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Potential Role of Protein Kinase B in Glucose Transporter 4 Translocation in Adipocytes¹

INSERM U 145, Faculté de Médecine (J-F.T., S.G., T.G., E.V.O., Y.L-M-B.), 06107, Nice CEDEX 02, France; Department Pulmonary Diseases, University Hospital Utrecht (P.J.C.), Heidelberglaan, 3584 CX Utrecht, The Netherlands

Address all correspondence and requests for reprints to: Jean-François Tanti, INSERM U145, Faculté de Médecine, Avenue de Valombrose, Nice, Cedex 02, 06107, France. E-mail: tanti{at}unice.fr

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phosphatidylinositol 3-kinase (PI 3-kinase) activation promotes glucose transporter 4 (Glut 4) translocation in adipocytes. In this study, we demonstrate that protein kinase B, a serine/threonine kinase stimulated by PI 3-kinase, is activated by both insulin and okadaic acid in isolated adipocytes, in parallel with their effects on Glut 4 translocation. In 3T3-L1 adipocytes, platelet-derived growth factor activated PI 3-kinase as efficiently as insulin but was only half as potent as insulin in promoting protein kinase B (PKB) activation. To look for a potential role of PKB in Glut 4 translocation, adipocytes were transfected with a constitutively active PKB (Gag-PKB) together with an epitope tagged transporter (Glut 4 myc). Gag-PKB was associated with all membrane fractions, whereas the endogenous PKB was mostly cytosolic. Expression of Gag-PKB led to an increase in Glut 4 myc amount at the cell surface. Our results suggest that PKB could play a role in promoting Glut 4 appearance at the cell surface following exposure of adipocytes to insulin and okadaic acid stimulation.

	Introduction

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INSULIN promotes glucose uptake in muscle and adipose tissue by increasing the translocation of glucose transporter 4 (Glut 4) from an intracellular compartment to the plasma membrane (PM). Among the proteins activated in response to insulin, phosphatidylinositol 3-kinase (PI 3-kinase) plays a crucial role in this process. Indeed, inhibition of PI 3-kinase blocks stimulation of glucose transport by insulin (1, 2, 3). Further, expression of a constitutively active form of this enzyme in 3T3-L1 adipocytes or in freshly isolated adipocytes fully mimicks the effect of insulin on Glut 4 translocation (4, 5, 6). However, the mechanism by which this enzyme regulates Glut 4 translocation remains unknown. Recently a series of downstream targets of PI 3-kinase have been identified. Among them is protein kinase B (PKB) or Akt, a serine/threonine kinase of 60 kDa whose catalytic domain is closely related to that of protein kinase C and cAMP-dependent protein kinase (7, 8, 9). Part of the N-terminal sequence of PKB is a pleckstrin homology domain (PH domain). Activation of PKB by tyrosine kinase receptors such as receptors of platelet-derived growth factor (PDGF) or insulin requires PI 3-kinase activation (10, 11, 12). Indeed, pharmacological inhibitors of PI 3-kinase prevent PKB activation by insulin or PDGF (10, 11, 12, 13). Mutated PDGF receptors lacking the PI 3-kinase binding site fail to activate PKB, and a dominant negative mutant of PI 3-kinase prevents PKB activation by PDGF (10, 12). The exact mechanism of PKB activation by PI 3-kinase is not fully determined, but growing evidence suggests that growth factors and insulin activate PKB through phosphorylation by (an) unknown upstream PKB kinase(s) (10, 11, 12, 14, 15).

Although PKB appears to be activated by insulin and growth factors, its exact role remains to be determined. So far it has been implicated in adipocyte differentiation (16), cell growth (9), and regulation of glycogen metabolism by insulin (13). Indeed, the only identified cellular substrate of PKB is glycogen synthase kinase 3 that is phosphorylated and thus inactivated by PKB (13). The aim of the present study was to determine whether PKB could play a role in the regulation of Glut 4 translocation. We first characterized the activation of PKB by insulin and okadaic acid, stimulators of glucose transport in adipocytes (17, 18, 19), and by PDGF, which does not affect Glut 4 translocation (20, 21). We then looked for an effect of a constitutively active PKB (Gag-PKB) on Glut 4 subcellular distribution. To this aim, rat adipocytes were transiently cotransfected with Gag-PKB and a Glut 4 molecule tagged with a myc epitope (Glut 4 myc) in its first extracellular loop (3, 4). The Gag-PKB is formed by fusion of the Gag protein in frame to the coding region of PKB (9). Glut 4 myc allows us to measure Glut 4 translocation in the fraction of transfected cells by binding of antibodies to myc.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Collagenase was from Boehringer (Mannheim, Germany). BSA was purchased from Intergen Co. (Purchase, NY). Polyvinylidene difluoride membranes were from Millipore Corp. (Bedford, MA). ¹²⁵I Igs against mouse immunoglobulins were from Amersham Corp (Buckinghamshire, UK). Restriction enzymes were from Biolabs (Richmond, CA). All other reagents were from Sigma (St. Louis, MO) or Merck (Darmstadt, Germany). PDGF BB was from Pepro (Tech Inc., Rocky Hill, NJ).

