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Originally published In Press as doi:10.1074/jbc.M604534200 on October 18, 2006

J. Biol. Chem., Vol. 281, Issue 51, 39273-39284, December 22, 2006
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Amino Acid Transporter ATA2 Is Stored at the trans-Golgi Network and Released by Insulin Stimulus in Adipocytes*

Takahiro Hatanaka{ddagger}, Yasue Hatanaka{ddagger}§, Jun-ichi Tsuchida, Vadivel Ganapathy||, and Mitsutoshi Setou{ddagger}§**1

From the {ddagger}Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511, Japan, §PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan, the Mitsubishi Pharma Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-0033, Japan, the ||Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912-2100, and the **National Institute of Physiological Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan

Received for publication, May 11, 2006 , and in revised form, October 12, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, we cloned the ATA/SNAT transporters responsible for amino acid transport system A. System A is one of the major transport systems for small neutral and glucogenic amino acids represented by alanine and is involved in the metabolism of glucose and fat. Here, we describe the cellular mechanisms that participate in the acute translocation of ATA2 by insulin stimulus in 3T3-L1 adipocytes. We monitored this insulin-stimulated translocation of ATA2 using an expression system of enhanced green fluorescent protein-tagged ATA2. Studies in living cells revealed that ATA2 is stored in a discrete perinuclear site and that the transporter is released in vesicles from this site toward the plasma membrane. In immunofluorescent analysis, the storage site of ATA2 overlapped with the location of syntaxin 6, a marker of the trans-Golgi network (TGN), but not with that of EEA1, a marker of the early endosomes. The ATA2-containing vesicles on or near the plasma membrane were distinct from GLUT4-containing vesicles. Brefeldin A, an inhibitor of vesicular exit from the TGN, caused morphological changes in the ATA2 storage site along with the similar changes in the TGN. In non-transfected adipocytes, brefeldin A inhibited insulin-stimulated uptake of {alpha}-(methylamino)isobutyric acid more profoundly than insulin-stimulated uptake of 2-deoxy-D-glucose. These data demonstrate that the ATA2 storage site is specifically associated with the TGN and not with the general endosomal recycling system. Thus, the insulin-stimulated translocation pathways for ATA2 and GLUT4 in adipocytes are distinct, involving different storage sites.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The significance of the muscle and liver in amino acid metabolism has been well recognized and the involvement of these two tissues in amino acid uptake has been investigated in detail (1). The adipose tissue also possesses multiple amino acid transport systems (2). In addition to the obvious role of amino acids in protein synthesis, available evidence indicates that amino acids may have other important functions in the adipose tissue. Adipose tissue uses amino acids to synthesize fatty acids, triglycerides, and glycerol-based phospholipids (3, 4). Furthermore, amino acids modulate the magnitude of insulin-stimulated translocation of the facilitated glucose transporter GLUT4 (5), glutamine modulates fat metabolism through a regulatory effect on hexosamine biosynthesis (6), and arginine enhances insulin-stimulated glycogen synthesis (7). Therefore, it seems that amino acid uptake may influence glucose and fat metabolism in adipose tissue.

Under physiological conditions, uptake of small neutral and gluconeogenic amino acids such as glutamine and alanine into adipocytes occurs primarily via the amino acid transport system A (2). System A is a Na+-dependent active transport system for neutral amino acids expressed in most tissues (8). A unique characteristic of this system is its ability to recognize N-alkylated amino acids as substrates (9). {alpha}-(Methylamino)isobutyric acid (MeAIB)2 is commonly used as a model substrate for system A. Among the various amino acid transport systems known to be expressed in mammalian cells, system A is best known for its regulation (10-12). However, most of the studies investigating the regulatory aspects of this system have been carried out only at the phenomenological level. Very little is known about the molecular events involved in the process. In the past, the lack of appropriate tools such as transporter-specific antibodies and cDNA probes precluded studies on the molecular mechanisms involved in the regulation of system A.

Recently, the molecular identity of amino acid transport system A has been established (13-20). These studies have identified three distinct transporter proteins that are responsible for system A transport activity in mammalian cells and all three transporters are capable of mediating the Na+-coupled uptake of the system A model substrate MeAIB. The three transporters are known as amino acid transporter A (ATA)1 (also known as SNAT1), ATA2 (SNAT2), and ATA3 (SNAT4). These transporters belong to the solute-linked carrier family SLC38 (21). ATA1 and ATA2 possess similar functional characteristics, but exhibit differential tissue expression patterns. ATA1 is expressed primarily in the placenta and brain, whereas ATA2 is expressed ubiquitously in mammalian tissues. ATA3 is functionally distinguishable from ATA1 and ATA2 and its expression is restricted to the liver. Among these transporters, it is generally believed that ATA2 represents system A, which is known for its regulatory features. There is evidence to indicate that ATA2 corresponds to system A activity in adipocytes (2, 22).

It is known that system A activity in adipocytes is influenced by long-time exposure to insulin (23, 24). However, the molecular mechanisms involved in this phenomenon remain unknown. Moreover, whether insulin has any role in the acute regulation of amino acid flux in this tissue is not known.

Here, we investigated the cellular events involved in the trafficking of ATA2 in response to insulin in 3T3-L1 adipocytes. The present study shows for the first time that the perinuclear storage site for ATA2 is identical to the trans-Golgi network (TGN), that insulin acutely up-regulates system A activity by facilitating the translocation of ATA2 in vesicles from the TGN to the plasma membrane, and that the ATA2-containing vesicles are different from the GLUT4-containing vesicles.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Mouse ATA2 (mATA2) cDNA was cloned from mouse kidney cDNA library using rat ATA2 cDNA as the hybridization probe, as described previously (13, 14, 16, 17, 19, 25). 3T3-L1 murine fibroblasts were from HSRRB (Osaka, Japan). CHO-K1 cells were provided by the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). Cell culture media, Lipofectamine 2000, Alexa 568-conjugated secondary antibodies, and Alexa 568-conjugated phalloidin were purchased from Invitrogen. [14C]MeAIB and L-[14C]tryptophan were from American Radiolabeled Chemicals (St. Louis, MO). 2-Deoxy-D-[14C]glucose ([14C]2-DG) was purchased from Moravek Biochemicals (Brea, CA). Wortmannin was purchased from Merck Calbiochem (Darmstadt, Germany). Latrunculin A, latrunculin B, and brefeldin A were purchased from Sigma. The anti-GFP polyclonal antibody was purchased from Medical Biological Laboratories (Nagoya, Japan). The horseradish peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). The polyclonal antibodies against EEA1, GM130, syntaxin 6, and Rab11 were obtained from BD Pharmingen. The anti-GLUT4 polyclonal antibody was from CHEMICON (Temecula, CA). The monoclonal antibody against transferrin receptor (TfnR) was from Zymed Laboratories Inc..

