Activation of Syntaxin 1C, an Alternative Splice Variant of HPC-1/Syntaxin 1A, by Phorbol 12-Myristate 13-Acetate (PMA) SuppressesGlucose Transport into Astroglioma Cells via the GlucoseTransporter-1 (GLUT-1)*

Received for publication, December 30, 2003, and in revised form, March 16, 2004Published, JBC Papers in Press, March 22, 2004, DOI 10.1074/jbc.M314297200

Takahiro Nakayama‡§¶, Katsuhiko Mikoshiba§�, Tetsuo Yamamori**, and Kimio Akagawa‡

From the ‡Department of Physiology, Kyorin University School of Medicine, Tokyo 181-8611, Japan, the §Laboratory forDevelopmental Neurobiology, Developmental Brain Science Group, Brain Science Institute, RIKEN, Saitama 351-0198,Japan, the �Department of Molecular Neurobiology, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639,Japan, and the **Division of Speciation Mechanisms, National Institute for Basic Biology, Aichi 444-8585, Japan

Syntaxin 1C is an alternative splice variant lackingthe transmembrane domain of HPC-1/syntaxin 1A. Wefound previously that syntaxin 1C is expressed as a sol-uble protein in human astroglioma (T98G) cells, andsyntaxin 1C expression is enhanced by stimulation withphorbol 12-myristate 13-acetate (PMA). However, thephysiological function of syntaxin 1C is not known. Inthis study, we examined the relationship between syn-taxin 1C and glucose transport. First, we discoveredthat glucose transporter-1 (GLUT-1) was the primaryisoform in T98G cells. Second, we demonstrated thatglucose uptake in T98G cells was suppressed followingan increase in endogenous syntaxin 1C after stimulationwith PMA, which did not alter the expression levels ofother plasma membrane syntaxins. We further exam-ined glucose uptake and intracellular localization ofGLUT-1 in cells that overexpressed exogenous syntaxin1C; glucose uptake via GLUT-1 was inhibited withoutaffecting sodium-dependent glucose transport. Thevalue of Vmax for the dose-dependent uptake of glucosewas reduced in syntaxin 1C-expressing cells, whereasthere was no change in Km. Immunofluorescence studiesrevealed a reduction in the amount of GLUT-1 in theplasma membrane in cells that expressed syntaxin 1C.Based on these results, we postulate that syntaxin 1Cregulates glucose transport in astroglioma cells bychanging the intracellular trafficking of GLUT-1. This isthe first report to indicate that a syntaxin isoform thatlacks a transmembrane domain can regulate the intra-cellular transport of a plasma membrane protein.

The protein machinery that regulates intracellular transportand vesicle formation, docking, and fusion has been the focus of

intense research over the last few years. The SNARE1 hypoth-esis (soluble N-ethylmaleimide-sensitive fusion protein (NSF)attachment protein receptor) constitutes a widely acceptedmodel in which dynamic interactions among proteins withinthe acceptor (t-SNARE: syntaxin and SNAP-25) and donor(v-SNARE: VAMP) compartments control exocytosis (1, 2). Re-cent studies have revealed that syntaxins function in a widevariety of cells and tissues, including neurons, endocrineglands, amphibian ectodermal cells, epithelial cells, cells of theimmune system, platelets, and yeast (3). Consequently, a uni-fied role for the SNARE complex in the docking and fusion ofvesicles during intracellular trafficking, as well as in nerveterminals, has been proposed.

To date, 18 members of the mammalian syntaxin family havebeen identified, all of which localize to specific membrane com-partments via a transmembrane domain at the C terminus. Incontrast to the localization of syntaxins 5–18 to different intra-cellular compartments, such as the Golgi and post-Golgi appa-ratus (4), syntaxins 1–4 are restricted predominantly to theplasma membrane, where they mediate constitutive and regu-lated vesicle trafficking to the cell surface (4). All syntaxinshave a coiled-coil helix domain (called H3 in syntaxin 1A) nextto the transmembrane domain at the C terminus. The H3domain is a highly conserved region that interacts with severaldifferent SNARE proteins, including SNAP-25, VAMP, and�-SNAP, and to some extent, nSec-1/Munc-18 (4).

Syntaxin 1C is an alternative splice variant of HPC-1/syn-taxin 1A. Syntaxin 1A is involved in the docking of synapticvesicles at active zones in neurons (5, 6), and is deleted hem-izygously in patients with the neurodevelopmental disorder,Williams syndrome (7, 8). In a previous study, we demon-strated that syntaxin 1C is expressed as a soluble protein inastroglioma cells (9). While the N-terminal domain of syntaxin1C is the same as that of syntaxin 1A, the functionality of theH3 and transmembrane domain has been lost, caused by thegeneration of a novel 34-residue C-terminal domain by theinsertion of a 91-bp splicing region. Several other isoforms ofsyntaxin that lack a transmembrane domain by alternative

* This study was supported by grants-in-aid from the Japan Societyfor the Promotion of Science for Japanese Junior Scientists (to T. N.)and the Promotion and Mutual Aid Corporation for Private Schools inJapan (to K. A.). The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the DDBJ/GenBankTM/EBI Data Bank with accession number(s)D37932 and AB086954M.

¶ A fellow of the Japan Society for the Promotion of Science forJapanese Junior Scientists. To whom correspondence should be ad-dressed: Dept. of Physiology, Kyorin University School of Medicine,6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. Fax: 81-422-47-4801; E-mail: nakayama@kyorin-u.ac.jp.

1 The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor; 2-DG, 2-deoxy-glucose; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calfserum; GLUT, glucose transporter; HA, hemagglutinin; PKC, proteinkinase C; PMA, phorbol 12-myristate 13-acetate; SGLT, sodium-de-pendent glucose transporter; Syn, syntaxin; PBS, phosphate-bufferedsaline; RT, reverse transcription; nt, nucleotide; CNS, central nervoussystem; SNAP, soluble NSF attachment protein; VAMP, vesicle-associ-ated membrane protein.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 22, Issue of May 28, pp. 23728–23739, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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splicing have been identified, namely syntaxin 2D, syntaxin3D, and syntaxin 16C (10, 11, 12), but the function of syntaxinisoforms that lack a transmembrane domain is unknown.