Eukaryotic expression vectors
pCIS2 and pCIS-Glut 4 myc.
pCIS2 is an expression vector containing a cytomegalovirus promoter and enhancer with a generic intron located upstream from the multiple cloning site (22). This vector gives high levels of protein expression in adipocytes. The complementary DNA (cDNA) coding for rat Glut 4 with the myc epitope inserted in the first exofacial loop (Glut 4 myc) was constructed and subcloned into pCIS2 (4).

pSG5-Gag-PKB and pSG5-Gag.
pSG5-Gag-PKB was constructed by ligating an 800-bp MoMuLV cDNA fragment encoding the p30 capsid protein in frame with the initiation codon of PKB as described before (12). pSG5-Gag encodes for the Gag protein only.

The plasmid DNAs were obtained using a maxi kit (Qiagen, SA, Courteboeuf, France), and their concentrations were determined by measuring the OD at 260 nm.

Antibodies
Antibody to PKB used in the immunoblotting experiment is directed against a C-terminal peptide of PKB (12). Antibody to PKB used for immunoprecipitation was from Santa Cruz Biotechnology (Santa Cruz, CA) and was directed against the N-terminal part of the protein. Antibody to Glut 4 was raised against the 12 amino acid peptide corresponding to the COOH-terminal sequence of Glut 4 (23). Antibody to the p85 subunit of the PI 3-kinase was from UBI (Lake Placid, NY) or was raised against a peptide corresponding to residues 500–519 of p85 protein. The monoclonal antibody (9E10) to the myc epitope was from Santa Cruz Biotechnology.

Preparation of isolated adipocytes and measurement of PKB activity
Adipose cells were isolated from epididymal fat pads of fed male Wistar rats (200–220 g) by collagenase digestion. PKB activation was determined either by a shift in its apparent molecular weight due to its phosphorylation (11, 14) or by measuring the phosphorylation of Crosstide (Neosystem, Strasbourg, France) (13). Adipocytes were incubated as a 50% (vol/vol) suspension in Krebs-Ringer bicarbonate buffer containing 30 mM HEPES (KRBH), 1% (wt/vol) BSA with insulin or with okadaic acid at the concentrations and durations given in the figure legends. At the end of the incubation, the cell suspensions were centrifuged through dinonylphthalate, and the cell cakes were solubilized in 3% (wt/vol) SDS buffer (24). Protein aliquots (50 µg) were separated by SDS/PAGE and immunoblotted with the antibody to PKB as described below. Alternatively, at the end of the incubation, the cell suspensions were solubilized for 40 min at 4 C in buffer A (20 mM Tris, pH 7.4, 5 mM EDTA, 10 mM Na₄P₂O₇, 100 mM NaF, 2 mM Na₃VO₄) containing 1% Nonidet-P40, 10 µg/ml aprotinin and 1 mM phenylmethanesulphonylfluoride. Lysates were centrifuged for 10 min at 12 000 x g, and the supernatants were immunoprecipitated for 4 h at 4 C with antibodies to PKB (5 µg) coupled to protein G Sepharose beads. Immune pellets were washed three times with buffer A containing 1% Nonidet-P40 and twice with 50 mM Tris, pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol. Kinase assay was performed on the immune pellets by addition of 50 mM Tris pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol, 5 µM ATP (2 µCi), and 30 µM Crosstide. After 30 min at room temperature, samples were adsorbed on phosphocellulose p81 paper, extensively washed in 1% orthophosphoric acid solution, and radioactivity associated to the paper was counted.

