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Overexpression of a Constitutively Active Form of Phosphatidylinositol 3-Kinase Is Sufficient to Promote Glut 4 Translocation in Adipocytes*

Open AccessPublished:October 11, 1996DOI:https://doi.org/10.1074/jbc.271.41.25227
      Insulin stimulates glucose transport in its target cells by recruiting the glucose transporter Glut 4 from an intracellular compartment to the cell surface. Previous studies have indicated that phosphatidylinositol 3-kinase (PI 3-kinase) is a necessary step in this insulin action. We have investigated whether PI 3-kinase activation is sufficient to promote Glut 4 translocation in transiently transfected adipocytes. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged Glut 4 and a constitutively active form of PI 3-kinase (p110*). The expression of p110* induced the appearance of epitope-tagged Glut 4 at the cell surface at a level similar to that obtained after insulin treatment, whereas a kinase-dead version of p110* had no effect. The p110* effect was observed over a wide range of the transfected cDNA. When subcellular fractionation of adipocytes was performed, p110* was found, similar to the endogenous PI 3-kinase, enriched in the low density microsomal compartment, which also contains the Glut 4 vesicles. This could suggest that a specific localization of PI 3-kinase in this compartment is required for the action on Glut 4. The observations made with PI 3-kinase are in contrast with those seen with the MAP kinase cascade. Indeed, a constitutively active form of MAP kinase kinase had no effect on Glut 4 translocation in basal conditions. At the highest degree of expression, the constitutively active form of MAP kinase kinase slightly inhibited the insulin stimulation of Glut 4 translocation. Taken together, our results indicate that Glut 4 translocation can be efficiently promoted by an active form of PI 3-kinase but not by the activation of the MAP kinase pathway.

