GLUT8 Subcellular Localization and Absence of Translocation to the Plasma Membrane in PC12 Cells and Hippocampal Neurons
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《内分泌学杂志》
Department of Physiology, University of Lausanne, 1005 Lausanne, Switzerland
Abstract
GLUT8 is a high-affinity glucose transporter present mostly in testes and a subset of brain neurons. At the cellular level, it is found in a poorly defined intracellular compartment in which it is retained by an N-terminal dileucine motif. Here we assessed GLUT8 colocalization with markers for different cellular compartments and searched for signals, which could trigger its cell surface expression. We showed that when expressed in PC12 cells, GLUT8 was located in a perinuclear compartment in which it showed partial colocalization with markers for the endoplasmic reticulum but not with markers for the trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles. To evaluate its presence at the plasma membrane, we generated a recombinant adenovirus for the expression of GLUT8 containing an extracellular myc epitope. Cell surface expression was evaluated by immunofluorescence microscopy of transduced PC12 cells or primary hippocampal neurons exposed to different stimuli. Those included substances inducing depolarization, activation of protein kinase A and C, activation or inhibition of tyrosine kinase-linked signaling pathways, glucose deprivation, AMP-activated protein kinase stimulation, and osmotic shock. None of these stimuli-induced GLUT8 cell surface translocation. Furthermore, when GLUT8myc was cotransduced with a dominant-negative form of dynamin or GLUT8myc-expressing PC-12 cells or neurons were incubated with an anti-myc antibody, no evidence for constitutive recycling of the transporter through the cell surface could be obtained. Thus, in cells normally expressing it, GLUT8 was associated with a specific intracellular compartment in which it may play an as-yet-uncharacterized role.
Introduction
THE TRANSPORT OF glucose through biological membranes is facilitated by several glucose transporter isoforms. GLUT1–5 have been studied in detail, and their physiological roles in various aspects of the control of glucose homeostasis have been well established (1, 2, 3, 4, 5). More recently screening gene banks for GLUT homologs revealed the existence of additional GLUT-like molecules in the rodent and human genomes. These sequences [GLUT6–12, HMIT, and GLUT14] have been cloned and studied in various experimental settings to determine their transport characteristics (6, 7, 8, 9, 10, 11, 12, 13). GLUT8, formerly named GLUTX1, was the first member of this extended GLUT family to be identified. It has a large extracellular, glycosylated loop between transmembrane domains 9 and 10, a characteristic of the type III glucose transporters, and it transports glucose with a relatively high affinity (Km = 2 mM) (14, 15).
GLUT8 expression is mostly restricted to the testis and some brain regions, primarily the hippocampus and hypothalamus (16, 17). It is also found at a lower level in insulin-sensitive tissues such as the liver, heart, white and brown fat, and adrenal glands. Interestingly, GLUT8 was shown to be present in nerve terminals in the supraoptic nucleus and to colocalize with vasopressin granules in neurons forming the posterior pituitary (16). In all these tissues as well as in transfected cells, GLUT8 was present in intracellular compartments and not at the plasma membrane. The retention in an intracellular compartment depends on the presence of an N-terminal dileucine motif; mutating this motif into dialanine results in the localization of GLUT8 to the cell surface (14).
The only instance in which GLUT8 was reported to appear to the cell surface in a regulated manner is in murine blastocysts after insulin stimulation (18). However, in primary rat adipocytes, insulin did not stimulate GLUT8 translocation to the cell surface (7).
Thus, at present, the identity of the intracellular compartment harboring GLUT8 is not well defined, and it is not known whether in physiological situations GLUT8 translocates to the cell surface to take up glucose and which signals could control this translocation. The aim of this study was thus to assess the subcellular localization of GLUT8 in PC12 cells and primary culture of hippocampal neurons, which normally express this transporter, and investigate whether translocation to the cell surface could be triggered by a variety of stimuli.
Materials and Methods
Cell culture and antibodies
PC12ES cells were grown in DMEM (Invitrogen Life Technologies, Inc., Paisley, Scotland, UK) containing 25 mM glucose and supplemented with 6% horse serum (Amimed, BioConcept, Allschwil, Switzerland), 6% fetal bovine serum (Life Technologies), 10 mM HEPES, 1 mM sodium pyruvate, and 2 mM glutamine at 37 C with 5% CO2. 293AD cells were grown in DMEM containing 10% fetal bovine serum. PC12 differentiation was obtained by changing the high-serum-containing medium to DMEM containing 1% horse serum and 50 ng/ml nerve growth factor 7S (Alomone Labs, Jerusalem, Israel). All experiments were done on differentiated PC12 cells.
