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

doi:10.1046/j.1432-1033.2003.03771.x

Eur. J. Biochem. 270, 3891–3903 (2003) (cid:2) FEBS 2003

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

findings

that

the

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.

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¢) kinase by wortman- nin. These (cid:1)classical(cid:2) indicate 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

sites for class I phosphatidylinositol-3¢ (PI-3¢) kinase that 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].

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

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, translocates tyrosine-phosphorylated c-Cbl 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, H2O2 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: J.A.Maassen@lumc.nl Abbreviations: IR(s), insulin receptor (substrates); IBMX, 1-methyl- 3-isobutylxanthine; PI-3¢, phosphatidylinositol-3¢-kinase; 2-DOG, 2-deoxy-D[14C]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)

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Experimental procedures

Materials

from Brunschwig, Amsterdam; bovine

(IBMX),

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.

Dulbecco’s modified Eagle’s medium (DMEM) was pur- chased from Life Technologies, Inc.; foetal bovine serum insulin, was 1-methyl-3-isobutylxanthine dexamethasone, 12-O-tetradecanoylphorbol 13-acetate (TPA) and 2-deoxy- glucose were obtained from Sigma. 2-deoxy-D-[14C]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

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.

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.

Reference Pharmacological inhibitor Concentration applied Described target IC50

Bisindolyl-maleimide I 5 lM

Chelerythrine Chloride 10 lM

100 nM Go¨ 6976

LY 294002 10 lM

Ro31–8220 0–20 lM

PKC-a, bI, bII, c, d, e 5-hydroxytryptamine3 receptor Glycogen synthase kinase 3b K(ACh)channels MAPK activated protein-kinase 1b p70 S6 kinase Mitogen and stress activated kinase-1 AMP-activated protein kinase Phosphorylase kinase PKC K(ACh) channels Trk A and B Conventional PKC-a Conventional PKC-bI PKC-l (PRK) Phosphatidylinositol 3¢-kinase Casein kinase 2 Conventional and novel PKC Atypical PKC-k Atypical PKC-f Calbiochem [49] [50] [51] [52] [52] [53] [53] [53] Calbiochema [51] [54] [55] [55] [56] [57,58] [53] [11,59,60] This study [11]

Glycogen synthase kinase 3b

Ro32–0432 10 lM

a Disputed by Davies et al. [53].

SB203580 Tyrphostin A25 Wortmannin 10 lM 25 lM 100 nM 10–100 nM 7 nM 170 nM 100 nM 50 nM 100 nM <1 lM 1 lM 1 lM 660 nM 490 nM 10–100 nM 2.3 nM 6.2 nM 20 nM 1.4 lM 6.9 lM 10–100 nM 5 lM 1 lM in vitro 4 lM in vivo 38 nM 2.8 nM 3 nM 15 nM 8 nM 9 nM 28 nM 108 nM 0.6 lM EGF 3 lM 2–5 nM [53] [50] [52] [52] [53] [61] [61] [61] [62,63] [64,65] [66,67] MAPK activated protein-kinase 1b p70 S6 kinase Mitogen and stress activated protein kinase Conventional PKC-a Conventional PKC-bI Novel PKC-e p38 MAP kinase (a and b) Tyrosine kinases Phosphatidylinositol 3¢-kinase

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Immunoprecipitations and Western blotting

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.

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 mM Na3VO4, 1 mM EGTA, 1 mM EDTA, 50 mM Tris/HCl pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, 5 mM NaF in the presence of protease inhibitors). Cell lysates were tumbled for half an hour at 4 (cid:3)C, cell lysate was cleared from cellular debris by spinning at 14 000 g, for 10 min at 4 (cid:3)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 (cid:3)C. Immunocomplexes were harvested by incubating with ProtG beads for 1.5 h at 4 (cid:3)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).

Cell culture

PI-3¢ kinase activity assay

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

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-32P- labelled ATP per reaction as described by Burgering et al. [34]. Incorporated radioactivity was quantified on a Molecular Dynamics phosphorimager.

PKC-k kinase assay

3T3-L1 adipocytes were stimulated as indicated in the figure legends. Subsequently cells were washed twice in ice-cold HES buffer (20 mM Hepes pH 7.4, 1 mM EDTA and 250 mM 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

Dishes (9 cm) of 3T3-L1 adipocytes were stimulated with 100 nM of insulin for 10 min or 0.5 mM arsenite for 30 min. Cells were lysed in an NP-40 based lysis buffer (see above) in the presence of 1 lM microcystin LR and immunopreci- pitated with 5 lg mouse monoclonal PKC-k for 1.5 h at 4 (cid:3)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 mM Hepes pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol). PtdSer (4 lg per sample) was dried under N2(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 lM, final concentration), 5 lCi c-32P-labelled ATP per reaction, dithiothreitol (1.5 mM), PKI (1 mM), the indicated concen- trations of Ro 31-8220, and PKCe-substrate (40 lM) were added. As a control, 10 lM 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 (cid:3)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.

3T3-L1 adipocytes, grown in 12-well plates (Costar), were subjected to an assay of 2-deoxy-D-[14C]glucose (0.075 lCi per well) uptake as described previously [33].

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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[14C]glucose (2-DOG) uptake was maximal at a 30-min preincubation period with 0.5 mM 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

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.

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 ((cid:1) 10 lg) (Fig. 2A,C). were subjected to immunoblot analysis 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.

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

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

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[14C]glucose (2-DOG) uptake. (B) 3T3-L1 adipocytes were stimulated with 0.5 mM arsenite for the indicated times and assayed for 2-DOG uptake. (C) 3T3-L1 adipocytes were stimulated as indicated with 100 nM insulin for 15 min (ins), 0.5 mM arsenite for 30 min (as), or 0.5 mM arsenite for 30 min combined with 100 nM 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.