Facilitative glucose transporters (GLUTs) are proteins thatregulate the entry of glucose into cells and maintain cell me-tabolism and homeostasis throughout the periphery and brain(13). There are at least six different GLUT genes with differ-ential tissue distributions, subcellular localizations, and kinet-ics for glucose uptake (14). In the brain, there are two GLUTisoforms, namely GLUT-1 and GLUT-3. GLUT-1 appears dur-ing early embryogenesis and is required for cell metabolismand homeostasis in glial cells (13, 15). GLUT-3 is found pri-marily in neurons (15). GLUT-4 is expressed only in muscleand fat cells, where it resides in an intracellular compartmentunder basal conditions and is translocated to the cell surfaceafter stimulation with insulin (15). Recently, it became clearthat syntaxin 4 and several SNARE-related molecules partici-pate in the translocation of GLUT-4 to the plasma membrane(16, 17). In addition, recent studies have revealed that glucosetransport is regulated though several signal transduction path-ways, including those that involve mitogen-activated proteinkinase, phosphatidylinositol 3-kinase, and protein kinase C(PKC) (18–20). We showed previously that astroglioma cellsexpress syntaxin 1C but not syntaxin 1A, and that the expres-sion of syntaxin 1C protein is up-regulated via a PKC signalingpathway by stimulating cells with phorbol 12-myristate 13-acetate (PMA) (9).

In the present study, we used a human astroglioma cell linethat expresses syntaxin 1C to determine whether syntaxin 1Cis involved in glucose transport. We found that the induction ofendogenous syntaxin 1C expression by PMA caused a reductionin GLUT-1 in the plasma membrane and suppressed glucoseuptake. Expression of exogenous syntaxin 1C in T98G cells hadthe same effects. These results suggest that the physiologicalfunction of syntaxin 1C in astroglioma cells is the regulation ofintracellular trafficking of GLUT-1.

EXPERIMENTAL PROCEDURES

Reagents—All tissue culture reagents were purchased from Invitro-gen, Life Technologies (Carlsbad, CA) with the exception of fetal calfserum (FCS), which was purchased from Sigma. Human insulin waspurchased from Roche Applied Science (Basel, Switzerland). Acrylam-ide/bis-acrylamide was obtained from WAKO Chemical (Osaka, Japan).All other reagents were purchased from Sigma or Calbiochem (SanDiego, CA), unless otherwise noted.

DNA Cloning—Total cellular RNA was extracted using the QIA-AmpRNA extraction kit from Qiagen (Valencia, CA), according to the man-ufacturer’s protocol. The reverse transcription (RT)-PCR was carriedout using an RNA PCR kit (Takara, Tokyo, Japan), according to themanufacturer’s protocol.

To clone the coding region of human syntaxins, we designed oligo-nucleotide primers based on the sequence of human (h) syntaxin 1A andsyntaxin 1C (DDBJ accession nos. D37932 and AB086954M, respec-tively), and syntaxin 4 (GenBankTM accession no. NM004604). Theprimers were used in RT-PCR cloning, using mRNA that was isolatedfrom the human brain library (BIO101) and T98G human astrogliomacells, as described below. RT was performed using the oligo(dT) primerand AMV reverse transcriptase (Takara), according to the manufactur-er’s instructions. The full-length human syntaxin cDNAs were insertedinto the BamHI/EcoRI site of a pcDNA3 expression vector (Invitrogen,Life Technologies), or cloned into the BamHI site of a pcDNA3 expres-sion vector as an N-terminal 5� HA-tagged version. The subclonedsyntaxins were confirmed by using an ABI 377 sequencer (AppliedBiosystems, Foster City, CA).

Semiquantitative PCR—GLUT gene expression was quantified ac-cording to the method of Schreiber et al. (21). The cDNA template (5 ng)that was synthesized from total RNA in cells was used for semiquanti-tative PCR, with primer pairs that were specific for hGLUT-1,hGLUT-2, hGLUT-3, and hGLUT-4 (GenBankTM accession nos.K03195, J03810, M20681, and M20747, respectively). Human smallintestine cDNA was a kind gift from Dr. Yoshikatsu Kanai (KyorinUniversity, Japan). The primer pairs were as follows: hGLUT-1 sense,

906–932 nt; hGLUT-1 antisense, 1519–1491 nt; hGLUT-2 sense, 2223–2243 nt; hGLUT-2 antisense, 2626–2607 nt; hGLUT-3 sense, 884–912nt; hGLUT-3 antisense, 1375–1349 nt; hGLUT-4 sense, 1485–1513 nt;and hGLUT-4 antisense, 2076–2048 nt. As a control, we used a pair ofprimers for �-actin (Maxim Biotech, South San Francisco, CA) thatamplifies a 540-bp DNA segment. The primers for �-actin span at leastone intron, and contamination of RNA samples by genomic DNA can bedetected according to the size of the amplified product (1116 bp forgenomic DNA). PCR was linear up to 30 cycles for each pair of primers(data not shown). The intensity of the SYBR-green (Molecular Probes,Eugene, OR) signals from scanned images of the gels was measuredusing NIH Image (rsb.info.nih.gov/nih-image/).

Northern Blot Analysis—Northern blot analysis was carried out,according to the method of Nagamatsu et al. (22). Total RNA (20 �g)isolated from native and transfected (see below) T98G cells was sepa-rated by electrophoresis in 1.0% formaldehyde-agarose denaturing gels.The EcoRI-digested 600-bp fragments of the GLUT-1 and GLUT-3cDNA were labeled with 32P by random priming. The GLUT-1 andGLUT-3 cDNAs were a kind gift from Dr. Shinya Nagamatsu (KyorinUniversity, Japan). The intensity of the autoradiographic signals wasmeasured directly from digital images (Bas 2000, Fuji, Tokyo, Japan).

Cell Culture and Transfection—Two human astroglioma cell lines,T98G and U87MG, were provided by Dr. Hiroki Sawa (Kyorin Univer-sity, Japan). Cells were grown on 90-mm in diameter plastic dishes inDulbecco’s modified Eagle’s medium (DMEM), supplemented with 10%(v/v) FCS, penicillin (100 �g/ml), and streptomycin (100 �g/ml). Fordrug stimulation, cells were treated for 3–48 h with 1–10 �M PMA(Sigma), 10 �M 4�-PMA (Sigma), or 10 �M forskolin (RBI).

T98G and U87MG cells were trypsinized, washed twice with phos-phate-buffered saline (PBS), and resuspended in 0.3 ml of serum-freeDMEM on ice. Approximately 1 � 106 T98G cells were transfected with150 �g/ml recombinant syntaxins by electroporation (Bio-Rad genepulsar, 0.75 kV/cm field strength, 960 microfarad capacitance). Thecells were then cultured in DMEM (that included 800 �g/ml neomycin)for 2 weeks, and the transfected cells were cloned as a single colony.