Measurement of PI 3-kinase and PKB activities in 3T3-L1 adipocytes
3T3-L1 cells were cultured and induced to differentiate into adipocytes as previously described (25). Sixteen hours before each experiment, 3T3-L1 adipocytes were changed to serum-free DMEM supplemented with 0.5% (wt/vol) BSA. Cells were incubated with insulin (100 nM) or PDGF (50 ng/ml) for 5 min at 37 C. The cells were solubilized for 40 min at 4 C in buffer A containing 1% Nonidet-P40, 10 µg/ml aprotinin, and 1 mM phenylmethanesulphonylfluoride. Lysates were centrifuged for 10 min at 12000 x g, and the supernatants were incubated for 4 h at 4 C with antibodies to the p85 subunit of the PI 3-kinase (10 µl) coupled to protein A Sepharose beads. PI 3-kinase activity was measured on the immune pellets as previously described (21). PKB activity was assayed as described above using Crosstide as substrate.

Assay for cell surface epitope-tagged Glut 4 measurement
Isolated adipocytes were transfected by electroporation using a double electric shock as previously described (4, 22). Adipocytes were transfected with 0.5 µg pCIS-Glut 4 myc and the amounts of pSG5-Gag-PKB indicated in the figure legends. In all cases, the amount of DNA was adjusted to 10 µg by the addition of pCIS. Cells from multiple cuvettes were pooled to obtain the necessary volume of cells for each experiment. After 16–20 h, electroporated adipocytes were washed twice and resuspended at a 10% (vol/vol) suspension in KRBH, 1% (wt/vol) BSA. Cells were then incubated for 30 min at 37 C in the absence or presence of 100 nM insulin. After insulin treatment, cells were incubated with potassium cyanide (2 mM) to prevent Glut 4 redistribution. The level of cell surface epitope-tagged Glut 4 was determined by using the anti-myc mouse monoclonal antibodies (9E10) in conjunction with sheep antimouse ¹²⁵I-Ig as previously described (4, 26). Radioactivity was normalized by measuring protein concentration in each sample using bicinchoninic acid assay (Pierce, Rockford, IL). Cells transfected with pCIS2 alone were used to determine nonspecific binding, which represents 30% of the total binding observed in cells transfected with pCIS Glut 4 myc in the absence of insulin stimulation. This value was substracted from all values.

Subcellular fractionation of adipocytes
Adipocytes transfected with pSG5-Gag PKB were washed three times in KRBH and homogenized in 2 vol 20 mM Tris, pH 7.4, 250 mM sucrose, 1 mM EDTA, and proteases inhibitors using a Thomas potter type C (Bioblock, Strasbourg, France). Plasma membranes (PM), high density microsomal membranes (HDM), and low-density microsomal membranes (LDM) were prepared by differential centrifugation as described (27). Fraction proteins (50–80 µg) were separated on SDS-PAGE, transferred to a polyvynilidene difluoride (PVDF) sheet. Immunodetection of PKB, Gag-PKB, Glut 4, and the p85 subunit of the PI 3-kinase was performed with specific antibodies. After washes, sheets were incubated with ¹²⁵I-labeled protein A, washed, and submitted to autoradiography.

Immunoprecipitation and immunoblotting of Glut 4 myc
Adipose cells were cotransfected with pCIS-Glut 4 myc and either pCIS (control) or pSG5-Gag-PKB (GAG-PKB), and cells were washed and homogeneized as described above. Total membranes were prepared by centrifugation at 300,000 x g for 1 h and solubilized in 20 mM Tris, pH 7.4, 1 mM EDTA, 1% Triton X-100, and proteases inhibitors. Then Glut 4 myc was immunoprecipitated using monoclonal antibodies to myc (5 µg) coupled to protein G Sepharose beads. After washes, Laemmli buffer was added to the pellets, the proteins were separated on SDS-PAGE, transferred to PVDF sheets, and immunoblotted with an antibody to Glut 4.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of insulin, okadaic acid, and PDGF on PKB activation
We first studied PKB activation in freshly isolated rat adipocytes in response to insulin and okadaic acid, which both stimulate glucose transport and Glut 4 translocation (18, 19). PKB was visualized after immunoblotting with an antibody to the C-terminus of the protein. As shown in Fig. 1, in basal condition, a doublet of about 60 kDa that corresponds to different phosphorylation states of the protein was observed (11, 14). Insulin or okadaic acid treatment caused the slowest migrating form to increase in intensity, and in parallel the intensity of the fastest migrating form decreased, suggesting that both agents increased the phosphorylation state of PKB, a process linked to its activation (10, 11, 12, 14, 15). To determine more directly the kinase activity of PKB, we used a kinase assay towards Crosstide, a peptide corresponding to the sequence of glycogen synthase kinase 3 surrounding the serine phosphorylated by MAP kinase activated protein kinase 1 and p70 S6 kinase (13). As shown in Table 1, both insulin and okadaic acid increased the kinase activity of PKB, but the effect of okadaic acid represented only 30% of the insulin effect. The time course of PKB activation by insulin was rapid with a maximal effect within 5 min of insulin treatment, and it was sustained for at least 20 min. The effect of insulin was maximal with about 0.1 nM insulin (Fig. 1).