      INTRODUCTION

      Insulin promotes glucose uptake in muscle and adipose tissue by increasing the translocation of the glucose transporter Glut 4 from an intracellular compartment to the plasma membrane, but the exact molecular mechanism is still undetermined (
      • Stephens J.M.
      • Pilch P.F.
      ,
      • Cheatham B.
      • Kahn C.R.
      ). Following insulin binding, the insulin receptor phosphorylates on tyrosine residues different substrates, including insulin receptor substrate 1 and 2 (IRS1
      The abbreviations used are: IRS1
      insulin receptor substrate 1
      PVDF
      polyvinylidene difluoride
      BSA
      bovine serum albumin
      MAP
      mitogen-activated protein
      MEK
      mitogen-activated kinase kinase
      PI 3-kinase
      phosphatidylinositol 3ʹ kinase.
      p110*
      constitutively active form of PI 3-kinase
      MEK*
      constitutively active MEK
      SH2
      Src homology 2
      PAGE
      polyacrylamide gel electrophoresis
      PBS
      phosphate-buffered saline
      PM
      plasma membrane
      LDM
      low density microsomal membrane
      HDM
      high density microsomal membrane
      IL
      interleukin.
      and IRS2) and Src homology collagen (SHC). The phosphorylated IRS1 docks proteins containing Src homology 2 (SH2) domains, such as phosphatidylinositol 3-kinase (PI 3-kinase), Nck, Syp, and Grb2 (
      • White M.F.
      • Kahn C.R.
      ). Thus, the p85 subunit of PI 3-kinase binds through its SH2 domains to the tyrosine phosphorylated YMXM motifs of IRS1 (
      • White M.F.
      • Kahn C.R.
      ); the consequences are an increase in the catalytic activity of the PI 3-kinase p110 subunit (
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.-J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ) and a rise in the intracellular level of PI 3,4-biphosphate and PI 3,4,5-triphosphate. A series of recent data indicates that PI 3-kinase is involved in insulin-induced Glut 4 translocation. For example, the blockade of Glut 4 translocation by pharmacological inhibitors of PI 3-kinase, such as wortmannin or LY294002 (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Le Marchand-Brustel Y.
      • Gautier N.
      • Cormont M.
      • Van Obberghen E.
      ), or by a dominant negative mutant of PI 3-kinase (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • Hawkins P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Quon M.J.
      • Chen H.
      • Ing B.L.
      • Miu M.-L.
      • Zarnowski M.J.
      • Yonezawa K.
      • Kasuga M.
      • Cushman S.W.
      • Taylor S.I.
      ) indicates that PI 3-kinase activation is a necessary step for the insulin stimulation of transporter translocation and glucose transport (
      • Shepherd P.R.
      • Reaves B.J.
      • Davidson H.W.
      ). However, other observations suggest that, although necessary, PI 3-kinase activation is not sufficient to promote glucose transporter translocation. Indeed, growth factors such as platelet-derived growth factor can stimulate PI 3-kinase strongly but have a minor effect on Glut 4 translocation (
      • Gould G.W.
      • Merrall N.W.
      • Martin S.
      • Jess T.J.
      • Campbell I.W.
      • Calderhead D.M.
      • Gibbs E.M.
      • Holman G.D.
      • Plevin R.J.
      ,
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ). Further, interleukin 4 (IL-4), which induces tyrosine phosphorylation of IRS1 and activation of PI 3-kinase, does not stimulate Glut 4 translocation in L6 myoblasts (
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ). However, it is also possible that PI 3-kinase has to be activated in a specific subcellular compartment (
      • Shepherd P.R.
      • Reaves B.J.
      • Davidson H.W.
      ,
      • Yang J.
      • Clarke J.F.
      • Ester C.J.
      • Young P.W.
      • Kasuga M.
      • Holman G.D.
      ,
      • Kelly K.L.
      • Ruderman N.B.
      ,
      • Ricort J.-M.
      • Tanti J.-F.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ), an effect that could occur with insulin but not with platelet-derived growth factor or IL-4.
      The aim of the present study was to determine whether PI 3-kinase activation was sufficient to promote Glut 4 translocation, using rat adipocytes transiently transfected with a constitutively active form of PI 3-kinase. Adipocytes are transfected with a Glut 4 molecule tagged with a Myc epitope in the first extracellular loop of the protein. By the binding of an anti-Myc antibody to intact cells, Glut 4 translocation is exclusively studied in the small fraction of the cells that have been transfected. This recently described system allows investigation of the signal transduction pathway involved in the translocation of Glut 4 because the epitope-tagged Glut 4 behaves similarly to the endogenous Glut 4 in response to insulin (
      • Quon M.J.
      • Butte A.J.
      • Zarnowski M.J.
      • Sesti G.
      • Cushman S.W.
      • Taylor S.I.
      ,
      • Quon M.J.
      • Guerre-Millo M.
      • Zarnowski M.J.
      • Butte A.J.
      • Em M.
      • Cushman S.W.
      • Taylor S.I.
      ). Taking advantage of this system, we looked directly at the effect of a constitutively active p110 PI 3-kinase (p110*) (
      • Hu Q.
      • Klippel A.
      • Muslin J.A.
      • Fantl J.W.
      • Williams T.L.
      ,
      • Klippel A.
      • Escobedo J.A.
      • Hirano M.
      • Williams L.T.
      ) on Glut 4 translocation, and we determined whether its targeting into the cell was similar to that of the endogenous enzyme. Our results indicate that the active p110* is present in the same intracellular compartment as the endogenous enzyme and is as efficient as insulin in promoting Glut 4 translocation.

      EXPERIMENTAL PROCEDURES

      Materials

      Collagenase was from Boerhinger Mannheim. Bovine serum albumin (BSA) was purchased from Intergen Co. (Purchase, NY). Polyvinylidene difluoride (PVDF) membranes were from Millipore Corp. (Bedford, MA). [125I]Ig against mouse immunoglobulins was from Amersham Corp. The antibody to Myc used for binding is a monoclonal antibody (9E10) and the antibody for blotting is a rabbit polyclonal antibody from Santa Cruz Biotechnology. Restriction enzymes were from Biolabs (Richmond, CA). All other chemical and biochemical products were from Sigma or Merck.

      DNA Vector Constructions

      pCIS2 and pCIS-Glut 4myc

      pCIS2 is an expression vector containing a cytomegalovirus promoter and enhancer with a generic intron located upstream from the multiple cloning site. This vector gives a high level of protein expression in adipocytes (
      • Quon M.J.
      • Zarnowski M.
      • Guerre-Millo M.
      • De La Luz Sierra M.
      • Taylor S.I.
      • Cushman S.W.
      ). A unique StuI site was introduced into the nucleotide sequence of the rat Glut 4 cDNA by changing GGTCCT to AGGCCT (coding for Glut 4 Gly65-Pro66) using site-directed mutagenesis (CLONTECH). The cDNA was cut with StuI to allow for the insertion of the oligonucleotides coding for the Myc epitope. The sense oligonucleotide was 5ʹ-GCA-GAG-GAG-CAA-AAG-CTT-ATT-TCT-GAA-GAG-GAC-TTG-CTT-AAG-3ʹ. The antisense oligonucleotide was 5ʹ-CTT-AAG-CAA-GTC-CTC-TTC-AGA-AAT-AAG-CTT-TTG-CTC-CTC-TGC-3ʹ. This resulted in a fusion gene encoding the peptide sequence (AEEQKLISEEDLLK) inserted between amino acids 65 and 66 in the first exofacial loop of Glut 4. The construction was verified by direct sequencing of the regions surrounding and including the oligonucleotide insert. Glut 4myc was subcloned into the pCIS2 vector.