Cell Culture—3T3-L1 fibroblasts (preadipocytes) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum, and differentiation was induced according to established protocols (26, 27). Briefly, cells were allowed to reach confluence at least 2 days prior to the induction of differentiation. Differentiation was induced (day 0) with high-glucose DMEM, 10% fetal bovine serum containing 0.25 µM dexamethasone, 5 µg/ml insulin, and 500 µM methylisobutylxanthine. After 48 h (day 2), the cells were fed with high-glucose DMEM, 10% fetal bovine serum containing 5 µg/ml insulin. After an additional 48 h (day 4), the cells were re-fed every 2 days with high-glucose DMEM, 10% fetal bovine serum. All media were supplemented with 2 mM glutamine, 50 units of penicillin/ml, and 50 µg of streptomycin/ml. Differentiation was monitored by noting the accumulation of lipid droplets, which typically began by day 4 of differentiation. Cells were considered fully differentiated between days 8 and 12. CHO-K1 cells were cultured in Ham's F-12 medium according to the protocol of the provider.

Reverse Transcription (RT)-PCR—ATA isoform-specific oligonucleotide primer pairs were synthesized to match bp 97-107 (ATA1 forward, 5'-ACGACTCTAATGACTTCACAG-3') and bp 785-764 (ATA1 reverse, 5'-ACTGACTGTCGAGTTCTGCTCC-3') of the mouse ATA1 sequence (NM 134086), bp 327-347 (ATA2 forward, 5'-AACTACTCATACCCCACCAAG-3') and 1031-1011 (ATA2 reverse, 5'-AAAGGTGCCGTTCACAGTTTC-3') of the mouse ATA2 sequence (BC 041108), and bp 1056-1075 (ATA3 forward, 5'-GAGTACGAAGCCCAGGGTGC-3') and 1773-1754 (ATA3 reverse, 5'-CCCGGGATTAGTGGTGATTG-3') of the mouse ATA3 sequence (NM 027052). Total RNA was isolated from 3T3-L1 cells and mouse brain and liver using TRI reagent (Sigma) and subsequent protocol. RT-PCR was performed using ReverTraAce (TOYOBO, Osaka, Japan) and the AmpliTaq Gold DNA polymerase (Applied Biosystems) according to the manufacturer's protocol using the following PCR amplification conditions: 94 °C for 1 min, 56.5 °C for 1 min, 72 °C for 2 min, repeated for 32 cycles. RT-PCR products were resolved on agarose/Tris acetate/EDTA gel and detected with ethidium bromide under UV light.

Uptake Experiments—Prior to uptake experiments, 3T3-L1 cells were fed serum-free DMEM for 4 h, and then incubated with or without insulin (1 µM) in uptake buffer (pH 7.4) for 30 min. Because we wanted to obtain the maximal effect of insulin for MeAIB uptake, we used 1 µM insulin according to the previous report showing that insulin concentration with the saturated maximal effect was several hundred nM (24). The uptake buffer was 25 mM Tris/HEPES (pH 8.0), for MeAIB (14, 16) or HEPES/Tris (pH 7.4), for 2-DG or L-tryptophan, and the buffer contained 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, and 0.8 mM MgSO4. The uptake experiment was performed in 3T3-L1 cells at 37 °C for 30 min with [14C]MeAIB, [14C]2-DG, or L-[14C]tryptophan as the substrate. The concentration of the radiolabeled substrate was 3.6, 10, and 30 µM for MeAIB, 2-DG, and L-tryptophan, respectively. The culture medium was removed by aspiration, and the cells were washed once with the uptake buffer. 0.25 ml of uptake buffer containing radiolabeled substrate (MeAIB, 2-DG, or L-tryptophan) was added to the wells and incubated for 30 min at 37 °C. Uptake was terminated by aspirating the buffer and subsequently washing the cells twice with ice-cold fresh uptake buffer. The cells were then lysed with 0.25 ml of 1% SDS in 0.2 N NaOH, and the lysate was transferred to scintillation vials for quantification of radioactivity. Carrier-mediated uptake of the substrate was calculated by subtracting the uptake measured in the presence of an excess amount of unlabeled substrate (10 mM) from the uptake measured in the absence of unlabeled substrate.

Transient Expression and Characterization of EGFP-tagged ATA2 in CHO-K1 Cells—CHO-K1 cells that transiently express mATA2 tagged with enhanced green fluorescent protein (EGFP) were established using the BD Living Colors pEGFP vector system from Clontech. The full-length cDNA of mATA2 was subcloned into the pEGFP-C2 vector. We have named this construct EGFP-ATA2. CHO-K1 cells were plated onto 24-well plates 1 day before transfection. EGFP-ATA2 cDNA was transfected into the cells using Lipofectamine 2000. The expression of EGFP-ATA2 protein in CHO-K1 cells was evaluated by confocal laser scanning fluorescence microscopy and Western blot with the anti-GFP antibody. The functional activity of the expressed EGFP-ATA2 protein was monitored by measurements of MeAIB uptake.

Stable Expression of EGFP-tagged ATA2 in 3T3-L1 Cells— 3T3-L1 cells that stably express EGFP-ATA2 protein were also established using the BD Living Colors pEGFP vector system (Clontech). EGFP-ATA2 vector was introduced into 3T3-L1 preadipocytes using Effectene reagent from Qiagen (Valencia, CA). The transfected cells were cultured in a medium that contained G418 (Sigma). After 2 weeks of culture, cells that formed single colonies were isolated, and stored individually. After that, we screened these stable cell lines for their ability to differentiate into adipocytes, and used only those that could differentiate for the subsequent experiments. We also verified by fluorescence-activated cell sorter analysis that all the cells expressed EGFP. We have named this cell line EGFP-ATA2 3T3-L1 cells.

