Tài liệu Báo cáo khoa học: Arsenite stimulated glucose transport in 3T3-L1 adipocytes involves both

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¢) kinase by wortman￾nin. These findings indicate that the
classical

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¢) 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].

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, 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: [email protected]

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)

Eur. J. Biochem. 270, 3891–3903 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03771.x