View larger version (36K): [in this window] [in a new window]   Figure 1. Activation of PKB by insulin and okadaic acid in freshly isolated adipocytes. Adipocytes were prepared as described in Materials and Methods and incubated without or with insulin (Ins) or okadaic acid (Oka) at concentrations and times indicated. Cells were solubilized in Laemmli buffer, and proteins were separated on SDS-PAGE and transferred to PVDF sheets. PKB was vizualized by immunoblotting with a specific antibody as described in Materials and Methods.

View larger version (25K): [in this window] [in a new window]   Figure 2. Activation of PI 3-kinase and PKB by insulin and PDGF in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated as described in Materials and Methods without (open bars) or with insulin (100 nM) (hatched bars) or PDGF (50 ng/ml) (grey bars) for 5 min. Following solubilization, PI 3-kinase and PKB were immunoprecipitated with specific antibodies, and kinase assays were performed on immune pellets as described in Materials and Methods. Results are expressed as percent of insulin effect and represent mean ± SEM of four different experiments.

View larger version (73K): [in this window] [in a new window]   Figure 3. Subcellular distribution of endogenous PKB, Gag-PKB, p85, and Glut 4 in isolated adipocytes. Isolated adipocytes were transfected with 9 µg pSG5-GagPKB as described in Materials and Methods. Subcellular fractionation of adipocytes were performed 24 h later to give PMs, LDMs, HDMs, and CYTOs. Proteins (50–80 µg) were separated by SDS-PAGE and immunoblotted with antibodies to PKB, p85 subunit of PI 3-kinase, and Glut 4.