Biotinylation of Cell Surface Proteins—Biotinylation of cell surface proteins was performed by the method described by Rotmann et al. (28) for the study of the internalization of the cationic amino acid transporter with slight modifications. Briefly, the preadipocytes stably expressing EGFP-ATA2 protein were grown to confluence and differentiated into adipocytes in 10-cm dishes. After the experimental treatment, the cells were rinsed with ice-cold phosphate-buffered saline (PBS) containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBS+), and incubated in the same solution supplemented with 0.5 mg/ml sulfosuccinimidobiotin (EZ-Link sulfo-NHS-SS-Biotin: Pierce) for 30 min at 4 °C. The cells were then rinsed with the Quenching Solution in the Cell Surface Protein Biotinylation and Purification Kit (Pierce) once and Tris-buffered saline twice to quench any unbound biotin. The cells were then lysed by the addition of 1 ml of radioimmunoprecipitation assay buffer (100 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibitors (Complete EDTA free: Roche Diagnostics) for 30 min at 4 °C. After removal of the cellular debris, protein concentrations of the lysates were determined using the Bradford method. 1 mg of the lysate proteins were batch-extracted overnight at 4 °C using avidin-coated agarose beads (immobilized NeutrAvidin, Pierce), and then released from the beads by incubation in SDS-PAGE sample buffer (50 mM Tris/HCl (pH 6.8), 2% SDS, 100 mM dithiothreitol, 10% glycerol, 0.001% bromphenol blue, 5 min at 95 °C).

Western Blot Analysis—Lysate proteins and cell surface proteins were separated in 8% SDS-PAGE and then blotted to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked with 10% skim milk, and probed with diluted primary antibodies (1:1000-2000) at 4 °C overnight. The bound antibodies were detected with diluted horseradish peroxidase-conjugated secondary antibodies (1:5000-10000) at room temperature for 1 h. Signals were visualized using the ECL kit (Amersham Biosciences). Quantification of signals was carried out using Scion Image imaging software. The values of signal intensity in the cell surface protein blot were normalized with those in the total lysate blot.

Imaging of Movement of EGFP-ATA2-containing Vesicles in Living Cells—The preadipocytes, which stably express EGFP-ATA2 protein, were grown to confluence, differentiated into adipocytes on a glass-bottom dish, and imaged by a specially devised incubation microscope (LCV100, Olympus Co., Ltd., Tokyo, and SANYO Electric Biomedical Co., Ltd., Tokyo). This device possesses the fluorescence microscopy capability to monitor fluorescence in living cells under normal culture conditions (temperature 36.8 ± 0.2 °C, humidity 90% or more, and CO2 5%). Simultaneous GFP and differential interference contrast images were collected at intervals of 4 min for time lapse experiments. The cells were visualized with a x40 objective lens (UAP040X/340, NA 0.9, Olympus) using standard filter sets and a mercury lamp. Sequential images were acquired with a cooled charge-coupled device camera (DP30BW, Olympus) with a 600-ms exposure time every 4 min. The images with several focuses along the z axis were acquired to catch the images depicting the movement of puncta or vesicles from the perinuclear site during insulin stimulation. Data were analyzed using Metamorph software from Universal Imaging Corp. (Downingtown, PA).

Immunofluorescent Analysis and Confocal Laser Scanning Microscopy—3T3-L1 adipocytes were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 in PBS for 15 min. Coverslips were incubated with diluted primary antibodies (1:200-500) for 1 h and with diluted Alexa 568-conjugated secondary antibodies (1:200) in 2% skim milk, 0.1% Triton X-100, 0.02% SDS, PBS for 30 min. Images were taken with upright confocal laser scanning microscope LSM5 PASCAL (Carl Zeiss, Oberkochen, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ATA2 Is Mostly Responsible for Uptake of MeAIB in 3T3-L1 Adipocytes and Preadipocytes—To confirm whether ATA2 mRNA is mainly expressed in 3T3-L1 cells, we performed RT-PCR using mouse-specific ATA1, ATA2, and ATA3 primers (Fig. 1A). We used total RNA of mouse brain and liver as positive control samples for detection of ATA1 and ATA2/3, respectively, according to our previous reports (13, 14, 16, 17, 19). The results from positive controls show that all three primer pairs function appropriately. 3T3-L1 adipocytes and preadipocytes generated the expected 705-bp PCR fragment for ATA2. In contrast, ATA1 mRNA was not detected. ATA3 mRNA was detected at very low levels in preadipocytes, compared with ATA2. As a negative control, an equal volume of water replaced reverse transcriptase in the RT-PCR, and no amplified products were obtained (results not shown).

Insulin Stimulates Carrier-mediated MeAIB Uptake in 3T3-L1 Adipocytes Partially by the Phosphatidylinositol 3'-Kinase-Actin Polymerization Signal Pathway—We first confirmed that acute treatment of adipocytes with insulin increases system A activity, as monitored by the Na+-dependent uptake of MeAIB, without involving de novo synthesis of the transporter protein (Fig. 1B, MeAIB). After the insulin stimulus, carrier-mediated uptake of MeAIB was markedly increased, uptake activity in insulin-treated cells being twice as that in untreated cells. Under identical conditions, insulin also stimulated the carrier-mediated uptake of 2-DG, attributable mainly to GLUT4 activity, to the level of more than 700% of control (Fig. 1B, 2-DG). Carrier-mediated uptake of L-tryptophan, an amino acid that is not recognized by system A, was not affected by insulin (Fig. 1B, L-Trp). We then investigated the molecular events involved in stimulation of MeAIB uptake by insulin. The insulin-stimulated uptake of MeAIB was inhibited to less than 30% of control by 100 nM wortmannin, an inhibitor of phosphatidylinositol 3'-kinase, whereas the insulin-stimulated uptake of 2-DG was blocked almost completely under identical conditions (Fig. 1C). Treatment of the cells with 0.2 µg/ml latrunculin A and 1 µg/ml latrunculin B, specific inhibitors of actin polymerization, decreased the insulin-stimulated MeAIB uptake to 40 and 30% of control, respectively. Under identical conditions, the insulin-stimulated uptake of 2-DG was reduced to 70% of control (Fig. 1D, a). To confirm that the treatment condition of latrunculin A and B is appropriate, we undertook immunohistochemical staining of the actin filament (Fig. 1D, b-d). The images show these reagents actually disrupted the actin structure as reported previously (29). Similar results for MeAIB uptake were obtained with 3T3-L1 undifferentiated preadipocytes (Fig. 1E).