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to arsenite-treatment

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 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

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).

Fig. 2. Arsenite-treatment induces GLUT1 and GLUT4 translocation to the plasma membrane in 3T3-L1 adipocytes. 3T3-L1 adipocytes were mock- treated (basal), stimulated for 15 min with 100 nM insulin (insulin/ins) or for 30 min with 0.5 mM 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 nM insulin (ins) or for 30 min with 0.5 mM 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.

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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 nM insulin for 5 min or 0.5 mM 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) as indicated. 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) or PKB (aPKB) as indicated.

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

MAPKAP-K2 on Thr334 (a direct target site of p38 MAP kinase activity [37]). Treatment with 10 lM 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

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

Fig. 4. Arsenite-induced in vitro PI-3¢ kinase activity. 3T3-L1 adipo- cytes were stimulated as indicated with 100 nM insulin for 5 min or 0.5 mM 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-32P]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 nM wortmannin. Subsequently, adipocytes were mock-treated (basal) or stimulated as indicated with 100 nM insulin for 15 min or with 0.5 mM 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).

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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 IC50 value of approximately 5 lM 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 IC50 of 5 lM) was observed (Fig. 7B). Co-incubation with 50 lM of a peptide resembling the PKC-k pseudosubstrate domain reduced 32P 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 IC50 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

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.

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 lM tyrphostin A25 as indicated. Subsequently, adipocytes were mock-treated (-), stimulated for 5 min with 100 nM insulin (INS) or for 30 min with 0.5 mM 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 lM tyrphostin A25. Subsequently, adipocytes were mock-treated (basal), stimulated for 15 min with 100 nM insulin or for 30 min with 0.5 mM 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.

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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.

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.

A PI-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,

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

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 lM SB203580 (A–D). Subsequently adipocytes were mock-treated (basal) or stimulated as indicated with 100 nM insulin for 15 min (insulin) or with 0.5 mM 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.

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as is the case for insulin. The identity of the tyrosine- kinase activity activated in response to arsenite remains to be resolved.

through these stages,

the progress

for

The term coined for the modulatory effect of SB203850 is (cid:1)intrinsic activity(cid:2) [35], possibly by altering the speed of transition between (cid:1)outward(cid:2) and (cid:1)inward(cid:2) conformations of the transporter [42]. The term (cid:1)occlusion(cid:2) 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 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 sensitivity to SB203580, may provide an a similar additional future research addressing these tool 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

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 lM SB203580 had no effect on GLUT4 (or GLUT1) translocation but did reduce arsenite-induced glucose uptake by (cid:1) 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. fully exclude a similar effect of Though we cannot 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).

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 nM insulin for 15 min (insulin) or 0.5 mM 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 lM Go¨ 6976, 5 lM bisindolylmaleimide I (BIM I), 10 lM chelerythrine chloride (Chel- erythrine), or 10 lM Ro 32–0432 as indicated. Subsequently, 3T3-L1 adipocytes were stimulated with 100 nM 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 nM insulin for 15 min (insulin), or 0.5 mM 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).

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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.

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.

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 IC50 of 5 lM suggesting an involvement of atypical PKCs. The IC50 of atypical PKC for BIM I is 5.8 lM, hence the significant reduction in glucose uptake measured using 5 lM (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.

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

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 nM 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 nM insulin (insulin) or for 30 min with 0.5 mM 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).

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insulin stimulation in rat adipocytes. Potential role in glucose transport. J. Biol. Chem. 272, 30075–30082.

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.

12. Kotani, K., Ogawa, W., Matsumoto, M., Kitamura, T., Sakaue, H., Hino, Y., Miyake, K., Sano, W., Akimoto, K., Ohno, S. & Kasuga, M. (1998) Requirement of atypical protein kinase C lambda for insulin stimulation of glucose uptake but not for Akt activation in 3T3-L1 adipocytes. Mol. Cellular Biol. 18, 6971– 6982.

13. Liu, J., Kimura, A., Baumann, C.A. & Saltiel, A.R. (2002) APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 trans- location in response to insulin in 3T3-L1 adipocytes. Mol. Cellular Biol. 22, 3599–3609.

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.

14. Chiang, S.H., Baumann, C.A., Kanzaki, M., Thurmond, D.C., Watson, R.T., Neudauer, C.L., Macara, I.G., Pessin, J.E. & Saltiel, A.R. (2001) Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10. Nature 410, 944–948.

15. Inoue, M., Chang, L., Hwang, J., Chiang, S.H. & Saltiel, A.R. (2003) The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature 422, 629–633.

Acknowledgements

16. Chen, D., Elmendorf, J.S., Olson, A.L., Li, X., Earp, H.S. & Pessin, J.E. (1997) Osmotic shock stimulates GLUT4 transloca- tion in 3T3L1 adipocytes by a novel tyrosine kinase pathway. J. Biol. Chem. 272, 27401–27410.

17. Barros, L.F., Marchant, R.B. & Baldwin, S.A. (1995) Dissection of stress-activated glucose transport from insulin-induced glucose transport in mammalian cells using wortmannin and ML-9. Biochem. J. 309, 731–736.

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).

18. Lund, S., Holman, G.D., Schmitz, O. & Pedersen, O. (1995) Contraction stimulates translocation of glucose transporter GLUT4 in skeletal muscle through a mechanism distinct from that of insulin. Proc. Natl Acad. Sci. USA 92, 5817–5821.

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