Overexpression of wild-type Akt1 promoted insulin-stimulated p70S6 kinase (p70S6K) activity and affected GSK3β regulation, but did not promote insulin-stimulated GLUT4 translocation or glucose transport in L6 myotubes

Overexpression of wild-type Akt1 promoted insulin-stimulated p70S6 kinase (p70S6K) activity and affected GSK3β regulation, but did not promote insulin-stimulated GLUT4 translocation or glucose transport in L6 myotubes

Satoshi Noda^a, Kazuhiro Kishi^b, Tomoyuki Yuasa^b, Hideki Hayashi^b, Tetuo Ohnishi^b,
Ikuko Miyata^b, Hiromu Nishitani^a, and Yousuke Ebina^b

^aDepartment of Radiology, The University of Tokushima School of Medicine;and ^bDivision of Molecular Genetics, Institute for Enzyme Research, The University of Tokushima, Japan

Abstract:We have developed a simple, direct and sensitive method to detect GLUT4 on the cell surface. Using this system, we found that PI3-kinase plays a key role in the signaling pathway of insulin-stimulated GLUT4 translocation. One of the down stream effectors of PI3-kinase is serine-threonine kinase Akt (protein kinase B, RAK-PK), but the involvement of Akt in insulin-stimulated GLUT4 translocation is controversial. To investigate whether Akt1 regulates insulin-stimulated GLUT4 translocation and glucose uptake in L6 myotubes, we established L6 myotubes stably expressing c-myc epitope-tagged GLUT4 (GLUT4myc) and mouse wild type (WT) Akt1. We found that overexpression of WT Akt1 promoted insulin-stimulated p70S6 kinase (p70S6K) activity and increased the basal activity of GSK3β, but did not promote insulin-stimulated GLUT4translocation or glucose uptake. These data supported the result that Akt is not a main signaling molecule to transmit the signal of insulin-stimulated GLUT4 translocation or glucose uptake from insulin-activated PI3-kinase. J. Med. Invest. 47:47-55, 2000

Keywords:AKT, p70S6 kinase, GSK3β, GLUT4

INTRODUCTION
Insulin elicits many biological responses such as cellular metabolism and gene expression. One of the most important metabolic responses induced by insulin is the stimulation of glucose uptake in muscle and adipose tissues. This effect occurs as a consequence of the translocation of GLUT4 from the intracellular pool to the cell surface (1-4). Defect of this function is thought to be one of the main causes of non-insulin-dependent diabetes mellitus (NIDDM). Therefore, elucidating the molecular mechanism of GLUT4 translocation is important for understanding the etiology of NIDDM.
To examine the mechanisms of GLUT4 trans-location, we developed a sensitive and quantitative method to measure directly c-myc epitope-tagged GLUT4 (GLUT4myc) on the cell surface (5). Using this system, we found that phosphatidylinositol (PI) 3-kinase (p85/p110 heterodimer type) plays a key role in GLUT4 translocation triggered by insulin, by platelet-derived growth factor, and by epidermal growth factor in cultured cells (6-8).
Activation of PI3-kinase is essential for insulin-stimulated GLUT4 translocation and glucose uptake (7). However, the downstream mediators of PI3-kinase for GLUT4 translocation are still unknown. One candidate molecule is Akt (Protein kinase B or RAC/PK) (9-11). Akt is the cellular homologue of a viral oncogene, v-Akt, and, has therefore also been termed c-Akt (12). It has a PH (pleckstrin homology) domain in the N-terminus which binds to other kinases (13). This PH domain shares structural similarity with PKC (protein kinase C) isozymes and PKA (cyclic AMP-dependent protein kinase) (12). There are three isoforms of Akt;Akt1 (PKBα), Akt2 (PKBβ), and Akt3 (PKBγ) (14-16).
Akt is rapidly activated by insulin and by certain growth factors (17, 18). It is fully activated by phosphorylation of two key regulatory amino acid residues, Thr308 and Ser473 (19). Phosphatidylinositol 3,4,5-triphosphate-dependent protein kinase (PDK1) phosphorylates Akt catalytic domain Thr308 and activates Akt (20). There are two types of constitu-tively active mutants of Akt ; viral Gag protein fused type (21, 22) and myristoylation signal sequence tagged type (23-25). Overexpression of these active Akt promoted p70S6 kinase activity (10, 26), GLUT4 translocation and glucose uptake (23, 24).Overexpression of wild type Akt inhibited GSK3β activity (17). However, Kitamura et al. reported that Akt is not required for insulin-stimulated GLUT4translocaiton or glucose uptake (27).
Therefore, we examined the effects of overex-pressed WT Akt1 on insulin-stimulated GLUT4 translocation, glucose uptake, p70 S6kinase activity, and GSK3β activity in L6 -GLUT4myc myotubes.