Figure 1
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  		FIGURE 1.
A, mRNA expression pattern of ATA isoforms in 3T3-L1 adipocytes and preadipocytes. We performed RT-PCR using mouse-specific ATA1, ATA2, and ATA3 primers. We used total RNA of mouse brain and liver as positive control samples for detection of ATA1 and ATA2/3, respectively, according to our previous reports (13, 14, 16, 17, 19). B, effect of acute treatment with insulin on the carrier-mediated uptake of MeAIB (3.6 µM), 2-DG (10 µM), or L-tryptophan (30 µM) in 3T3-L1 adipocytes. Values are mean ± S.E. from three determinations. C, effect of wortmannin (100 nM), a phosphatidylinositol 3'-kinase inhibitor, on insulin-stimulated uptake of MeAIB (3.6 µM) or 2-DG (10 µM) in 3T3-L1 adipocytes. Adipocytes were incubated with wortmannin for 40 min prior to and during insulin treatment. Values are mean ± S.E. from three determinations. D, panel a, effect of latrunculin A and B (0.2 µg/ml and 1 µg/ml, respectively), inhibitors of actin polymerization, on insulin-stimulated uptake of MeAIB (3.6 µM) or 2-DG (10 µM) in 3T3-L1 adipocytes. Adipocytes were incubated with latrunculin A or B for 40 min prior to and during insulin treatment. Values are mean ± S.E. from three determinations. Panels b-d, immunohistochemical staining of actin with Alexa 568-conjugated phalloidin in 3T3-L1 adipocytes treated with none (b), latrunculin A (c), and latrunculin B (d). Adipocytes were incubated with latrunculin A or B for 40 min prior to and during insulin treatment. E, effect of acute treatment with insulin on MeAIB (3.6 µM) uptake and of wortmannin (100 nM) on the insulin-stimulated uptake of MeAIB (3.6 µM) in 3T3-L1 preadipocytes. Adipocytes were incubated with wortmannin for 40 min prior to and during insulin treatment. Values are mean ± S.E. from three determinations.

 
Characterization of EGFP-tagged ATA2 Transiently Expressed in CHO-K1 Cells—To carry out the cell biological approach for direct observation of ATA2 translocation stimulated by insulin, we first constructed the plasmid for EGFP-tagged mATA2 (EGFP-ATA2), transfected the construct into CHO-K1 cells, and evaluated the expression and function of the fusion transporter, because CHO-K1 cells show much higher transfection efficiency and lower basal ATA activity than 3T3-L1 preadipocytes and adipocytes. Furthermore, there is information available in the literature for the transfection of the myc- and GFP-tagged GLUT4 constructs into CHO-K1 cells (5). EGFP tagging is a powerful tool to observe the molecular traffic, especially in real time; however, we have to make certain that the tagging does not perturb the native function and property of the molecule, especially in a case of membrane proteins. Because of the lack of a good antibody against ATA2 to detect endogenous ATA2 protein in 3T3-L1 cells, we analyzed the property of the EGFP-ATA2 construct by three criteria: first, the EGFP signal is membrane-associated, not diffuse or aggregative; second, the construct produces appropriate molecular weight bands detected in the Western blot; and third, the construct retains the native transporter activity in the cells. Localization of EGFP-ATA2 protein was evaluated with a confocal laser scanning microscope (Fig. 2A). EGFP-ATA2 was localized primarily on the plasma membrane, whereas EGFP was expressed uniformly throughout the cytoplasm in CHO-K1 cells. The expression of the fusion protein was also evaluated by Western blot using an anti-GFP antibody with cell lysates (Fig. 2B). We detected a major band at an approximate size of 74 kDa only with the lysate of CHO-K1 cells transfected with EGFP-ATA2. This band was not detected with the lysate of non-transfected control cells. We also evaluated the transport function of the EGFP-tagged transporter by measuring the uptake of MeAIB (Fig. 2C). The uptake was 80% higher in cells transfected with the EGFP-mATA2 plasmid compared with cells transfected with the control EGFP plasmid. Thus, this construct successfully met the three criteria, establishing the appropriateness of the EGFP-ATA2 fusion transporter for further studies using stable expression in 3T3-L1 cells.

Insulin Stimulates Cell Surface Recruitment of EGFP-tagged ATA2 from the Intracellular Compartment in 3T3-L1 Adipocytes—We established a 3T3-L1 cell line that stably expressed the EGFP-tagged mATA2 protein. These cells were differentiated into adipocytes and used for the analysis of insulin action on ATA2. We selected the image of each cell with similar expression levels of EGFP-ATA2 from respective representative images for comparison, because the amount of EGFP-ATA2 on the surface of cells detected by microscopy is dependent on the total amount expressed in cells; those that express the highest levels of the reporter appears to have the most on the surface. EGFP-ATA2 protein was predominantly localized in the perinuclear site and the plasma membrane in differentiated 3T3-L1 adipocytes, and acute insulin treatment promoted plasma membrane recruitment of the fusion protein (Fig. 3A). Increased numbers of intracellular vesicles containing EGFP-tagged ATA2 were also observed after insulin treatment (Fig. 3A). To quantify the cell surface recruitment of EGFP-ATA2, we performed Western blots with cell surface proteins and total lysates. The blots were probed with an anti-GFP antibody (Fig. 3B). The values of signal intensity in cell surface protein blots were normalized with those in the total lysate blot. The data show that insulin treatment to the cells increased the cell surface recruitment of EGFP-ATA2 ~2-fold (2.38 ± 0.26, n = 4) compared with untreated control cells. For comparison, we analyzed the insulin-stimulated translocation of TfnR by re-blotting the polyvinylidene difluoride membranes used for quantification of EGFP-ATA2 translocation. The results show that cell surface TfnR is increased by 1.71 ± 0.21-fold. The -fold increase was greater for EGFP-ATA2 than for TfnR in all four samples, and the difference between EGFP-ATA2 and TfnR is significant in paired t tests (p = 0.034).