View larger version (39K): [in this window] [in a new window]   Figure 4. Constitutively active PKB (Gag-PKB) promotes Glut 4 myc translocation in transiently transfected adipocytes. Cells were transfected as described in Materials and Methods with pCIS Glut 4 myc (0.5 µg) and as indicated with 9.5 µg pCIS (CONTROL) or 1 or 9.5 µg pSG5-Gag-PKB. A, After 24 h, adipocytes were incubated for 30 min in absence (white bars) or in presence (hatched bars) of 100 nM insulin before measuring binding of antibodies to myc at the cell surface as detailed in Materials and Methods. Results are expressed as a fold of basal values obtained in cells transfected with pCIS Glut 4 myc and pCIS and incubated without insulin. Values are presented as mean ± SEM of five experiments performed with different cell preparations. B, Adipocytes were homogeneized 24 h after transfection, and total membranes fractions were prepared and solubilized with 1% Triton X-100. Proteins (100 µg) were immunoprecipitated with 5 µg anti-myc antibodies, separated by SDS-PAGE, and immunoblotted with antibodies to Glut 4 as described in Materials and Methods. Triplicate determinations were performed under each condition. Two typical experiments are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
As reviewed in our introduction, several studies have shown that PI 3-kinase is required for insulin effect on glucose transport and Glut 4 translocation. Because PKB is downstream of PI 3-kinase, we looked for its potential role in Glut 4 translocation. We found that insulin activates PKB in freshly isolated adipocytes or 3T3-L1 adipocytes, with a sensitivity and a time course compatible with the hormonal effect on glucose transport (29). These results indicate that the activation of PKB by insulin occurs not only in cells overexpressing insulin receptors (11, 12), but also in a physiological target of the hormone. Okadaic acid, which stimulates Glut 4 translocation without stimulating PI 3-kinase (30), activated PKB, indicating that okadaic acid could increase glucose transport by activating PKB without PI 3-kinase activation. It should be observed that the activation of PKB by okadaic acid (this study) and its effect on Glut 4 translocation (17, 18, 31) were comparable. Activation of PKB through a PI 3-kinase-independent pathway was reported following heat shock or hyperosmolarity stress (32). In 3T3-L1 adipocytes, PDGF, which stimulated PI 3-kinase as efficiently as insulin, was only half as potent as insulin in activating PKB. Further, PDGF did not stimulate Glut 4 translocation (20, 21). The lack of correlation between the levels of PI 3-kinase and PKB activation by PDGF was unexpected because PI 3-kinase was reported to be involved in PKB activation (10, 12). The mechanism underlying PKB activation is not yet completely understood. Indeed, it seems that this activation requires both the production of phosphatidylinositol 3,4,5 triphosphate and the phosphorylation of PKB by (an) unknown protein kinase(s) (10, 11, 12, 14, 15). Phosphatidylinositol 3,4,5 triphosphate produced following PI 3-kinase activation could prime PKB, perhaps by recruiting PKB to membranes, for phosphorylation and full activation (33). The relatively low PDGF effect on PKB compared with insulin could have at least the following two explanations. First, PDGF does not produce the phosphoinositides in the correct membrane fraction. Indeed, in 3T3-L1 adipocytes, PDGF stimulates PI 3-kinase only in PMs (21, 34), whereas insulin increases its activity in PMs and LDMs (21, 34), the fraction enriched in Glut 4-containing vesicles. It is conceivable that the kinase(s) involved in PKB activation would be present only in the LDM fraction. Second, PDGF does not stimulate the upstream kinase(s) involved in PKB phosphorylation. The observation that PDGF, although activating PKB, had no effect on Glut 4 translocation remains unclear. PDGF was as efficient as okadaic acid in activating PKB. Because okadaic acid partly mimicks insulin effect on Glut 4 translocation, whereas PDGF was inactive, it suggests that PKB is not activated in the right subcellular compartment by PDGF. This hypothesis of a difference in the subcellular activation of PKB between insulin and PDGF remains to be tested.

To look more directly for a role of PKB, we tested whether a constitutively active form of PKB (Gag-PKB) could activate Glut 4 translocation. To this aim, we transiently transfected adipocytes with a Glut 4 myc construct. Because the behavior of the epitope-tagged Glut 4 is similar to that of endogenous Glut 4, the use of an epitope-tagged Glut 4 is a reporter for Glut 4 subcellular distribution exclusively in the fraction of transfected cells, which was about 10% in our experimental conditions (4). Our results indicate that expression of Gag-PKB was sufficient to promote Glut 4 translocation to the cell surface as efficiently as insulin. Gag-PKB was localized in membrane fractions, whereas the endogenous PKB was mainly cytosolic. The membrane localization was likely to be due to the presence of a myristoylation site in the sequence of the Gag protein, which allows membrane binding (28). Indeed, addition of a myristoylation site in the PKB sequence is sufficient to target the protein to the membrane leading to its activation (14). This suggests that the constitutive activity of Gag-PKB is probably not due to a conformational change induced by the Gag protein but is the more likely consequence of the localization of the protein close to its upstream membrane activator(s). Interestingly, Gag-PKB was present in the LDM, the fraction enriched in Glut 4-containing vesicles. It has been recently suggested that activation of PI 3-kinase in this fraction was crucial for insulin-induced Glut 4 translocation (21, 34, 35). In accordance with the suggestion that Gag-PKB targeting was important for its action, are the very recently published data demonstrating that a PKB protein targeted to the membranes by addition of a myristoylation motif was sufficient to promote Glut 4 translocation in 3T3-L1 adipocytes (36).

Our results indicate that a constitutively active form of PKB was sufficient to promote Glut 4 translocation to the cell surface. Whether the stimulatory effect of PI 3-kinase on insulin-induced Glut 4 translocation requires PKB activation is not yet proven, because neither a pharmacological inhibitor of PKB nor a dominant negative construct of PKB are available (12). However, the overall picture observed with okadaic acid, PDGF, and insulin suggests the existence of a direct link between PKB activation and Glut 4 translocation.

	Acknowledgments

 
We thank M. Cormont and Carol Sable for scientific discussions. We acknowledge G. Visciano for illustrations. We thank Genentech for the gift of pCIS2 cDNA.

