements
We thank Drs Hans Joost and Annette Schu
¨
rmann (Aachen and
Potsdam, Germany) for their kind gift of antibodies against GLUT1
and 4 and Dr Ken Siddle (Cambridge, UK) for antibodies against the
insulin receptor. We thank R. van de Ven for excellent technical
assistance with some of the glucose uptake experiments and valuable
discussions. We would also like to acknowledge Drs P. J. A. van den
Broek and J. van der Zee for critical reading of this manuscript. M. B.
was supported by a grant from the Dutch Diabetes Foundation (DFN
98.106).
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