Figure 2
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  		FIGURE 2.
Characterization of EGFP-tagged ATA2 protein expressed transiently in CHO cells. A, images were captured with a confocal laser scanning microscope. Scale bar,10 µm. The data are from a representative experiment and similar results were obtained from two other experiments. B, Western blots with an anti-GFP antibody. C, uptake of MeAIB (100 µM) in control cells and in cells transfected with EGFP-mATA2 cDNA. Values are mean ± S.E. from three determinations. Ctrl, control.

 
EGFP-ATA2-containing Vesicles Shift from the Perinuclear Pool to the Plasma Membrane after Insulin Stimulus in 3T3-L1 Adipocytes—To observe the protein translocation in living cells in a time-lapse manner after insulin stimulus, we employed the newly developed fluorescence microscope with CO2 incubator for cell culture (Fig. 4). Prior to insulin addition, intense fluorescence signals of EGFP-ATA2 were observed in the perinuclear vesicle storage site. After insulin stimulus, the vesicles with the fluorescence signals seemed to be released from the perinuclear vesicle storage site, and headed for the plasma membrane. The onset of response to insulin stimulus seemed a little delayed, because the method of insulin addition into cell culture dishes was different from other experiments and it took some time for stabilization of insulin concentration in the medium.


Figure 3
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  		FIGURE 3.
A, cell surface recruitment of EGFP-tagged ATA2 stimulated by insulin in 3T3-L1 adipocytes. Images were captured with a confocal laser scanning microscope. We selected the image of each cell with the similar expression levels of EGFP-ATA2 from respective representative images for comparison, because the amount of EGFP-ATA2 on the surface of cells detected by microscopy is dependent on the total amount expressed in cells; those that express the highest levels of the reporter appears to have the most on the surface. Scale bar, 10 µm. The data are from a representative experiment and similar results were obtained from two other experiments. B, quantification of the cell surface recruitment of EGFP-tagged ATA2 by acute insulin stimulus in 3T3-L1 adipocytes. Western blots were performed with cell surface proteins and total lysates were separated by 8% SDS-PAGE. The blots were probed with an anti-GFP antibody. Arrows indicate EGFP-tagged ATA2. We also quantified the insulin-stimulated translocation of TfnR by re-blotting the polyvinylidene difluoride membranes used for quantification of EGFP-tagged ATA2 translocation. In the lower panel, the values in the graph are mean ± S.E. from four determinations. The -fold increase was greater for EGFP-ATA2 than for TfnR in all four samples, and the difference between EGFP-ATA2 and TfnR is significant in paired t test (p = 0.034).

 
Immunofluorescent Analysis with Organelle-specific Markers and GLUT4 in EGFP-ATA2 3T3-L1 Adipocytes—To characterize the intracellular location of EGFP-ATA2 in detail, immunofluorescent staining was performed using antibodies against the organelle-specific markers such as EEA1, Rab11, GM130, syntaxin 6, and GLUT4 (Fig. 5). EGFP-ATA2 was found to co-localize most with the TGN marker syntaxin 6 (30, 31), partially with the recycling endosome marker Rab11 (32), and to a lesser extent with the Golgi apparatus marker GM130 (33) in the perinuclear site, but not with the early endosome marker EEA1 (30, 31). EGFP-ATA2 also co-localized partially with GLUT4 in the perinuclear site and the plasma membrane. With insulin treatment, intracellular vesicles containing EGFP-ATA2 or GLUT4 on or near the plasma membrane were clearly observed (indicated with arrowheads for vesicles containing EGFP-ATA2 and arrows for GLUT4-containing vesicles in the bottom rows of the columns of Fig. 5, respectively). These vesicles containing EGFP-ATA2 and GLUT4 did not overlap with each other on or near the plasma membrane, indicating that ATA2 and GLUT4 proteins are loaded into separate vesicles through insulin-stimulated translocation.

Effect of Brefeldin A on the Morphology of the Perinuclear EGFP-ATA2 Storage Site and the Uptake Function of Endogenous ATA2—To confirm that the intracellular ATA2 storage site is localized in the TGN, we studied the effects of brefeldin A (BFA) on 3T3-L1 adipocytes. BFA is an inhibitor of the guanine nucleotide-exchange protein for ADP-ribosylation factor 1, a monomeric GTPase; thus, BFA treatment of intact cells inhibits vesicular exit of proteins from the TGN (34). Treatment with BFA causes rapid disassembly of protein trafficking between the endoplasmic reticulum and the Golgi apparatus and redistributes these proteins into the endoplasmic reticulum (35), and also causes membrane proteins resident in the TGN to disassemble and redistribute to the microtubule organizing center (36). Therefore, BFA can be used to determine whether a protein of interest is associated with the Golgi complex or with the TGN (37). First, we examined the effect of BFA on the morphology of the perinuclear EGFP-ATA2 storage site and its co-localization with syntaxin 6, as a TGN marker. In the cells treated with BFA, we observed more concentrated spherical distribution of syntaxin 6 to the microtubule organizing center in a dose-dependent manner (Fig. 6A), as reported previously (37, 38). Even in these BFA-treated cells, EGFP-ATA2 was found to co-localize mostly with syntaxin 6 at the perinuclear site. BFA decreased insulin-stimulated translocation of EGFP-ATA2 to the plasma membrane. The effect of brefeldin A, either with or without insulin, on TGN morphology was similar to that on the EGFP-ATA2 storage site (data not shown). We also examined the effect of BFA on insulin-stimulated MeAIB uptake via endogenous ATA2 in non-transfected 3T3-L1 adipocytes (Fig. 6B). BFA significantly decreased the insulin-stimulated MeAIB uptake to 55 and 26% of control at concentrations of 10 and 100 µg/ml, respectively. Insulin-stimulated 2-DG uptake was reduced only to 76% of control even at 100 µg/ml BFA (not significant, n = 3). The uptake of L-tryptophan was not affected by brefeldin A treatment either in the presence or absence of insulin. The effect of BFA on insulin-stimulated TfnR translocation was also analyzed by Western blot. Insulin-stimulated TfnR translocation was not affected at 10 µg/ml BFA, and was only to 68% of control even at 100 µg/ml BFA.