MATERIALS AND METHODS
Cells and materials
The parent cell line used in this study was L6-GLUT4myc (28, 29). The HA-tagged mouse wild-type Akt1 (30) was subcloned into a mammalian expres-sion vector, pCXN (31). This plasmid was cotransfected into L6-GLUT4myc cells with pSV2-hph (32), a hygromycin B phosphotransferase expression plasmid using lipofectamine reagent, and selected with hygromycin B (Sigma). Two independent clones were established, #203 and #2, to avoid clonal deviations.

Cell surface anti-c-myc antibody binding assay (GLUT4myc translocation assay)
Cells in24-well plates were incubated in 500μl of Krebs-Ringer-HEPES buffer (KRHB) (5) for 20 min at 37°C and then with given concentrations of ligands for 10 min at 37°C. GLUT4myc translocation was measured as described (29).

2-Deoxyglucose Uptake Measurement
Cells in 24-well plates were treated with given concentrations of ligands for 10 min at 37°C. 2-Deoxyglucose uptake was measured as described previously (5).

Cell lysate and Immunoprecipitation
For Akt phosphorylation and kinase assays, cells in6 -well plates were incubated in KRH buffer for30 min at 37°C, incubated in the absence or presence of 10-7M insulin for 5 min at 37°C, lysed with buffer containing 1% Nonidet P-40, sonicated and centrifuged at 15,000 rpm for 15 min as described previously(33), and the supernatants were incubated with appropriate polyclonal antibodies for 2h at 4°C. The immunocomplexes were precipitated with protein A-Sepharose CL-4B (Amersham Pharmacia Biotech). For p70S6K, GSK3β phosphorylation and kinase assays, cells in 6 -well plates were deprived of serum for4h, incubated in KRH buffer for 30min at 37°C, incubated in the absence or presence of 10-7M insulin for10min at37°C, lysed in a solution containing50mM Hepes (pH7.5), 150mM NaCl, 1% TritonX-100, 20μM P-APMSF, 1mg/ml Bacitracin, 5mM EDTA, 5mM EGTA, 1mM Na pyrophosphate, 1mM Na3VO4, and 20mM NaF, and immunoprecipitated as described above.

Immunoblotting
Cell lysates were boiled for5min in Laemmli sample buffer and subjected to SDS-polyacrylamide gel electrophoresis;the separated proteins were transferred to nitrocellulose filter, probed with the antibodies indicated below and detected as described previously (34).

Antibodies
A monoclonal antibody (9E10) against human c-myc was obtained from the American Type Culture Collection. Phosphospecific Akt (Ser473), p70S6K (Thr389), and GSK3α/β (Ser21/9) antibody were purchased from New England Biolabs, Inc.. Anti-Akt1, anti-p70S6K, and anti-GSK3β were prepared by im-munizing rabbits with keyhole limpet hemocyanin-coupled peptides, the COOH-terminal 20 amino acids of Akt1 (CVDSERRPHFPQFSYSASGTA), COOH-terminal 31 amino acids of p70S6K (MAGVFDIDLDQPEDAGSEDELEEQQNLNESC), and NH3-terminal23amino acids of GSK3β (CDTNAGDRGQTNNAASASASNST).

Analysis of Akt, p70S6K, and GSK3α/β Phos-phorylations
Cells lysates were separated by 7% SDS-polyacrylamide gel electrophoresis and immunoblotted with each Phosphospecific antibody as described above.