      pCG-p110* and pCG-p110*Δkin

      pCG p110* encodes for a constitutive form of p110 in which the inter-SH2 domain of p85 was ligated to the NH2 terminus of p110 (
      • Hu Q.
      • Klippel A.
      • Muslin J.A.
      • Fantl J.W.
      • Williams T.L.
      ). The p110*Δkin is a kinase-deficient version of p110* in which the ATP binding site was mutated. Both proteins were tagged at the COOH terminus with the Myc epitope. The engineering of the constructs and description of the vector driving the expression of the proteins have been published previously (
      • Hu Q.
      • Klippel A.
      • Muslin J.A.
      • Fantl J.W.
      • Williams T.L.
      ,
      • Klippel A.
      • Escobedo J.A.
      • Hirano M.
      • Williams L.T.
      ).

      pCEP-MEK*

      A constitutively active form of MEK (MEK*) was obtained by deleting the region encompassing amino acids 32-51 and by mutating the two serine residues (218 and 222) to aspartic acid. This form of MEK has been shown to possess high level of kinase activity (
      • Mansour S.J.
      • Matten W.T.
      • Hermann A.S.
      • Candia J.M.
      • Rong S.
      • Fukasawa K.
      • Vande Woude G.F.
      • Ahn N.G.
      ). This cDNA was subcloned into the mammalian expression vector pCEP (Invitrogen).

      pCIS2-v-RAS

      The cDNA for human RAS with activating mutation substituting Arg for Val at position 12 (a gift from Dr. de Gunzburg, INSERM, Paris, France) was subcloned in pCIS2.

      pADneo-fosluci

      pADneo-fosluci is a plasmid containing the c-fos promoter inserted upstream of the luciferase reporter gene and was used to assess the function of the transfected constructs. It was kindly provided to us by Dr. Czernilofsky (Bender Co., Boehringer Ingelheim, Vienna, Austria).
      The plasmid DNAs were obtained using a Qiagen maxi kit (QIAGEN), and their concentration was determined by measuring the A at 260 nm.

      Preparation of Isolated Rat Adipocytes and Electroporation

      Adipose cells were isolated from epididymal fat pads of male Wistar rats (170-200 g) by collagenase (Boehringer Mannheim) digestion (
      • Cormont M.
      • Tanti J.-F.
      • Zahraoui A.
      • Van Obberghen E.
      • Tavitian A.
      • Le Marchand-Brustel Y.
      ). Isolated adipocytes were transfected by electroporation as described (
      • Quon M.J.
      • Butte A.J.
      • Zarnowski M.J.
      • Sesti G.
      • Cushman S.W.
      • Taylor S.I.
      ,
      • Quon M.J.
      • Guerre-Millo M.
      • Zarnowski M.J.
      • Butte A.J.
      • Em M.
      • Cushman S.W.
      • Taylor S.I.
      ,
      • Quon M.J.
      • Zarnowski M.
      • Guerre-Millo M.
      • De La Luz Sierra M.
      • Taylor S.I.
      • Cushman S.W.
      ), with some modifications. Isolated adipocytes were resuspended at a 50% (v/v) cell suspension in Dulbecco's modified Eagle's medium. Cell suspension (400 μl) was placed in a 0.4-cm gap cuvette along with the plasmid DNAs (0.25 μg pCIS-Glut 4myc and the amounts of the various construct DNAs as indicated in the figure legends). In all cases, the total amount of DNA was adjusted to 10 μg by pCIS addition. Electroporation was performed with a double electric shock (800 V, 25 microfarads; 200 V, 1050 microfarads) using an Easyject electroporator (Eurogentec). Cells were diluted in 1.5 ml of Dulbecco's modified Eagle's medium containing 5% BSA (w/v), 25 mM Hepes, pH 7.4, 200 nM (R)-N6-1-methyl-2-phenylethyl adenosine and 100 μg/ml gentamycin. The cells were incubated for 16-24 h at 37°C in 5% CO2/95% air prior to further study.