Figure 4
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  		FIGURE 4.
Time lapse fluorescent microscopic observation in insulin-treated 3T3-L1 adipocytes that stably expressed EGFP-tagged ATA2. Images were captured with a specially devised incubation fluorescent microscope as described under "Experimental Procedures." Scale bar, 10 µm. The data are from a representative experiment and similar results were obtained from two other experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The influence of insulin on the activity of amino acid transport system A has been the topic of several studies in the past, but all of these studies were done before the establishment of the molecular identity of the gene/protein that is responsible for this transport activity (23, 24). Since then, the transporter ATA2 (also known as SNAT2 or SLC38A2) has been shown to be responsible for the activity of system A in most cells including the adipocytes (2, 21, 22). We also confirmed this using 3T3-L1 adipocytes and preadipocytes by RT-PCR in this study (Fig. 1A). It is well known that insulin stimulates glucose uptake acutely without relying on de novo synthesis of the transporter protein in adipocytes and muscle cells (39-41). This involves the translocation of the insulin-responsive facilitative glucose transporter GLUT4 from an intracellular pool to the plasma membrane. The present study was undertaken to investigate the cellular mechanisms involved in regulation of system A by acute insulin stimulus in adipocytes, with emphasis on the intracellular trafficking of ATA2 transporter.

We first confirmed that acute treatment of adipocytes with insulin increases system A activity, as monitored by the Na+-dependent uptake of MeAIB, without involving de novo synthesis of the transporter protein. The insulin-stimulated translocation of ATA2 in 3T3-L1 adipocytes depends mainly on the phosphatidylinositol 3'-kinase-actin filament rearrangement signal pathway similar to what has been shown to occur with GLUT4 (26, 42, 43) (Fig. 1, B-E). We attempted to monitor this insulin-stimulated translocation of ATA2 with a cell biological approach by using an expression system of EGFP-ATA2 fusion protein. In 3T3-L1 adipocytes, EGFP-ATA2 protein was predominantly localized in the perinuclear site and the plasma membrane (Fig. 3A). After acute insulin stimulus in 3T3-L1 adipocytes expressing the EGFP-ATA2, the fluorescent signals derived from EGFP-ATA2 became brighter and clearer on the plasma membrane of the cells (Fig. 3A). The insulin-stimulated translocation of the fusion protein was confirmed by quantification of the cell surface-associated transporter protein following biotinylation of plasma membrane proteins with a membrane-impermeable biotinylation reagent (Fig. 3B). The 2-fold increase of cell surface EGFP-ATA2 by insulin stimulus in the stably transfected adipocytes agrees well with the magnitude of the increase of MeAIB uptake by the insulin-stimulated translocation of endogenous ATA2 in non-transfected adipocytes, indicating both endogenous and EGFP-tagged ATA2 possess the same characteristics concerning intracellular trafficking. These studies demonstrate that EGFP-tagged ATA2 protein can be a useful tool to investigate the intracellular events involved in the trafficking of ATA2 in response to insulin in 3T3-L1 adipocytes. However, there are several caveats associated with this experimental approach. One important assumption here is that the tagged protein behaves identical to the native protein; but this may not be true. The EGFP tag increases the molecular size of the ATA2 transporter protein significantly and the change in the size of the protein may influence sites and cellular components involved in its intracellular trafficking. It is therefore difficult to ascertain unequivocally that the observed features of storage and cellular trafficking of the tagged protein are the same as those for the native protein. Furthermore, we have no information at this time on the relative levels of the exogenously expressed tagged protein compared with the levels of the native protein; but such information may be necessary to make conclusions on the applicability of the data from the tagged protein to the native protein. The rationale for this caveat is that the participation of different intracellular compartments in storage and trafficking may vary depending on the expression levels of the protein in the cell. These issues cannot be addressed directly at this time because of the lack of specific antibodies for the native mouse ATA2 protein in 3T3-L1 cells.

We were also able to detect the insulin-stimulated MeAIB uptake in 3T3-L1 adipocytes stably transfected with EGFP-ATA2, but not in CHO-K1 cells transiently transfected with the same fusion protein (data not shown). We suggest that this cell type-specific differential response to insulin is due to the distinct localization at a perinuclear site of EGFP-ATA2 in CHO-K1 cells and 3T3-L1 cells as described above. Regarding GLUT4, perinuclear localization and insulin response was observed in CHO-K1 cells transfected with myc- and GFP-tagged GLUT4 (5). Therefore, we speculate that the perinuclear storage pool for ATA2 is unique and necessary for insulin-stimulated translocation of this transporter in adipocytes. We focused on ATA2 localization and translocation from the initial storage location to the plasma membrane after insulin stimulus. To monitor this translocation in living cells in a time-lapse manner after insulin stimulus, we employed the fluorescence microscope with CO2 incubator for cell culture (Fig. 4). The observation in living cells revealed that perinuclear localization is a storage site for ATA2, and that ATA2 is released in vesicles from this perinuclear storage site toward the plasma membrane. The differences in the morphological images in Figs. 3, 4, 5 are due to the fact that the incubator microscope is not confocal.


Figure 5
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  		FIGURE 5.
Localization of EGFP-tagged ATA2 protein after the addition of insulin in 3T3-L1 adipocytes. The cells were treated with or without insulin similar to the protocol in the uptake experiments. Because the images of the insulin-treated cells and non-treated cells are similar in terms of co-localization, we decided to use only the insulin-treated cells for data presentation. Fluorescent immunohistochemical analysis with a confocal laser scanning microscope shows that EGFP-tagged ATA2 is co-localized partially with GM130, syntaxin 6, Rab11, and GLUT4, but not with EEA1. EGFP-tagged ATA2 is shown in green, and organelle-specific markers and GLUT4 are shown in red.Inthe lowest column, arrowheads and arrows indicate the ATA2-containing vesicles and GLUT4-containing vesicles, respectively. Scale bar, 10 µm. The data are from a representative experiment and similar results were obtained from two other experiments.