Immunoblot Analysis—Equal amounts of protein from each samplewere separated on 12% SDS-polyacrylamide gels as described previ-ously (9). Following antibodies were used as a primary antibody: amonoclonal antibody (14D8) that had been raised against the N-termi-nal of syntaxin 1A (9), a polyclonal anti-syntaxin 1C antibody (9), apolyclonal anti-syntaxin 2 antibody (Stressgen Biotech, Victoria, BC,Canada), a polyclonal anti-syntaxin 3 antibody (Sigma), a monoclonalanti-syntaxin 4 antibody (BD-Transduction Laboratory, San Jose, CA),anti-GLUT-1 antiserum, anti-GLUT-3 antiserum (Chemicon, Te-mecula, CA), or anti-HA monoclonal antibody (3F10, Roche AppliedScience). After washing, the membranes were incubated with horserad-ish peroxidase-conjugated anti-mouse IgG, anti-rabbit IgG (Cappel,Irvine, CA), or anti-rat IgG (Jackson Laboratories, Bar Harbor, ME).

For GLUT immunoblotting, a total cell membrane preparation wasmade, as described previously (23). The cell surface biotinylation assayfor GLUT-1 was carried out, according to the method of McMahonet al. (24).

Quantitative analysis of the syntaxin immunoblots was carried outas described previously (9). After drug application, cells were treatedwith 10% trichloroacetic acid. After centrifugation, the precipitate wassolubilized in 8 M urea, 1% SDS, 10 mM Tris-Cl (pH 7.5). The proteinconcentration was measured by using a DC-protein assay (Bio-Rad).The intensities of the immunoblotted signals were measured using NIHImage and normalized to that with anti-�-tubulin IgG (DM1A) (Sigma).

Immunocytofluorescence—Immunostaining was carried out essen-tially as described previously (9). Briefly, to study GLUT-1 localizationin cells treated with PMA or transfected with HA-tagged syntaxin, cellswere fixed and permeabilized with acetone/methanol (1:1). After treat-ment with a blocking solution, the cells were then incubated with amonoclonal antibody (14D8) (9) or an anti-HA monoclonal antibody(3F10) and an anti-GLUT-1 polyclonal antibody. After another washwith PBS, the cells were exposed to either anti-mouse IgG, anti-rat IgGcoupled to Cy-3, or anti-rabbit IgG coupled to fluorescein isothiocya-nate. The immunostained cells were examined using a confocal scan-ning laser microscope (Zeiss LSM 410, Jena, Germany) that wasequipped with a triple band-pass filter set.

Cell Growth and Cell Cycle Analysis—T98G cells that had beenstarved of serum for 24 h were stimulated with DMEM containing 10%FCS for 0–96 h. Control and syntaxin-expressing cells were seeded in90-mm in diameter culture dishes (5 � 104 cells/dish). The number ofliving cells was counted up to 96 h following DMEM/FCS treatment.The growth rate in the logarithmic growth phase (48–96 h) was calcu-lated for each cell line.

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FIG. 1. Identification of GLUT isoforms in the human glioma cell line, T98G. A, dose-dependent 2-DG uptake in T98G cells. Uptake of2-DG was measured for 13 min. Values are the mean � S.E. of three independent experiments. R2 � 0.999. The value of Km (2.42 � 0.44 mM)suggested that a low affinity glucose transporter, GLUT-2, did not participate in glucose uptake. Eadie-Hofstee plots from A are shown in the upper

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FIG. 2. Relationship between 2-DG uptake via GLUT-1 and expression of syntaxin 1C in T98G astroglioma cells treated with PMA.A, expression of syntaxin 1C in PMA-treated T98G cells. The amount of syntaxin 1C expressed in T98G cells that were treated for 24 h with 10�M PMA, 10 �M forskolin, or 10 �M 4�-PMA was quantified using 12% SDS-PAGE. Trichloroacetic acid lysates (25 �g) were immunoblotted withmonoclonal antibody 14D8 (35 kDa). Immunoblotted membranes were reprobed with anti-tubulin IgG (55 kDa). Densitometric analysis wasperformed using NIH Image. Expression of syntaxin 1C in response to treatment with 10 �M PMA increased 6.82 � 0.52-fold, compared with thecontrol (Cont., 0.1% Me2SO). B, 2-DG uptake in PMA-treated T98G cells. Cells were treated for 24 h with 10 �M PMA, 10 �M forskolin, or 10 �M

4�-PMA. Glucose uptake via GLUTs was measured as described under “Experimental Procedures.” The values are the mean � S.E. for threeindependent experiments. Glucose uptake in response to 10 �M PMA, 10 �M 4�-PMA, and 10 �M forskolin was, 8.84 � 0.62 (black bar), 10.49 �0.31 (gray bar), and 11.13 � 1.16 (striped bar) nmol/13 min/mg, respectively, versus the control (Cont., 10.87 � 0.24; 0.1% Me2SO; white bar). GLUTuptake was significantly reduced by treatment with PMA. **, p � 0.01 compared with control. C, time course of syntaxin 1C expression in T98Gcells treated with 10 �M PMA. Cells were treated with 10 �M PMA for 12, 24, or 48 h. Expression of syntaxin 1C was quantified as for A. The valuesare the mean � S.E. for three independent experiments. Expression of syntaxin 1C increased 4.53 � 0.51-, 7.34 � 1.31-, and 8.06 � 1.52-fold aftertreatment with PMA for 12, 24, and 48 h, respectively. D, time course of 2-DG uptake in T98G cells treated with 10 �M PMA. Cells were treatedwith 10 �M PMA for 12, 24, or 48 h. Glucose uptake was quantified as for B. The values are the mean � S.E. for three independent experiments.Glucose uptake decreased 0.94 � 0.02-, 0.86 � 0.05-, and 0.79 � 0.03-fold after treatment with PMA for 12, 24, and 48 h, respectively.