      Assay for Cell Surface Epitope-tagged Glut 4 Measurement

      Electroporated adipocytes were washed twice with Krebs-Ringer bicarbonate buffer containing 30 mM Hepes, pH 7.4 (KRBH) and resuspended at a 10% (v/v) suspension in KRBH, 1% (w/v) BSA. Cells were then incubated for 30 min at 37°C in the absence or presence of insulin (100 nM). After insulin treatment, KCN (final concentration, 2 mM) was added for 5 min to prevent Glut 4 redistribution, and adipocytes were incubated at 25°C for 1 h with 0.5 μg/ml mouse monoclonal antibodies to Myc (9E10). Cells were washed three times with KRBH, 1% BSA and incubated in triplicate for 1 h at 25°C with 125I-labeled sheep anti-mouse IgG (10 μCi/μg; final dilution, 1/200) (Amersham Corp.). Then, samples (300 μl) were placed on 100 μl of dinonylphthalate and centrifuged to separate cells from the medium. The fat cake was boiled in Laemmli buffer (3% SDS, 70 mM Tris, 10% glycerol) and radioactivity associated with the cells was counted in a gamma counter. Radioactivity was normalized by measuring protein concentration in each sample using BCA (Pierce). Nonspecific binding of the antibodies, which represented 30% of the total binding observed in cells transfected with pCIS-Glut 4myc in the absence of insulin stimulation, was obtained with cells transfected with pCIS2 alone and was subtracted from all values.

      Subcellular Fractionation of Adipocytes

      Adipocytes transfected with pCIS2, pCG-p110*, or pCG-p110*Δkin were washed three times with KRBH and homogenized in 2 volumes of 20 mM Tris, pH 7.4, 1 mM EDTA, 250 mM sucrose, and inhibitors of proteases using a Thomas potter type C. Plasma membranes (PMs), low density microsomal membranes (LDMs), and high density microsomal membranes (HDMs) were prepared by differential ultracentrifugation as described (
      • Cormont M.
      • Tanti J.-F.
      • Zahraoui A.
      • Van Obberghen E.
      • Tavitian A.
      • Le Marchand-Brustel Y.
      ). Fraction proteins (50 μg) were separated by SDS-PAGE using a 7.5% resolving gel and electrotransferred to a PVDF sheet (Millipore). Immunodetection of epitope-tagged p110* and p110*Δkin was performed with rabbit anti-Myc Ig (Santa Cruz), and p85 immunodetection was carried out with polyclonal anti-p85 antibodies (UBI). After washes, sheets were incubated with 125I-labeled protein A, washed, submitted to autoradiography, and quantified by Molecular Imager analysis (Bio-Rad).

      Immunoprecipitation and Immunoblotting of Glut 4myc

      Adipose cells were cotransfected with pCIS-Glut 4myc and either pCIS, p110*, or p110*Δkin, and cells were washed and homogenized as described above. Total membranes were prepared by centrifugation at 300,000 x g for 1 h and solubilized in 30 mM Hepes, pH 7.4, 30 mM NaCl, 1% Triton X-100, and proteases inhibitors. Then Glut 4Myc was immunoprecipitated using monoclonal anti-Myc antibodies (2 μg) coupled to protein G-Sepharose beads. After the washes, the pellets were boiled in Laemmli buffer, and the proteins were separated by SDS-PAGE and transferred on PVDF sheets. Glut 4Myc was immunodetected with a rabbit antipeptide antibody directed against the 12-amino acid peptide corresponding to the COOH-terminal sequence of Glut 4 (
      • Le Marchand-Brustel Y.
      • Olichon-Berthe C.
      • Grémeaux T.
      • Tanti J.F.
      • Rochet N.
      • Van Obberghen E.
      ).

      Luciferase Assay

      Adipocytes were transfected with pADneo-fosluci (1 μg) and 9 μg of pCIS, p110*, v-RAS, or pCEP-MEK* as described above. Cells were maintained for 16-20 h in 5% CO2/95% air at 37°C; cells were then resuspended at 10% (v/v) in KRBH, 1% BSA without or with insulin (100 nM) for 6 h. Cells were washed three times in KRBH, and luciferase activity was measured with the Promega kit according to the manufacturer's instructions.