	Footnotes

 
¹ This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (France), the University of Nice, the Institut Benjamin Delessert, and the Association pour la Recherche contre le Cancer (ARC 2111).

Received October 23, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M 1994 Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J Biol Chem 269:3568–3573[Abstract/Free Full Text]
2. Cheatham B, Vlahos CJ, Cheatham L, Wang L, Blenis J, Kahn CR 1994 Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol 14:4902–4911[Abstract/Free Full Text]
3. Quon MJ, Chen H, Ing BL, Miu M-L, Zarnowski MJ, Yonezawa K, Kasuga M, Cushman SW, Taylor SI 1995 Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells. Mol Cell Biol 15:5403–5411[Abstract]
4. Tanti J-F, Grémeaux T, Grillo S, Calleja V, Klippel A, Williams LT, Van Obberghen E, Le Marchand-Brustel Y 1996 Overexpression of a constitutively active form of phosphatidylinositol 3-kinase is sufficient to promote Glut 4 translocation in adipocytes. J Biol Chem 271:25227–25232[Abstract/Free Full Text]
5. Katagiri H, Asano T, Ishihara H, Inukai K, Shibasaki Y, Kikuchi M, Yazaki Y, Oka Y 1996 Overexpression of catalytic subunit p110 of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3–L1 adipocytes. J Biol Chem 271:16987–16990[Abstract/Free Full Text]
6. Martin SS, Haruta T, Morris AJ, Klippel A, Williams LT, Olefsky JM 1996 Activated phosphatidylinositol 3-kinase is sufficient to mediate actin rearrangement and GLUT4 translocation in 3T3–L1 adipocytes. J Biol Chem 271:17605–17608[Abstract/Free Full Text]
7. Coffer PJ, Woodgett JR 1991 Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. Eur J Biochem 201:475–481[Medline]
8. Jones PF, Jakubowicz T, Pitossi FJ, Maurer F, Hemmings BA 1991 Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci USA 88:4171–4175[Abstract/Free Full Text]
9. Bellacosa A, Testa JR, Staal SP, Tsichlis PN 1991 A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science 254:274–277[Abstract/Free Full Text]
10. Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR, Tsichlis PN 1995 The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81:727–736[CrossRef][Medline]
11. Kohn AD, Kovacina KS, Roth RA 1995 Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase. EMBO J 14:4288–4295[Medline]
12. Burgering BMT, Coffer PJ 1995 Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376:599–602[CrossRef][Medline]
13. Cross DAE, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA 1995 Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785–789[CrossRef][Medline]
14. Kohn AD, Takeuchi F, Roth RA 1996 Akt, a pleckstrin homology domain containing kinase, is activated primarily by phosphorylation. J Biol Chem 271:21920–21926[Abstract/Free Full Text]
15. Andjelkovic M, Jakubowicz T, Cron P, Ming X-F, Han J-W, Hemmings BA 1996 Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors. Proc Natl Acad Sci USA 93:5699–5704[Abstract/Free Full Text]
16. Magun R, Burgering BMT, Coffer PJ, Pardasani D, Lin Y, Chabot J, Sorisky A 1996 Expression of a constitutively activated form of protein kinase B (c-Akt) in 3T3–L1 preadipose cells causes spontaneous differentiation. Endocrinology 137:3590–3593[Abstract]
17. Corvera S, Jaspers S, Pasceri M 1991 Acute inhibition of insulin-stimulated glucose transport by the phosphatase inhibitor, okadaic acid. J Biol Chem 266:9271–9275[Abstract/Free Full Text]
18. Tanti J-F, Grémeaux T, Cormont M, Van Obberghen E, Le Marchand-Brustel Y 1993 Okadaic acid stimulates IGF II receptor translocation and inhibits insulin action in adipocytes. Am J Physiol 264:E868–E873
19. Haystead TAJ, Weiel JE, Litchfield DW, Tsukitani Y, Fischer EH, Krebs EG 1990 Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes. The role of protein phosphatase 2A in attenuation of the signal. J Biol Chem 265:16571–16580[Abstract/Free Full Text]