 
Studies with organelle-specific markers show that EGFP-ATA2 does not co-localize with EEA1, a marker for early endosomes. Instead, the fusion protein co-localizes partially with TGN marker syntaxin 6, the Golgi marker GM130, and the recycling endosome marker Rab11 at the perinuclear site (Fig. 5). The co-localization with syntaxin 6 is most significant among these three organelle-specific markers. GLUT4 also partially co-localizes with EGFP-ATA2 at the perinuclear site, suggesting that both transporter proteins have proximate storage sites. In insulin-treated cells, the vesicles containing ATA2 seem to be distinct from those containing GLUT4. These vesicles are found on and near the plasma membrane (Fig. 5). However, we cannot tell whether the ATA2-containing vesicles and the GLUT4-positive vesicles are on the way to the plasma membrane or on the way to the intracellular site only from the images in Fig. 5. From the results in Figs. 3 and 4, we suggested insulin-stimulated increases of cell surface ATA2 by the translocation of ATA2-containing vesicles similar to GLUT4. Therefore, these studies were carried out using insulin-treated cells to shift the equilibrium of the endocytosis/exocytosis process toward exocytosis (i.e. movement toward the plasma membrane). Based on this rationale, we assume that most of the ATA2-containing vesicles are moving toward the plasma membrane. The same is true for GLUT4-positive vesicles. Because the ATA2-containing vesicles are distinct from the GLUT4-positive vesicles, we conclude that each transporter is packaged in different and transporter-specific vesicles to target these transporters to the plasma membrane.

To confirm the localization of the ATA2 storage site in the TGN, we used BFA (Fig. 6). As described above, treatment with BFA causes membrane proteins resident in the TGN to disassemble and redistribute to the microtubule organizing center, and consequently results in inhibition of vesicular exit of the proteins from the TGN. In the present study, we first confirmed that BFA caused morphological changes in the TGN as evidenced in the immunofluorescent staining of syntaxin 6, a TGN marker (Fig. 6A). Such changes have been described previously by other investigators (50, 51). EGFP-ATA2 was found to co-localize with syntaxin 6 even after treatment with BFA. BFA also inhibited insulin-stimulated MeAIB uptake in non-transfected 3T3-L1 cells, indicating that endogenous ATA2 is also translocated at least via the TGN in response to insulin stimulus (Fig. 6B). Thus, we showed that the majority of EGFP-ATA2 are in the TGN as the storage site by immunofluorescent analysis, and that ATA2 and EGFP-ATA2 at least go through TGN after insulin stimulation by the inhibition study of the TGN exit with BFA. Therefore, it seems that both the storage site and the insulin-responsive site of ATA2 are the TGN. On the other, it is possible that ATA2 is detected at this site when it may be passing through this site to some distinct location, and additional studies may be needed to confirm this. Moreover, TGN has heterogeneous functional domains, and therefore whether both sites are identical within the TGN remains to be established.


Figure 6
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  		FIGURE 6.
A, effect of TGN disruption by brefeldin A on the morphology of ATA2 storage sites and their co-localization with syntaxin 6. Adipocytes were incubated with brefeldin A at concentrations of 10 or 100 µg/ml for 1 h prior to and during insulin treatment. The cells were treated with insulin similar to the protocol in the uptake experiments. EGFP-tagged ATA2 is shown in green, and syntaxin 6 in red. Images were captured with a confocal laser scanning microscope. Scale bar,10 µm. The data are from a representative experiment and similar results were obtained from two other experiments. B, effect of TGN disruption by brefeldin A on MeAIB (3.6 µM), 2-DG (10 µM), and L-tryptophan (30 µM) uptake and cell surface TfnR localization in 3T3-L1 adipocytes treated with or without insulin. Adipocytes were incubated with brefeldin A at concentrations of 10 or 100 µg/ml for 1 h prior to and during insulin treatment. Values are mean ± S.E. from three determinations.

 
BFA showed a relatively smaller, but not statistically significant, effect on insulin-stimulated 2-DG uptake, in accordance with the previous reports that BFA had no effect on insulin-stimulated GLUT4-containing vesicle trafficking to the cell surface (44, 45). Taken together, these data strongly indicate that the major ATA2 storage site is in the TGN and that the contribution of TGN as the storage site of ATA2 is much greater than as the storage site of GLUT4. GLUT4 also co-localizes with markers of the TGN, the recycling endosomes, and the Golgi complex in adipocytes by static microscopic analysis (46, 47). However, the location of the insulin-responsive compartment for GLUT4 has not completely been clarified (46, 47). In the present study, the main ATA2 storage site that responds to insulin acutely is found to be in the TGN, distinct from that of GLUT4.

Studies with organelle-specific markers show that EGFP-ATA2 does not co-localize with EEA1, a marker for early endosomes (Fig. 5). In the present study, we also compared insulin-stimulated translocation of ATA2 with that of TfnR, a marker for general endosomes including early endosomes and recycling endosomes. Insulin treatment is known to facilitate nearly 2-fold translocation of TfnR to the plasma membrane in 3T3-L1 adipocytes (48-50). This translocation pathway occurs through insulin-stimulated general endosomal recycling. In this study, the -fold increase of insulin-stimulated translocation was significantly greater for EGFP-ATA2 than for TfnR (Fig. 3B). BFA affected the insulin-stimulated translocation of ATA2 to a greater extent than it did the insulin-stimulated translocation of TfnR (Fig. 6B). Thus, we suggested that the translocation of ATA2 is distinct from that of TfnR both in quantitative and qualitative (sensitivity to brefeldin A) aspects. The differences observed in the comparisons may not reflect only differences in the pathway followed by these membrane proteins to the plasma membrane, but also in the rate constants characterizing their fate within the plasma membrane, etc. We have not found any report that insulin and/or BFA affect the degradation/sequestration of these proteins. Also in our recent study (51), we clarified the degradation/sequestration of ATA2 regulated with the ubiquitination of the transporter by Nedd4-2. We did not find any effect of insulin on the sequestration/degradation of ATA2. There is neither report of the regulation of Nedd4-2 by insulin and BFA. Therefore, these data for the comparisons of cell surface ATA2 and TfnR are supportive for the determination of the translocation pathway of ATA2, although they may not be convincing only by themselves. These results suggest that insulin-stimulated translocation of ATA2 is mediated by a specialized insulin-responsive compartment, not by a generalized increase in endosome recycling.