      Immunofluorescence Studies

      Adipocytes were transfected with pCIS or pCIS-Glut 4myc as described above and incubated for 16-24 h at 37°C in 5% CO2/95% air. Then adipocytes were washed twice with PBS and fixed for 15 min in PBS containing 2% formaldehyde. After permeabilization with PBS, 0.2% Triton X-100 for 15 min, cells were resuspended in PBS, 1% BSA and incubated with antibodies to Myc (final concentration, 10 μg/ml) for 1 h. Cells were washed three times with PBS, 1% BSA and incubated for 1 h in the same buffer containing fluorescein-conjugated donkey anti-mouse IgG (final dilution, 1/200) (Jackson Laboratory). Cells were then washed before visualization under a fluorescence microscope. Fluorescent cells were counted and expressed as a percentage of the total adipocyte number in the same fields.

      Statistics

      In each experiment, triplicate determination of the binding of the antibodies to Myc was performed. The experiments were repeated 5 or 6 times with different adipocyte preparations. Statistical significance was assessed using Student's t test for paired data.

      DISCUSSION

      Several studies have focused on the role of PI 3-kinase in the stimulation of glucose transport by insulin. The use of pharmacological inhibitors of the enzyme or of a dominant negative mutant clearly indicates that PI 3-kinase activation is required for the insulin stimulation of Glut 4 translocation (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Le Marchand-Brustel Y.
      • Gautier N.
      • Cormont M.
      • Van Obberghen E.
      ,
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • Hawkins P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Quon M.J.
      • Chen H.
      • Ing B.L.
      • Miu M.-L.
      • Zarnowski M.J.
      • Yonezawa K.
      • Kasuga M.
      • Cushman S.W.
      • Taylor S.I.
      ). However, in these studies, the question of whether PI 3-kinase was sufficient to promote glucose transporter translocation was not addressed. To directly investigate whether PI 3-kinase can promote glucose transporter translocation, we have taken advantage of a form of PI 3-kinase (p110*) that was constitutively active in a growth factor-independent manner (
      • Hu Q.
      • Klippel A.
      • Muslin J.A.
      • Fantl J.W.
      • Williams T.L.
      ,
      • Klippel A.
      • Escobedo J.A.
      • Hirano M.
      • Williams L.T.
      ). We used transfected adipocytes that transiently express epitope-tagged Glut 4 and the other proteins of interest. This system allows for a direct measurement of the level of cell surface Glut 4Myc by the binding of anti-Myc antibodies on intact cells (
      • Quon M.J.
      • Butte A.J.
      • Zarnowski M.J.
      • Sesti G.
      • Cushman S.W.
      • Taylor S.I.
      ,
      • Quon M.J.
      • Guerre-Millo M.
      • Zarnowski M.J.
      • Butte A.J.
      • Em M.
      • Cushman S.W.
      • Taylor S.I.
      ,
      • Quon M.J.
      • Zarnowski M.
      • Guerre-Millo M.
      • De La Luz Sierra M.
      • Taylor S.I.
      • Cushman S.W.
      ). Previous studies have demonstrated that Glut 4Myc behavior is similar to that of endogenous Glut 4, and thus the cell surface Glut 4Myc level reflects the translocation of endogenous Glut 4. The use of an epitope-tagged Glut 4 is thus a reporter for Glut 4 subcellular distribution exclusively in the fraction of cells that are transfected, which was about 10% in our transfection conditions. The level of Glut 4 overexpression was thus estimated to be increased only 2-3-fold in the Glut 4Myc-expressing cells. With this moderate level of overexpression, it is unlikely that the normal sorting mechanisms were saturated. Indeed, in adipocytes of transgenic mice that markedly overexpressed Glut 4 (15-20 fold) in fat tissue, the transporters appeared to be targeted to the same unique structurally defined vesicles as in native cells (
      • Tozzo E.
      • Kahn B.B.
      • Pilch P.F.
      • Kandror K.V.
      ).