inset. B, PCR analysis of GLUT isoforms in T98G cells. Schematic representation of the GLUT isoforms is shown in the upper panel. Open boxesindicate the coding region. The bars indicate the 3�-untranslated regions. Arrowheads indicate the position of the primer pairs (1 � 2). PCR wascarried out using the primers indicated in the upper panel. First strand cDNA templates from T98G astrocytoma cells (human glial cell line), NB-1cells (human neurobrastoma cell line), and 3T3L1 cells (mouse adipocytes cell line) were analyzed on a 2.5% agarose gel. Semiquantitative RT-PCRwas carried out for 30 cycles using 5 ng of each cDNA template. Relative expression of GLUT-3 versus GLUT-1 (GLUT-3/GLUT-1) � 0.355 � 0.076.C, expression of GLUT-1 and GLUT-3 mRNA in T98G cells. Northern blot analysis of GLUT-1 and GLUT-3 mRNA expression revealed a 2.8-kbfragment that corresponded to GLUT-1 mRNA and a small amount of a GLUT-3 transcript. The amount of GLUT-1 transcript was normalized tothe intensity of the 28 S tRNA band. Densitometric analysis revealed that the amount of GLUT-1 mRNA was �9-fold (949 � 79%; p � 0.0001)greater than that of GLUT-3. D, functional characterization of GLUT isoforms in T98G cells. Measurement of 2-DG uptake in T98G cells, culturedwith low and high concentrations of glucose, is shown in the white and black bar on the left, respectively. The concentration of D-glucose in theculture medium was 5.5 mM (low glucose condition, white bar) or 25 mM (high glucose condition, black bar). Glucose uptake was measured for 13min. Glucose uptake was 18.46 � 0.32 and 13.05 � 0.34 nmol/13 min/mg for the low and high glucose condition, respectively. Uptake of 2-DG wasincreased by reducing the concentration of glucose. Measurement of 2-DG uptake value in T98G cells, cultured with low and high concentrationsof insulin, is shown in the white and black bar on the right, respectively. Glucose uptake before 2-DG uptake measurement was 12.27 � 0.75 and12.77 � 0.55 nmol/13 min/mg for stimulation with 0 (white bar) and 4 �g/ml (black bar) insulin, respectively. Insulin did not affect 2-DG uptakein T98G cells, which suggested that there is no insulin-responsible GLUT-4 in T98G cells. The data in A–D support the conclusion that GLUT-1is the main isoform in T98G cells.

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The cell cycle analysis was carried out as reported previously (25).Growth of T98G cells that had been plated at a density of 2 � 105 cellsper 90-mm in diameter culture dish was arrested by removing serum for24 h. The cells were restimulated for 40 h with medium containing 10%serum. Thereafter, the cells (1 � 106 cells/ml) treated with 0.5 mg/mlRNase (Nippon Gene, Tokyo, Japan) were analyzed immediately afterpropidium iodide (Sigma) staining using fluorescence-activated cellsorting (FACS, BD Biosciences Epics Profile II) with argon laser exci-tation (488 nm) and a 588-nm (FL2) emission filter. At least 10,000 cellswere collected for each sample (excluding the gated cells). The percent-age of cells in the G0/G1, S, and G2/M phase was estimated fromFL2-height histograms using ModFit (Verity Software, Topsham, ME).

Measurement of Glucose Uptake—Glucose transport was assayed bymeasuring the uptake of 2- deoxy-[3H]glucose (2-DG), essentially asdescribed previously (22), but with slight modifications. The uptakeassay was carried out 48–72 h after cell passaging. Cells (1 � l05) inHanks’ balanced salt solution (HBSS), containing 0.03 g/100 ml bovineserum albumin, 136.9 mM NaCl, 5.6 mM KC1, 0.34 mM NaHPO4, 0.44mM KH2PO4, 1.27 mM CaCl2, and 4.20 mM NaHCO3, 20 mM HEPES, pH7.4, were incubated on 12-well multiplates at 37 °C, for 30 min. Glucoseuptake was initiated by adding 0.5 �Ci of 2-deoxy-D-[1,2-3H(N)]glucose(2-[3H]DG; PerkinElmer Life Sciences) to 0.5 ml of HBSS buffer, in thepresence of 0.1 mM 2-deoxy-D-glucose, in 35-mm in diameter wells. After13 min at room temperature, uptake was terminated by rapid washingwith 1 ml of ice-cold PBS. The uptake of 2-DG was linear between 0 and20 min of incubation (data not shown). For the kinetic analysis, we used0.1–100.0 mM 2-DG (0.0064–6.4 �M 2-[3H]DG). The cells were solubi-

lized in 1% SDS, and the amount of radioactivity was measured. Proteincontent was measured using the DC protein assay from Bio-Rad. Thedifference in the amount of uptake in the presence and absence of 0.5mM cytochalasin B (a transport inhibitor) was calculated; this repre-sented glucose transporter-dependent activity. In each experiment, glu-cose uptake was assayed in triplicate.

We studied the kinetics of glucose uptake using different concentra-tions of D-glucose, as described previously (26). Briefly, confluent cul-tures of T98G cells were incubated with either 5.5 or 25.0 mM D-glucosefor 7 days. The culture medium was changed daily to maintain arelatively constant concentration of glucose.

To study basal glucose uptake via sodium-dependent glucose trans-porters (SGLTs), cells were treated with Na�-free HBSS buffer contain-ing 0.03 g/100 ml bovine serum albumin, 138 mM N-methyl-D-(�)-glucamine (NMDG), 5.6 mM KC1, 0.34 mM KHPO4, 0.44 mM KH2PO4,1.27 mM CaCl2, and 20 mM HEPES (pH 7.4).

Statistical Analysis—Data are expressed as mean � S.E. and wereanalyzed using one-way analysis of variance. A p value of � 0.05 wasconsidered to be statistically significant.

RESULTS

Measurement of 2-DG Uptake via GLUTs and SGLTs inT98G Cells—To examine the relationship between syntaxin 1Cexpression and glucose transport, we first determined whetherthere was glucose uptake via GLUT in T98G cells. The amountof 2-DG uptake via GLUTs and SGLTs was measured in the

FIG. 3. Expression of syntaxins andGLUTs in T98G astroglioma cellstreated with PMA. A, syntaxin (Syn)expression. Cells were treated for 24 hwith 0–10 �M PMA and then analyzed by12% SDS-PAGE. Trichloroacetic acid ly-sates (25 �g) were immunoblotted withmonoclonal antibody 14D8 and then rep-robed with anti-tubulin IgG. Densitomet-ric analysis was performed using NIH Im-age. Only syntaxin 1C expressionappeared to increase in a dose-dependentmanner in T98G cells that were stimu-lated with 10 �M PMA. Syntaxin 1C ex-pression increased 7.37 � 1.34-fold, com-pared with the control (0.1% Me2SO). *,p � 0.05. **, p � 0.01, compared withcontrol. B, GLUT-1 mRNA expression.Cells were treated for 24 h with 10 �M

PMA, 10 �M forskolin, or 10 �M 4�-PMA.Total RNA (20 �g) was analyzed byNorthern blot. The amount of GLUT-1and GLUT-3 mRNA was quantified bynormalizing the band intensity to that ofthe 28 S tRNA band. There was no signif-icant change in the amount of GLUT-1and GLUT-3 mRNA in T98G cells thatwere treated with PMA. C, GLUT-1 pro-tein expression. Cells were treated for24 h with 10 �M PMA, 10 �M forskolin, or10 �M 4�-PMA. Membrane fractions (35�g) were immunoblotted with anti-GLUT-1 polyclonal antibody. The level ofGLUT-1 protein expression (47 kDa) wasnot affected by PMA.