20. Gould GW, Merrall NW, Martin S, Jess TJ, Campbell IW, Calderhead DM, Gibbs EM, Holman GD, Plevin RJ 1994 Growth factor-induced stimulation of hexose transport in 3T3–L1 adipocytes: evidence that insulin-induced translocation of Glut4 is independent of activation of MAP kinase. Cellular Signalling 6:313–320[CrossRef][Medline]
21. Ricort J-M, Tanti J-F, Van Obberghen E, Le Marchand-Brustel Y 1996 Different effects of insulin and platelet-derived-growth-factor on phosphatidylinositol 3-kinase at the subcellular level in 3T3–L1 adipocytes. A possible explanation for their specific effects on glucose transport. Eur J Biochem 239:17–22[Medline]
22. Quon MJ, Zarnowski M, Guerre-Millo M, De La Luz Sierra M, Taylor SI, Cushman SW 1993 Transfection of DNA into isolated rat adipose cells by electroporation. Evaluation of promoter activity in transfected adipose cells which are highly responsive to insulin after one day in culture. Biochem Biophys Res Commun 194:338–346[CrossRef][Medline]
23. Le Marchand-Brustel Y, Olichon-Berthe C, Grémeaux T, Tanti JF, Rochet N, Van Obberghen E 1990 Glucose transporter in insulin sensitive tissues of lean and obese mice. Effect of the thermogenic agent BRL 26830A. Endocrinology 127:2687–2695[Abstract]
24. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
25. Ricort JM, Tanti JF, Van Obberghen E, Le Marchand-Brustel Y 1995 Alterations in insulin signalling pathway induced by prolonged insulin treatment of 3T3–L1 adipocytes. Diabetologia 38:1148–1156[Medline]
26. Quon MJ, Butte AJ, Zarnowski MJ, Sesti G, Cushman SW, Taylor SI 1994 Insulin receptor substrate 1 mediates the stimulatory effect of insulin on GLUT4 translocation in transfected rat adipose cells. J Biol Chem 269:27920–27924[Abstract/Free Full Text]
27. Cormont M, Tanti J-F, Zahraoui A, Van Obberghen E, Tavitian A, Le Marchand-Brustel Y 1993 Insulin and okadaic acid induce Rab4 redistribution in adipocytes. J Biol Chem 268:19491–19497[Abstract/Free Full Text]
28. Ahmed NN, Franke TF, Bellacosa A, Datta K, Gonzales-Portal M-E, Taguchi T, Testa JR, Tsichlis PN 1993 The proteins encoded by c-akt and v-akt differ in post-translational modification, subcellular localization and oncogenic potential. Oncogene 8:1957–1963[Medline]
29. Simpson IA, Cushman SW 1986 Hormonal regulation of mammalian glucose transport. Annu Rev Biochem 55:1059–1089[CrossRef][Medline]
30. Jullien D, Tanti J-F, Heydrick SJ, Gautier N, Grémeaux T, Van Obberghen E, Le Marchand-Brustel Y 1993 Differential effects of okadaic acid on insulin-stimulated glucose and amino acid uptake and phosphatidylinositol-3-kinase activity. J Biol Chem 268:15246–15251[Abstract/Free Full Text]
31. Lawrence JC, Jr, Hiken JF, James DE 1990 Stimulation of glucose transport and glucose transporter phosphorylation by okadaic acid in rat adipocytes. J Biol Chem 265:19768–19776[Abstract/Free Full Text]
32. Konishi H, Matzuzaki H, Tanaka M, Ono Y, Tokunaga C, Kuroda S, Kikkawa U 1996 Activation of RAC-protein kinase by heat shock and hyperosmolarity stress through a pathway independent of phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 93:7639–7643[Abstract/Free Full Text]
33. James SR, Downes CP, Gigg R, Grove SJA, Holmes AB, Alessi DR 1996 Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-triphosphate without subsequent activation. Biochem J 315:709–713
34. Navé BT, Haigh RJ, Hayward AC, Siddle K, Shepherd PR 1996 Compartment-specific regulation of phosphoinositide 3-kinase by platelet derived growth factor and insulin in 3T3–L1 adipocytes. Biochem J 318:55–60
35. Heller-Harrison RA, Morin M, Guilherme A, Czech MP 1996 Insulin-mediated targeting of phosphatidylinositol 3-kinase to GLUT4-containing vesicles. J Biol Chem 271:10200–10204[Abstract/Free Full Text]
36. Kohn AD, Summers SA, Birnbaum MJ, Roth RA 1996 Expression of a constitutively active Akt Ser/Thr Kinase in 3T3–L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271:31372–31378[Abstract/Free Full Text]

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