Akt, p70S6K, and GSK3β kinase assays
For Akt kinase assays, the immunoprecipitates were washed three times with washing buffer (i)(140mM NaCl, 20mM Tris-HCl (pH 8.0), 1% Nonidet P-40, and 1mM DTT), and once with kinase buffer (50mM Tris-HCl (pH7.5), 10mM MgCl2, 1mM DTT), and incubated for 30min at 30°C in a reaction mixture containing1μM Protein kinase inhibitor, 160μM"Crosstide"(GRPRTSSFAEG) as substrate, 50mM Tris-HCl (pH7.5), 10mM MgCl2, 1mM DTT, 5μM unlabeled ATP, and 3.0μCi/μl [γ-32P] ATP. The reaction was stopped by adding 10μl of 250mM EDTA, and 5mM ATP, and the reaction mixture centrifuged at15,000rpm for1min.. The resulting supernatant (25μl) was spotted onto 2×2cm P81(Whatman) filter paper, dryed and washed15min×4 times with 75mM phosphoric acid, and once with 99.5% Ethanol for1min. The 32P incorporation into the peptide was determined by liquid scintillation spectroscopy.
For p70S6 kinase assays, the immunoprecipitates were washed three times with buffer containing2M LiCl, 82mM NaCl, 12mM Tris-HCl (pH8.0), 0.58% Nonidet P-40, and580μM DTT, and washed twice with kinase buffer (25mM MOPS (pH7.2), 1mM DTT, 6mM MgCl2, 1mM EDTA, and 0.05% Triton X-100), and incubated for 30min at 30°C in a reaction mixture (25μl) containing 1μM Protein kinase inhibitor, 200μM 40SR-pep20 (KRRRLASLRASYSKSESSQK) as substrate, 25mM MOPS (pH7.2), 1mM DTT, 6mM MgCl2, 1mM EDTA, and 0.05% Triton X-100, 5μM unlabeled ATP, and3.0μCi/μl [γ-32P] ATP. The kinase activity was determined as described above for Akt kinase assays.
For GSK3β kinase assays, the immunoprecipitates were washed once with buffer (i), as described above, washed twice with buffer (ii) (100mM Tris-HCl (pH7.4), 0.5M LiCl, and 1mM DTT), and washed twice with kinase buffer (20mM Hepes, 10mM MgCl2, and 1mM DTT), and incubated for 30min at 30°C in a reaction mixture (25μl) containing 2μM Protein kinase inhibitor, 80μM p-CREB (KRREILSRRP(p)SYR) as substrate, 16mM Hepes, 8mM MgCl2, 0.8mM DTT, 35μM unlabeled ATP, and3.0μCi/μl [γ-32P]ATP. The kinase activity was deter-mined as described above in Akt kinase assays.

RESULTS
Overexpression of WT Akt1 promoted the basal and insulin-stimulated kinase activity of Akt in L6myotubes
We transfected HA-tagged mouse WT Akt1 into parent L6GLUT4myc cells, and established two independent stable clones, #203 and #2. To confirm the overexpression of WT Akt1, the cell lysate was immunoblotted with a polyclonal anti-Akt1 anti-body (Fig. 1A) and anti-HA antibody (Fig. 1B). The exogenous mouse WT Akt1 was overexpressed at about 5-fold the level of endogenous rat Akt1 in L6GLUT4myc. Fig. 1B shows that the lysates from parent cells did not immunoreact with the HA anti-body. To examine the activity of the exogenously overexpressed WT Akt1 in L6 myotubes, we assessed Akt phosphorylation (Fig.2A) and kinase activity after insulin stimulation (Fig. 2B, C). Overexpression of WT Akt1 promoted the insulin-stimulated Akt phosphorylation of Ser 473 about 2-3 fold compared to that of parent L6GLUT4myc cells (Fig.2A). Overexpression of WT Akt1 elevated basal Akt activity about 2-fold, and insulin-stimulated Akt activity about 2-3 fold after immunoprecipitation with anti-Akt1 antibody (Fig.2B). The activation of exogenously expressed Akt1 was detected after immunoprecipitation with anti-HA antibody (Fig. 2C).
Overexpression of WT Akt1 promotes the insulin-stimulated kinase activity of p70S6K in L6 myotubes
To examine the effect of overexpressed WT Akt1 on one of the downstream effectors, p70S6K, in L6 myotubes, we assayed p70S6K phosphorylation and the kinase activity after insulin stimulation (Fig.3A, B). Overexpression of WT Akt1promoted the insulin-stimulated p70S6K phosphorylation of Thr 389 about 3-fold compared to that of parent L6GLUT4myc cells (Fig. 3A). The insulin-stimulated p70S6 kinase activity was promoted by the over-expression of WT Akt1 about 2-3 fold compared to that of parent cells (Fig.3B).
Overexpression of WT Akt1 affects the basal kinase activity of GSK3β in L6 myotubes
GSK3β is a down stream target of Akt, and nega-tively regulated by Akt-dependent phosphorylation(17, 35). We assayed the phosphorylation and activity of GSK3β kinase to examine whether the exogenously overexpressed WT Akt1 affects the GSK3β activity in the insulin signaling pathway. The phosphorylations of GSK3α (Ser21) and GSK3β (Ser9) were increased after insulin stimulation, and were promoted by the overexpression of WT Akt1 in L6 myotubes (Fig.4A). Stable overexpression of WT Akt1 results in about a1.5 - 1.7 fold increase of basal GSK3β activity relative to that of parent cells (Fig. 4B) (see"Discussion"). Insulin-stimulated GSK3β kinase activity was reduced about 43% in parental cells (L6GLUT4myc) and about 28% in the cells overexpressing WT Akt1 (#203, #2) (Fig.4B). These results indicated that overexpression of WT Akt1 affected the insulin-induced phosphorylation and the inactivation of GSK3β.
WT Akt1 overexpression did not affect the GLUT4translocation or glucose uptake in L6 myotubes
Exogenously overexpressed Akt in L6myotubes promoted insulin-stimulated Akt and p70S6K activities. Overexpression of WT Akt1 promoted insulin-stimulated the phosphorylation (Ser9) and reduced the activity of GSK3β. We therefore exam-ined insulin-stimulated GLUT4 translocation and glucose uptake in L6 myotubes overexpressing WT Akt1. Despite that exogenously overexpressed WT Akt1 promoted insulin-stimulated Akt activity, promoted p70S6K activity and reduced insulin-stimulated GSK3β activity, no significant differences of insulin-stimulated GLUT4 translocation or insulin-stimulated glucose uptake were found between L6GLUT4myc myotubes overexpressing WT Akt1 and parent L6GLUT4myc myotubes (Fig. 5A, B).