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presence of cytocharasin B (an antagonist of GLUTs) and inNa�-free medium. GLUT activity accounted for �80% of total2-DG uptake in T98G cells. By contrast, SGLT activity ac-counted for less than 20% of total 2-DG uptake (data notshown). Uptake of 2-DG was linear for up to 20 min of incuba-tion (data not shown). These observations indicate that GLUTsare expressed in T98G cells.

Identification of GLUT Isoform in T98G Cells—There are

several reports that the GLUT isoforms GLUT-1 and GLUT-3are the main components of several types of glioma (22, 27, 28).However, whether GLUTs are expressed in T98G cells has notbeen determined. To determine which GLUT isoform(s) is ex-pressed in T98G cells, we investigated the kinetics of 2-DGuptake. As shown in Fig. 1A, the Km of 2-DG uptake in T98Gcells was 2.42 � 0.12 mM. GLUT-1, GLUT-3, and GLUT-4 arehigh affinity glucose transporters, whereas GLUT-2 is a low

FIG. 4. Localization of GLUT-1 andexpression of syntaxin 1C in T98Gcells treated with PMA, 4�-PMA, orforskolin. Cells were treated for 24 hwith 10 �M PMA (B and F), 10 �M 4�-PMA (C and G), or 10 �M forskolin (D andH). Cells were fixed and permeabilizedwith acetone/methanol (1:1) and double-stained with anti-GLUT-1 polyclonal an-tibody and monoclonal antibody 14D8, asdescribed under “Experimental Proce-dures.” The figures are confocal scanninglaser microscopic images of GLUT-1(A–D) and 14D8 (E–H) immunofluores-cence. Treatment of cells with PMA in-creased syn1C and decreased GLUT-1 ex-pression in the plasma membrane.

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affinity transporter (29). Because the value of Km in the presentstudy is far smaller than that of GLUT-2 (Km: 20–40 mM), it islikely that the GLUT isoform functioning in T98G cells is notGLUT-2, but rather a high affinity transporter, i.e. GLUT-1,GLUT-3, or GLUT-4.

As shown in Fig. 1B, semiquantitative RT-PCR revealed thatGLUT-1 and GLUT-3 were expressed in T98G cells; GLUT-2and GLUT-4 expression was undetectable except under satu-rated PCR conditions (data not shown). To examine the expres-sion levels of endogenous GLUT-1 and GLUT-3, we studiedexpression of the mRNA of these GLUT isoforms in T98G cellsby Northern blot analysis. As shown in Fig. 1C, GLUT-1 mRNAwas more abundant than that of GLUT-3. We also confirmedexpression of GLUT-1 protein by immunoblotting (see Fig. 3C)and localization in plasma membrane in T98G cells by cellsurface biotinylation assay (data not shown).

It has been reported that glucose uptake via GLUT-1 in cellscultured in low glucose medium is higher than in the presenceof high concentrations of glucose (26). We investigated glucoseuptake in T98G cells that were cultured with different concen-trations of glucose. As expected, 2-DG uptake was �1.5 timesgreater in low-glucose medium (5.5 mM glucose), comparedwith high glucose medium (25 mM glucose) (Fig. 1D). Anotherproperty of GLUT-4 is that it translocates to the plasma mem-brane in cells that have been stimulated with insulin, whichresults in an increase in glucose uptake (16). Consequently, wetested whether the amount of 2-DG uptake in T98G cells would

increase after stimulation with insulin; this was not the case(Fig. 1D). The results shown in Fig. 1, B and D suggest thatthere is no functional GLUT-4 in T98G cells. The aforemen-tioned results demonstrate that the major isoform of GLUT inT98G cells is GLUT-1.

Activation of Endogenous Syntaxin 1C by PMA SuppressesTranslocation of GLUT-1 to the Plasma Membrane in T98GCells—Our previous study revealed that T98G astrogliomacells express syntaxin 1C, but not syntaxin 1A, and that syn-taxin 1C expression can be activated by PMA (9). In the presentstudy, we investigated whether a change in the level of syn-taxin 1C expression might affect glucose transport.

Fig. 2, A and B shows the change in syntaxin 1C expressionand 2-DG uptake in T98G cells that were treated with eitherPMA, forskolin, or 4�-PMA (a nonfunctional analog of PMA).Uptake of 2-DG in PMA-treated cells was reduced by �85%,compared with control cells (Fig. 2B), whereas 2-DG uptakewas not affected by either forskolin or 4�-PMA (Fig. 2B). Noneof the aforementioned treatments had any effect on the uptakeof 2-DG via SGLTs (data not shown). In addition, an analysis ofthe time course of glucose uptake (Fig. 2, C and D) revealedthat 2-DG uptake in T98G cells was reduced as the PMA-induced level of syntaxin 1C expression increased. Becausesyntaxin 1A mRNA is not found in astroglioma cells, irrespec-tive of whether cells are treated with PMA (9), the observedchange in glucose uptake in PMA-treated T98G cells was notcaused by the actions of syntaxin 1A. To determine whether the