Figure 7
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  		FIGURE 7.
Analysis of the expression profile of ATA2 mRNA in adipose tissues in humans and rodents. We performed a data base search and statistical analysis on ATA2 mRNA and GLUT4 mRNA expression among various tissues in humans, mice, and rats that were profiled by Affymetrix GeneChip technology and stored in the commercial Gene Logic BioExpress data base system. We identified human adipose samples accompanied with disease information of the donors for the present study. These samples were divided into two groups in which the donors were without and with type II diabetes mellitus (DMII) and/or metabolic syndrome X (MSX), named as normal group (n = 26) and DMII/MSX group (n = 24), respectively. The expression values were normalized by the Affymetrix global scaling method. A, box plots of differential expression of ATA2 mRNA in adipose tissues from patients with type II diabetes mellitus and/or metabolic syndrome X (DMII/MSX) and their controls. The expression level of ATA2 mRNA in type II diabetes mellitus and/or metabolic syndrome X (DMII/MSX) groups was significantly lower than in the normal groups (p = 0.0002).The middle horizontal solid and dashed line in each box indicates the median value and mean value of each group, respectively. Each box with error bars indicates 10th, 25th, 75th, and 90th percentiles. B, correlation between ATA2 mRNA expression levels in fat tissues and BMI of patients with type II diabetes mellitus and/or metabolic syndrome X (DMII/MSX) and their controls in A. Each line indicates the regression of the scattered plot for each group (upper, solid, normal; and lower, dashed, DMII/MSX). Each r2 value shows that there is no significant correlation between BMI and ATA2 mRNA expression levels. C, differential expression of ATA2 and GLUT4 mRNA in adipose tissues in human, mouse, and rat. The expression levels of ATA2 in human adipose tissue are quite high. In contrast, in rat and mouse adipose tissue samples, the expression levels of ATA2 are modest, whereas the expression levels of GLUT4 are high. In all human tissue samples, the expression levels of GLUT4 mRNA were lower than those of ATA2 mRNA. Values are mean ± S.D. n = 26, 5 and 22 in humans, mice and rats, respectively.

 
Recently, Hyde et al. (52) reported that insulin-stimulated ATA2 translocation in L6 rat myotubes is sensitive to chloroquine, an agent believed to impair endosomal function through its action as an acidotropic weak base. However, the effect of chloroquine is not specific for endosomal recycling. It is simply a disruptor of endosomal/lysosomal pH and thus may interfere with the function of the late endosomes and lysosomes (53, 54). Romanek et al. (55) also reported that chloroquine inhibits GLUT4 recruitment to the cell surface by insulin in rat adipocytes and the inhibitory effect is independent of its action on endomembrane pH in GLUT4-containing vesicles. Specific ablation of endosomes (56-58) and analysis of its effect on ATA2 translocation in response to insulin may lead to a better understanding of the role of the endosomal compartment in the process. In this study, the distinct perinuclear localization and response to insulin stimulus of EGFP-ATA2 were observed in 3T3-L1 adipocytes, but not in CHO-K1 cells. Thus, this storage pool for ATA2 is unique and necessary for the insulin-stimulated translocation of this transporter in adipocytes. The differences of the response to insulin between the cells such as adipocytes and myotubes need further investigation.

The results of the present study show that insulin influences the translocation of ATA2 to the plasma membrane in adipocytes and thus alters the functional activity of the transporter. Because diabetes and metabolic syndrome X represent insulin-resistant states in adipose tissues, we speculated that these disease states may be associated with changes in ATA2 expression in adipocytes in vivo. To determine whether this is true, we queried Gene Logic BioExpress, a commercial data base, which stores mRNA expression data in Affymetrix GeneChip format for a large number of human and disease-model animal samples. The results show that the expression of ATA2 is down-regulated in type II diabetes and metabolic syndrome X (Fig. 7A). The data base has information on body mass index (BMI) for all the patients included in the data base. BMI provides a reliable indicator of obesity and is used to screen for weight categories that may lead to health problems. We analyzed the data to see if there is any correlation between BMI and ATA2 mRNA expression levels and found no significant correlation (Fig. 7B). Thus, these data suggest a connection between ATA2 expression and diseases such as type II diabetes and MSX, but not between ATA2 expression and obesity. These results represent a chronic effect of the lack of insulin action on ATA2 function. This chronic effect is associated with changes in the steady-state levels of transporter mRNA. In contrast, the acute effects of insulin involve the trafficking of the preformed transporter protein from an intracellular pool to the plasma membrane. Taken together, these data show that insulin or the lack of its action may have acute as well as chronic effects on the activity of the amino acid transporter ATA2. Because ATA2 is the primary transporter responsible for uptake of small neutral and glycogenic amino acids in adipocytes, the insulin-dependent regulation of this transporter has significant implications in adipocyte biology and function and in disease states such as diabetes. We also show that the expression levels of ATA2 are quite high in the adipose tissue in humans (Fig. 7C). The high levels of expression of ATA2, the insulin-dependent regulation of its expression, and the significant changes in its expression observed in type II diabetic patients suggest an important role for this amino acid transporter in adipocyte function. ATA2 is a highly active transporter and has the ability to concentrate its amino acid substrates inside the cells. These amino acids can potentially influence various functions of the adipocyte, including the synthesis of protein, fatty acids, triglycerides, and glycerol-based phospholipids, in addition to their role in GLUT4 trafficking. Therefore, the acute and chronic effects of insulin on the expression of this amino acid transporter have physiological and pathological consequences. We suggest that ATA2 or proteins involved in its regulation may serve as potential therapeutic targets in the development of new anti-diabetic drugs.


   FOOTNOTES
 
* This work was supported by PRESTO from the Japan Science and Technology Agency and Grant-in-aid WAKATE-A (to M. S.) and Grant-in-aid WAKATE-B (to T. H.) from the Ministry of Education, Culture, Sports, Science, and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511, Japan. Tel.: 81-42-724-6259; Fax: 81-42-724-6316; E-mail: setou{at}nips.ac.jp.

2 The abbreviations used are: MeAIB, {alpha}-(methylamino)isobutyric acid; ATA, amino acid transporter A; TGN, trans-Golgi network; 2-DG, 2-deoxy-D-glucose; TfnR, transferrin receptor; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; BFA, brefeldin A; mATA, mouse amino acid transporter A; RT, reverse transcriptase; BMI, body mass index. Back


   ACKNOWLEDGMENTS
 
We thank M. Kawanami and K. Ohtsu at MITILS for technical assistance. We also thank Olympus Co., Ltd. for use of a prototype of the incubation microscope and their technical assistance for its use.



   REFERENCES
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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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