DISCUSSION
Overexpression of a constitutively active mutant of Akt promoted GLUT4 translocation and glucose uptake (21, 23, 24), while overexpression of a dominant negative mutant of Akt inhibited the translocation and uptake (23, 36). However, Kitamura et al. reported that overexpression of a constitutively active mutant of Akt and dominant negative mutant of Akt using an adenovirus expression system did not affect insulin-stimulated GLUT4 translocation or glucose uptake (27). Therefore, the relationship between Akt and insulin-stimulated GLUT4 translocation and glucose uptake is still controversial. Further-more, the effects of the dominant negative mutant of Akt, in which at two phosphorylation sites (Thr308 and Ser473) the residue was changed to alanine, are also controversial (25, 27, 36). We established stable CHO cells which overexpressed the mutant Akt at ten- fold the level of endogenous Akt, but a10- fold overexpression was not sufficient to inhibit endog-enous insulin-stimulated Akt activation (data not shown). Stable overexpression of functional dominant negative mutant Akt might be difficult because Akt is a mediator of cell survival which prevents apoptosis (37). Therefore, we overexpressed wild type Akt in this study. Although Akt negatively regu-lated the activity of GSK3β by the phosphorylation of Ser9, the stable overexpression of WT Akt1increased basal activity of GSK3β about 1.7-fold compared to parent L6GLUT4myc cells (Fig.4B). The same results were obtained in CHO clones (data not shown). The inconsistency of these results may be explained by the functions of Akt. There are several effectors of Akt and the regulation of GSK3β activity might be quite complicated. We found that overexpression of WT Akt1 in L6GLUT4myc affected downstream effectors, such as p70S6 kinase and GSK3β, but not GLUT4 translocation or glucose uptake. These results, however, do not indicate wheth-er Akt1 is a mediator of insulin-stimulated GLUT4 translocation and glucose uptake or not. Exogenous expression of Akt1 does not affect the insulin-stimulated GLUT4 translocation or glucose uptake, because the level of endogenous Akt1 is sufficient mediate this effect. This is not necessarily the case, however. An approximately 2-fold increase in Akt activity was observed in L6GLUT4myc cells after stimulation by insulin (Fig.2B). The basal Akt activities in the cells overexpressing WT Akt1 were two-fold higher than the basal Akt activity of the parent cells (L6GLUT4myc) (Fig.2B). However, there was no effect on the basal level of GLUT4 translocation or glucose uptake (Fig.5B).
Our results indicated that Akt is not a main signaling molecule to transmit between PI3-kinase and insulin-stimulated GLUT4 translocation.

ACKNOWLEDGMENT
We thank Dr. A. Bellacosa for kindly providing plasmids, as well as Y. Mitsumoto and A. Klip for kindly providing L6 cells.

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Received for publication November 30, 1999;accepted December 17, 1999.

Address correspondence and reprint requests to Yousuke Ebina, M.D., Ph.D., Division of Molecular Genetics, Institute for Enzyme Research, The University of Tokushima, Kuramoto-cho, Tokushima770-8503, Japan and Fax:+81-88-633-7437.