FIG. 5. GLUT-1 mRNA and protein expression and glucose uptake via GLUT-1 in T98G cells transfected with syntaxin 1A, syntaxin1C, or syntaxin 4. A, expression of HA-tagged syntaxins. Trichloroacetic acid lysates were immunoblotted with an anti-HA monoclonal antibody(3F10). The level of expression of the transfected syntaxins was similar for each type of syntaxin. T98G-Syn1A, T98G-Syn1C, and T98G-Syn4indicate T98G cells that were transfected with syntaxin 1A, syntaxin 1C, and syntaxin 4, respectively. B, expression of GLUT-1 mRNA. Arepresentative autoradiograph of a Northern blot is shown. The positions of 28 and 18 S rRNA are indicated on the right. The amount of GLUT-1mRNA was quantified by normalizing the band intensity to that of the 28 S tRNA band. The amounts of 2.8-kb GLUT-1 transcript in 20 �g of totalRNA were similar in T98G cells that were transfected with either the expression vector alone (T98G-Mock), syntaxin 1A (T98G-Syn1A), syntaxin1C (T98G-Syn1C), or syntaxin 4 (T98G-Syn4). C, immunoblot for GLUT-1. The membrane fraction (35 �g) of each of the transfected T98G cell lineswas immunoblotted with anti-GLUT-1 polyclonal antibody. Expression of GLUT-1 protein (47 kDa) was not affected by the overexpression ofsyntaxin 1A, syntaxin 1C, or syntaxin 4. D, glucose uptake via GLUTs (black bars) or SGLTs (gray bars) was measured, as described under“Experimental Procedures.” The values are the mean � S.E. for three independent experiments. Glucose uptake via GLUTs in T98G cells that weretransfected with either the expression vector alone (T98G-Mock), syntaxin 1A (T98G-Syn1A), syntaxin 1C (T98G-Syn1C), or syntaxin 4 (T98G-Syn4) was 11.39 � 0.47, 7.19 � 0.50, 6.80 � 0.41, and 10.85 � 0.96 nmol/13 min/mg, respectively. Glucose uptake via SGLT in T98G cells that weretransfected with either the expression vector alone (T98G-Mock), syntaxin 1A (T98G-Syn1A), syntaxin 1C (T98G-Syn1C), or syntaxin 4 (T98G-Syn4) was 1.66 � 0.51, 1.57 � 0.42, 1.57 � 0.56, and 1.37 � 0.43 nmol/13 min/mg, respectively. ***, p � 0.001, compared with control (T98G-Mock).GLUT-1 uptake was suppressed in cells that were transfected with syntaxin 1A or syntaxin 1C.

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reduction in glucose uptake was caused by the presence ofsyntaxin 1C, we examined the expression of syntaxin 2, syn-taxin 3, and syntaxin 4 in the plasma membrane. In contrast tothe expression of syntaxin 1C, which is increased in a dose-de-pendent manner by PMA treatment (7.37 � 1.34-fold increasein response to 10 �M PMA), the expression of syntaxin 2,syntaxin 3, and syntaxin 4 was not affected by PMA (Fig. 3A).

Furthermore, PMA had no effect on the expression of GLUT-1and GLUT-3 mRNA and protein in T98G cells (Fig. 3, B and C).These results suggest that the treatment of T98G cells withPMA did not alter the level of expression of GLUT-1, GLUT-3,or syntaxins other than syntaxin 1C.

FIG. 6. Dose-dependent glucose uptake in untransfected and transfected T98G cell lines. The values are the mean � S.E. for threeindependent experiments. T98G (Native cell) (untransfected cells): Vmax � 10.00 � 0.86, Km � 2.01 � 0.46 mM, R2 � 0.996. T98G-Mock (cellstransfected with the expression vector alone): Vmax � 10.00 � 0.90, Km � 2.66 � 0.31 mM, R2 � 0.998. T98G-Syn1C (syntaxin 1C-transfected cells):Vmax � 6.04 � 0.72, Km � 2.71 � 0.29 mM, R2 � 0.999. The value of Vmax was reduced in cells that were transfected with syntaxin 1C (T98G-Syn1C),whereas the value of Km was the same as that in the untransfected (T98G (Native cell)) and expression vector-transfected (T98G-Mock) cells.

TABLE IGrowth rate of T98G cells after transfection

Cells were grown in DMEM supplemented with 10% (v/v) FCS for0–96 h. The number of cells was counted, and the growth rate wascalculated during the logarithmic phase (48–96 h). Results are pre-sented as growth rate per 48 h (mean � S.E.; n � 4). The growth ratewas the same in each of the transfected cell lines.

Cell line Growth rate p value versus T98G-Mock

per 48 h

T98G 3.05 � 0.39 0.442T98G-Mocka 3.26 � 0.33T98G-Syn1Ab 3.19 � 0.14 0.706T98G-Syn1Cc 3.23 � 0.19 0.856T98G-Syn4 3.25 � 0.26 0.931

a Mock, control cells (transfected with the expression vector alone).b Syn1A, syntaxin 1A.c Syn1C, syntaxin 1C.

TABLE IIDistribution of cell cycle phases in T98G cells after transfection

After growth had been arrested by the removal of serum, cells wererestimulated for 40 h with medium that contained 10% serum. Afterstaining with propidium iodide, the fluorescence intensity of individualcells was measured using fluorescence-activated cell sorting (at least10,000 cells per sample, excluding the gated cells). Results are pre-sented as the percentage of cells in the G0/G1, S, and G2/M phase(means � S.E.; n � 3). The distribution of cells for each of the phaseswas the same in each of the transfected cell lines. Abbreviations are asfor Table I.

Cell lineCell cycle stage

G0/G1 S G2/M

%

T98G 73.59 � 3.50 23.88 � 4.69 2.53 � 1.21T98G-Mock 77.97 � 0.69 19.32 � 1.99 2.70 � 1.37T98G-Syn1A 77.91 � 6.08 21.28 � 3.81 2.48 � 0.94T98G-Syn1C 76.41 � 2.22 20.11 � 1.43 3.49 � 0.99T98G-Syn4 76.79 � 3.78 20.08 � 4.72 2.13 � 1.02

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We also analyzed the effect of PMA using immunofluores-cence and found that in cells in which the expression of endog-enous syntaxin 1C had been enhanced by PMA, GLUT-1 ex-pression in the plasma membrane decreased, whereasexpression in the intracellular fraction increased (Fig. 4, B andF). By contrast, neither forskolin (Fig. 4, D and H) nor 4�-PMA(Fig. 4, C and G) had any effect on GLUT-1 expression.

Glucose Uptake via GLUT-1 in T98G Cells Transfected withSyntaxin 1A, Syntaxin 1C, or Syntaxin 4—To determinewhether syntaxin 1C expression affects glucose transport inastroglioma cells, we introduced exogenous syntaxin 1A, syn-taxin 1C, or syntaxin 4 tagged with HA into at least three linesof T98G cells for each syntaxin. Western blot analysis revealedthat the level of expression of each type of transfected syntaxinwas similar (Fig. 5A). There was also no difference among thecell lines with respect to the level of GLUT-1 mRNA and pro-tein expression (Fig. 5, B and C). These observations indicatethat the overexpression of exogenous syntaxin did not affectGLUT-1 expression in T98G cells.