      Using transiently transfected adipocytes, we found that p110* promoted Glut 4Myc translocation very efficiently. Indeed, the level of Glut 4Myc at the cell surface was comparable to the level of Glut 4Myc present at the cell surface of control adipocytes stimulated with insulin. The level of p110* overexpression could not be appreciated by comparison with the endogenous protein because no antibody was able to recognize the rat p110 PI3 kinase subunit. However, it could be observed that the p110* effect on Glut 4Myc subcellular distribution was observed at various levels of p110* expression. The increase in Glut 4Myc at the cell surface was dependent on the p110* kinase activity because it was not observed when a kinase-dead form of p110* was expressed. This effect was not due to a change in Glut 4Myc expression. We can also exclude the possibility that it is related to a RAS pathway activation because the p110* overexpression was not able to activate the c-fos promoter. This result differs from the observations in Chinese hamster ovary cells, where this construct activated the RAS pathway (
      • Hu Q.
      • Klippel A.
      • Muslin J.A.
      • Fantl J.W.
      • Williams T.L.
      ). It should be noted that the relationship between PI 3-kinase and RAS is a matter of debate. Some observations indicate that PI 3-kinase could be upstream, downstream, or independent of RAS; these observations are perhaps related to cell-type differences (for a review, see
      • Carpenter C.L.
      • Cantley L.C.
      ). In contrast to the p110* effect, transfection of a constitutively active MEK did not modify basal Glut 4Myc level at the cell surface. These data reinforce the idea that stimulation of the MAP kinase cascade is not involved in Glut 4 translocation (
      • Lazar D.F.
      • Wiese R.J.
      • Brady M.J.
      • Mastick C.C.
      • Waters S.B.
      • Yamauchi K.
      • Pessin J.E.
      • Cuatrecasas P.
      • Saltiel A.R.
      ,
      • Wiese R.J.
      • Mastick C.C.
      • Lazar D.F.
      • Saltiel A.R.
      ,
      • Robinson L.J.
      • Razzack Z.F.
      • Lawrence Jr., J.C.
      • James D.E.
      ). Using the same approach, v-RAS was able to promote an increase in Glut 4Myc at the cell surface (
      • Quon M.J.
      • Chen H.
      • Ing B.L.
      • Miu M.-L.
      • Zarnowski M.J.
      • Yonezawa K.
      • Kasuga M.
      • Cushman S.W.
      • Taylor S.I.
      ). The difference with our results suggests that the RAS effect was not due to activation of MEK or MAP kinase but was more likely due to the stimulation of another branching signaling pathway. The high expression of MEK* reduced insulin effect on Glut 4Myc translocation, an effect that was not observed with a lower expression. These observations suggest that the inhibitory effect of MEK* is likely to be due to the saturation of downstream molecules and not to its high kinase activity. However, it remains possible that the MAP kinase pathway is involved in a negative feedback loop.
      Our present demonstration that a constitutively active PI 3-kinase is sufficient to increase Glut 4 level at the plasma membrane, together with the observation that inhibition of PI 3-kinase blocks insulin stimulation of glucose transport translocation, suggests that the insulin effect on glucose transporter translocation is mainly due to activation of PI 3-kinase. Our results are in agreement with the observation that overexpression of IRS1 in adipocytes is able to trigger glucose transporter translocation in the absence of insulin stimulation (
      • Quon M.J.
      • Butte A.J.
      • Zarnowski M.J.
      • Sesti G.
      • Cushman S.W.
      • Taylor S.I.
      ). In this case the high level of IRS1 expression could compensate the low level of tyrosine kinase activity of the insulin receptor, leading to a sufficient tyrosine phosphorylation of IRS1 and thus to activation of PI 3-kinase. However, our results are at variance with two recent studies using different approaches, which led to the conclusion that activation of PI 3-kinase was not sufficient and that an alternative signaling pathway was required for stimulation of glucose uptake. In one study (
      • Herbst J.J.
      • Andrews G.C.
      • Contillo L.G.
      • Singleton D.H.
      • Genereux L.G.
      • Gibbs E.M.
      • Lienhard G.E.
      ), although PI 3-kinase was activated in 3T3-L1 adipocytes by thiophosphorylated peptides corresponding to the binding motif of PI 3-kinase to IRS1, only a minor effect on Glut 4 translocation was observed. In the other study (
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ), IL-4 stimulation of L6 myoblasts expressing the IL-4 receptor induced IRS1 tyrosine phosphorylation and increased PI 3-kinase activity but had no effect on glucose uptake. It should be noted that only the cytosolic (
      • Herbst J.J.
      • Andrews G.C.
      • Contillo L.G.
      • Singleton D.H.
      • Genereux L.G.
      • Gibbs E.M.
      • Lienhard G.E.
      ) or total (