Next, we studied 2-DG uptake via GLUTs in each type ofsyntaxin-transfected cell line. The results indicated that 2-DGuptake was reduced to �60% in the syntaxin 1C-transfectedcell line (T98G-Syn1C; 6.80 � 0.41 nmol/13 min/mg), comparedwith cells that were transfected with the expression vectoralone (Fig. 5D, T98G-Mock; 11.39 � 0.47). Similar results wereobtained for syntaxin 1A-transfected cell lines (Fig. 5D, T98G-Syn1A; 7.19 � 0.50). By contrast, there was no change in 2-DGuptake in syntaxin 4-transfected cells (Fig. 5D, T98G-Syn4;10.85 � 0.96). Uptake of 2-DG via SGLTs was not altered byoverexpression of exogenous syntaxin (Fig. 5D). Similar obser-vations were obtained for cell lines in which other syntaxinswere expressed, and in cells that were transfected with exoge-nous syntaxin without an HA tag (data not shown), suggestingthat the suppression of 2-DG uptake was not caused by eitherclonal variation or the presence of the HA tag motif. Finally, weobtained similar results with another astroglioma cell line,namely U87MG (data not shown).

Dose-dependent Glucose Uptake in Untransfected T98G Cellsand T98G Cells Transfected with Syntaxin 1C—To determinewhether glucose uptake in syntaxin 1C-transfected T98G cellswas dose-dependent, we studied kinetic analysis of 2-DG up-take (Fig. 6). The value of Vmax (6.041 � 0.72 mM) was reducedin the HA-tagged syntaxin 1C-expressing cell line (T98G-Syn1C), whereas the value of Km (2.708 � 0.29 mM) was un-changed relative to the untransfected cells (T98G (Native cell):Km � 2.009 � 0.46 mM, Vmax � 10.002 � 0.86 mM) and cells thatwere transfected with the expression vector alone (T98G-Mock:Km � 2.657 � 0.31 mM, Vmax � 10.001 � 0.90 mM) (Fig. 6).These results suggest that the decrease in glucose uptake thatis associated with the expression of syntaxin 1C might becaused by a decrease in amount of GLUT-1 in the plasmamembrane, whereas the rate of glucose transport by individualGLUT-1 remains the same.

Overexpression of Syntaxin Did Not Affect the Cell Growth orMitogenesis—It has been reported that glucose transport isassociated closely with mitogenic properties of glioma cells,such as cell growth (22). Therefore, we investigated whethersyntaxin expression might affect cell proliferation. We meas-ured the growth rate of cells during the logarithmic growthphase (48–96 h). As shown in Table I, the growth rate of cellstransfected with syntaxin was not significantly different to thatof cells that were transfected with the expression vector alone.To examine cells in each phase of the cell cycle, cell growth wasarrested (by withdrawing serum for 24 h) to synchronize thecell cycle; cells were then restimulated for 40 h with mediumcontaining 10% serum. As shown in Table II, there was no

difference after restimulation between the cell cycles of cellsthat had been transfected with syntaxin, compared with cellsthat were transfected with the expression vector alone. These

FIG. 7. Intracellular localization of GLUT-1 in T98G cellstransfected with non-HA-tagged syntaxins. A–E, representativeimmunofluorescence images of T98G cells, which were transfected withsyntaxins that did not have an HA tag. Cells were fixed, permeabilizedwith acetone, stained with an anti-GLUT-1 polyclonal antibody, andobserved with a confocal laser microscope. F–J, higher magnification(�800) images of A–E. The amount of GLUT-1 in the plasma membraneappeared to be reduced in cells that were transfected with syntaxin 1Aor syntaxin 1C.

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results indicate that the overexpression of syntaxin did notaffect the mitogenic properties of T98G cells. Thus, the sup-pression of glucose uptake by syntaxin 1C was not caused by analteration in mitogenesis.

Intracellular Localization of GLUT-1 in T98G Cells Trans-fected with Syntaxin 1C—We examined the intracellular local-ization of endogenous GLUT-1. Immunofluorescence images ofthe various syntaxin-transfected cell lines are presented in Fig.7. There was little GLUT-1 expression in the plasma mem-brane of T98G cells that had been transfected with syntaxin 1C(T98G-Syn1C), and most GLUT-1 appeared to be localized tothe intracellular compartment of these cells (Fig. 7). A similarresult was obtained in the case of syntaxin 1A-transfected cells(T98G-Syn1A) (Fig. 7). By contrast, in cells that had beentransfected with either syntaxin 4 (T98G-Syn4) or the expres-

sion vector alone (T98G-Mock), almost all GLUT-1 was presentin the plasma membrane, and there was little or no GLUT-1within the cell (Fig. 7). The same result was obtained for cellsthat were transfected with HA-tagged syntaxins (data notshown). These observations are consistent with the results ofthe analysis of glucose and 2-DG uptake (see Figs. 5 and 6,respectively).

We also examined immunostaining in T98G cells that hadbeen transiently transfected with HA-tagged syntaxins (Fig. 8).Only cells that expressed HA-tagged syntaxin 1A (Fig. 8, A, D,and G) and HA-tagged syntaxin 1C (Fig. 8, B, E, and H)exhibited a reduction in GLUT-1 expression in the plasmamembrane and an increase in GLUT-1 within intracellularcompartments. However, there was no change in localization ofGLUT-1 in cells that had been transfected with HA-tagged

FIG. 8. Intracellular localization of GLUT-1 in T98G cells that were transiently transfected with syntaxin. T98G cells that expressedsyntaxins transiently were fixed and permeabilized with acetone/methanol (1:1) and then double-stained with anti-HA monoclonal antibody (3F10)and anti-GLUT-1 polyclonal antibody, as described under “Experimental Procedures.” A–C, 3F10 (anti-HA) immunostaining. D–F, GLUT-1immunostaining. G–I, merged images. Asterisks indicate transfected cells that expressed exogenous syntaxin. Arrowheads indicate cells that didnot express exogenous syntaxin, but did express GLUT-1 in the plasma membrane. The amount of GLUT-1 in the plasma membrane was reducedin cells that expressed syntaxin 1A (A, D, and G) or syntaxin 1C (B, E, and H). Syntaxin 4 had no effect on the intracellular localization of GLUT-1(C, F, and I).

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syntaxin 4 (Fig. 8, C, F, and I). Syntaxin 1A and syntaxin 4,both of which are membrane-bound, were not localized to theplasma membrane in astroglioma cells. This might be causedby the absence of the regulatory molecules (e.g. nSec-1/Munc18) that was necessary for localization of plasma mem-brane syntaxin (30, 31). These findings suggest that solublesyntaxin 1C might prevent the localization of GLUT-1 to theplasma membrane, which would ultimately reduce glucosetransport.

DISCUSSION

Recent studies have revealed that several signal transduc-tion mechanisms participate in the glucose metabolism, theregulation of GLUT expression and localization, and glucosetransport (18–20). We demonstrated previously that syntaxin1C expression was up-regulated by PMA via PKC signaling inastroglioma cells (9). In this report, we demonstrated thatglucose transport and the amount of GLUT-1 in the plasmamembrane were suppressed in astroglioma cells by stimulationwith PMA. It is likely that the suppression of glucose transportby PMA was caused by a decrease in the number of GLUT-1molecules that is present in the plasma membrane, and thatthis was caused by an increase in the syntaxin 1C expressionbecause: 1) PMA increased the endogenous expression of syn-taxin 1C without changing the total expression of syntaxin2–4, or GLUT-1 in astroglioma cells and 2) overexpression ofexogenous syntaxin 1C caused the similar phenomenon to thatby PMA. These suggest that the PKC signaling, which affectsthe syntaxin 1C expression, may (at least partly) regulate theglucose metabolism through changing the GLUT-1 expressionin the plasma membrane.

However, it was not clear whether the reduced expression ofGLUT-1 in the plasma membrane was caused by a suppressionof translocation of GLUT-1 to the plasma membrane or anincrease in the amount of GLUT-1 that was internalized fromthe plasma membrane, because most GLUT-1 in the plasmamembrane is recycled constitutively between the plasma mem-brane and intracellular vesicles. Recent studies of GLUT trans-location suggest two possibilities. First, syntaxin 1C may playa role in membrane fusion. Although the intracellular trans-port pathway of GLUT-1 is unknown, studies of GLUT-4 vesic-ular translocation suggest that the machinery that is used forthe intracellular trafficking of GLUT is similar to that in neu-roendocrine systems (16). In this model, GLUT-4-containingvesicles are primed to the plasma membrane where fusion isdriven by the formation of a stable heterotrimeric complex ofsyntaxin 4, SNAP23, and VAMP-2. In addition, it has beenproposed that the formation of the SNARE complex is regu-lated by several suppressive molecules, including Munc18c,synip, pantophysin, and Rab4 (17). Syntaxin 1C is distinct fromsyntaxin 1A in that the C-terminal of syntaxin 1C is convertedby alternative splicing to a novel proline-rich region of 35residues; this results in an absence of both the transmembranedomain and the latter half of the H3 domain (residues 191–267in syntaxin 1A). Therefore, the C-terminal of syntaxin 1C doesnot have the capacity to bind most SNAREs or accessory mol-ecules, except nSec-1/Munc18 and SNAP-25. Therefore, unlikesyntaxin 4, syntaxin 1C may not be able to form a SNARE corecomplex. We showed previously that syntaxin 1C is found onlyin the soluble fraction of astroglioma cells (9), which indicatesthat there is no binding with membrane-bound SNAREs. How-ever, since syntaxin 1C is able to bind Munc18b in vitro (7), itis possible that syntaxin 1C may be in competition with thefactors including Munc18b that are able to bind its N-terminal,thereby changing the constitutive intracellular transport ofGLUT-1 vesicles to the plasma membrane. Second, syntaxin 1Cmay act on the cytoskeleton. It was demonstrated recently that

components of the cytoskeleton, such as microtubules and actinfilaments, are necessary for the translocation of GLUT-1 andGLUT-4 to the plasma membrane (32–34). From this view-point, it is interesting that syntaxin 1C possesses a tubulinbinding motif in the N-terminal region. An analysis of micro-tubule reassembly showed that a peptide for the tubulin-bind-ing motif that is found in both syntaxin 1A and syntaxin 1Ccould directly bind tubulin subunits, and the N-terminal pep-tide involving this motif could decrease tubulin polymerizationin vitro (35, 36), suggesting that syntaxin 1C might affect thelocal structure of microtubules. The aforementioned observa-tions suggest that the N-terminal region of syntaxin 1C mightplay an important role in regulating the local structure of thecytoskeleton in astroglioma cells. This might explain why theoverexpression of syntaxin 1A (which binds the plasma mem-brane) produced similar effects on glucose transport as did theoverexpression of syntaxin 1C.

In the CNS, energy metabolism via GLUT-1 plays a centralrole in the function of astroglia, which modulates the distribu-tion and metabolism of glucose in the brain (13). The regulationof glucose transport in the CNS is particularly important in thehippocampus and frontal cortex, because these regions areintegration centers that are crucial to learning, memory, andpersonality traits. Several studies have suggested that disrup-tion of glucose metabolism in the CNS is associated with neu-ronal dysfunction. For example, neuronal activity (particularlycognitive function) may be adversely affected in metabolic dis-orders, such as diabetes mellitus, in which glucose delivery orutilization in the CNS is disrupted (37–39). Impaired cognitivefunction can be ameliorated to an extent by administeringglucose and insulin. We reported previously that in patientswith Williams syndrome, which is characterized by cognitivemalfunction that produces hyperactivity, poor attention, rela-tively intact linguistic function, and visual spatial deficits, thesyntaxin 1C gene is located within a region that is deletedhemizygously (8, 40). An examination of glucose metabolism inthe CNS of patients with Williams syndrome would be a po-tentially fruitful avenue of investigation.

In conclusion, we have shown in the present study thatsyntaxin 1C, a nonmembrane-bound syntaxin, can affect theintracellular transport of a plasma membrane protein. Furtherstudies will enable us to better understand the mechanism ofuptake and membrane transport of glucose.

Acknowledgments—We thank Dr. Shinya Nagamatsu for useful ad-vice about analyzing GLUT expression and glucose uptake andDr. Ryo Takahashi for advice about analyzing the cell cycle. We aregrateful to Masumi Sanada for assistance with molecular biologytechniques.

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Takahiro Nakayama, Katsuhiko Mikoshiba, Tetsuo Yamamori and Kimio AkagawaAstroglioma Cells via the Glucose Transporter-1 (GLUT-1)

Phorbol 12-Myristate 13-Acetate (PMA) Suppresses Glucose Transport into Activation of Syntaxin 1C, an Alternative Splice Variant of HPC-1/Syntaxin 1A, by

doi: 10.1074/jbc.M314297200 originally published online March 22, 20042004, 279:23728-23739.J. Biol. Chem. 

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