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ) PI 3-kinase activity was measured. However, activation of PI 3-kinase and extensive tyrosine phosphorylation of IRS1 occur in the LDM fraction (
      • Yang J.
      • Clarke J.F.
      • Ester C.J.
      • Young P.W.
      • Kasuga M.
      • Holman G.D.
      ,
      • Kelly K.L.
      • Ruderman N.B.
      ,
      • Ricort J.-M.
      • Tanti J.-F.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Heller-Harrison R.A.
      • Morin M.
      • Czech M.P.
      ), and PI 3-kinase activation has been observed in Glut 4-containing vesicles (
      • Heller-Harrison R.A.
      • Morin M.
      • Guilherme A.
      • Czech M.P.
      ). Interestingly, platelet-derived growth factor increased total PI 3-kinase activity but did not stimulate PI 3-kinase activity in the LDM and did not stimulate Glut 4 translocation in comparison with insulin (
      • Ricort J.-M.
      • Tanti J.-F.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ). Therefore, it is possible that in IL-4-stimulated myoblasts, IRS1, which is thought to be tyrosine phosphorylated by activation of Jak 3 (
      • Witthuhn B.A.
      • Silvennoinen O.
      • Miura O.
      • Lai K.S.
      • Cwik C.
      • Liu E.T.
      • Ihle J.N.
      ), is not phosphorylated in the LDM fraction, and that stimulation of glucose transport may require activation of PI 3-kinase in this particular fraction. In accordance with the hypothesis that the activation of PI 3-kinase has to occur in the appropriate subcellular compartment to promote Glut 4 translocation, we found that p110* was present at a high level in the LDM compartment. The localization of p110* paralleled that of endogenous PI 3-kinase. Thus, it suggests that overexpression of p110* did not induce a generalized change in the subcellular distribution of proteins. Furthermore, the lack of SH2 and SH3 domains of p85 in p110* construct did not alter the subcellular localization of PI 3-kinase and the association of PI 3-kinase with membrane fractions. These data would imply that p85 has no targeting role as far as the association of p110 to these membranes is concerned. Interestingly, insulin treatment of cells transfected with p110* led to a further increase in Glut 4 translocation. A possible explanation is that insulin activates a PI 3-kinase-independent signaling pathway. Alternatively, this effect could be due to the activation of endogenous PI 3-kinase by insulin, suggesting that activation of PI 3-kinase is a rate-limiting step for glucose transporter translocation.
      Future advances in the understanding of the mechanism of Glut 4 translocation will come from the identification of the steps following PI 3-kinase activation. Several studies have focused on the relationship between PI 3-kinase and the small GTPases Rho and Rac. However, a role of Rac in the stimulation of glucose transport seems unlikely because a dominant negative form of this protein did not inhibit this insulin effect, although it blocked insulin-induced ruffling (
      • Marcusohn J.
      • Isakoff S.J.
      • Rose E.
      • Symons M.
      • Skolnik E.Y.
      ). Novel targets of PI 3-kinase have been identified, such as the Akt/PKB kinase (
      • Cross D.A.E.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ,
      • Kohn A.D.
      • Kovacina K.S.
      • Roth R.A.
      ,
      • Burgering B.M.T.
      • Coffer P.J.
      ) and atypical protein kinase Cζ and protein kinase Cλ (
      • Akimoto K.
      • Takahashi R.
      • Moriya S.
      • Nishioka N.
      • Takayanagi J.
      • Kimura K.
      • Fukui Y.
      • Osada S.-I.
      • Misuno K.
      • Hirai S.-I.
      • Kazlauskas A.
      • Ohno S.
      ,
      • Berra E.
      • Diaz-Meco M.T.
      • Dominguez I.
      • Municio M.M.
      • Sanz L.
      • Lozano J.
      • Chapkin R.S.
      • Moscat J.
      ). These kinases are activated by phosphatidylinositol 3-phosphate and hence appear as potential players in the Glut 4 translocation machinery. Although the link between PI 3-kinase activation and the production of phosphatidylinositol 3-phosphate and translocation of Glut 4-containing vesicles remains to be determined, our results show that the PI 3-kinase signaling pathway is sufficient to stimulate glucose transporter translocation.

      Acknowledgments

      We thank M. Cormont for scientific discussion. We acknowledge G. Visciano for illustrations. We thank Genentech for the gift of pCIS2 cDNA.

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