GLUT8myc was detected with anti-myc antibodies (9E10 clone; Roche, Basel, Switzerland). Polyclonal antibodies used for double-labeling studies were the following: anti-TGN38 (gift of Professor Grenningloh, Lausanne, Switzerland), anti-early endosome autoantigen (EEA)-1 (Affinity BioReagents, Golden, CO), anti-NEEP21 (gift of Professor Hirling, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland), anti-lysosome-associated membrane protein (Lamp)1 (Santa Cruz Biotechnology, Santa Cruz, CA), anticalregulin (Santa Cruz), antisynaptophysin (Santa Cruz). Monoclonal antibodies against hemagglutinin (HA) were from Boehringer (Mannheim, Germany). All other reagents were from Sigma (St. Louis, MO) except A23187 ionophore (Calbiochem, La Jolla, CA), insulin (Novo Nordisk, Copenhagen, Denmark), and brain-derived neurotrophic factor (BDNF) (Alomone Labs).
Preparation and infection of hippocampal neurons
Hippocampi were dissected from E17 mouse brains of the C57BL6/J strain (Iffa-Credo, Lyon, France) (19, 20). Neurons were isolated by gentle trituration as described and then immediately plated on 10-mm round poly-L-lysine-coated (30–70 kDa; Sigma) glass coverslips in Neurobasal medium (Invitrogen Life Technologies) containing 2% B-27 supplement (Invitrogen Life Technologies), 0.5 mM glutamine, and 25 μM glutamate. Cell viability was assessed before plating by trypan blue staining. After 7–12 d of culture at 37 C and 5% CO2, neurons were infected during 48 h with high doses [multiplicity of infection (MOI) > 100] of adenovirus.
Expression of GLUT8myc in PC12 cells and hippocampal neurons
A human GLUT4 cDNA-containing seven c-myc epitope (GLUT4myc) in the first exofacial loop was kindly provided by Jonathan S. Bogan (Yale University, New Haven, CT) and Harvey F. Lodish (Whitehead Institute, Cambridge, MA). A fragment containing the c-myc epitopes was amplified from this plasmid by using specific primers containing additional 5' and 3' extensions corresponding to positions 1076–1095 and 1096–1114 of the rat GLUT8 cDNA sequence. A second PCR fragment (position 272-1095 of rat GLUT8 cDNA) was amplified by PCR from a rat GLUT8ss cDNA with specific primers containing additional 3' extension corresponding to position 193–208 of GLUT4myc cDNA. Finally, a third fragment (position 1096–1590) of GLUT8 cDNA containing a 5' extension corresponding to position 345–360 of GLUT4myc cDNA was obtained by PCR. A mix of 10 ng of each fragment was used as a template for a fourth PCR to generate a fragment of 1318 bp corresponding to position 272-1590 of GLUT8 cDNA with one c-myc epitope inserted into the exofacial loop between transmembrane domains 9 and 10. This PCR product digested by SacII and BglII enzymes was subcloned in pcDNA3 (Invitrogen Life Technologies) vector containing the wild-type and mutant forms of GLUT8 digested with the same enzymes. The cDNA for rat GLUT8myc was then subcloned into adenoviral vectors pAdTrack-CMV for determination of cell surface GLUT8 and pAdShuttle-CMV for colocalization studies (referred to as AdTrack-GLUT8myc and AdShuttle-GLUT8myc). High-titer stocks of adenovirus were prepared by infecting 293 cells as described in details elsewhere (21). Viral titers were evaluated by counting green fluorescent protein (GFP)-positive cells for AdTrack-GLUT8myc adenovirus and positive cells after anti-myc and anti-HA immunofluorescence for AdShuttle-GLUT8myc and AdShuttle-HA-dyn1K44A, respectively. PC12 cells were infected at an MOI of 5. Plasmid coding for mutant GLUT8 cDNA was transiently transfected into PC12 cells using Lipofectamine 2000 (Invitrogen Life Technologies) following the manufacturer’s instructions. Adenoviruses coding for HA-tagged mutant dynamin1 K44A were a kind gift of J. Pessin (State University of New York, Stony Brook, NY) (22).
Subcellular fractionation
PC12 cells infected with AdTrack-GLUT8myc were homogenized by 200 strokes in a dounce homogenizer in 2 ml of a solution consisting of 0.25 M sucrose, 20 mM HEPES (pH 7.4), 10 μg/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride (homogenization buffer). The homogenate was then centrifuged at 1500 rpm (270 x g), the pellet was discarded, and the supernatant loaded on top of a 10–50% continuous sucrose gradient containing 5 mM HEPES (pH 7.4). The homogenate was centrifuged at 4 C for 18 h at 30,000 rpm (160,000 x g) in an SW40Ti rotor (Beckman, Palo Alto, CA). Fractions (700 μl) were collected and the protein and sucrose concentrations were measured. An equal volume of 80 μl of each fraction was then separated by electrophoresis of 10% sodium dodecyl sulfate-polyacrylamide gels and the proteins were transferred onto nitrocellulose membranes for immunodetection using different antibodies as described. These procedures were as described previously (23).
Endogenous expression of GLUT8 in hippocampal neurons
The endogenous expression of GLUT8 in hippocampal neurons was assessed by RT-PCR. Total RNA was extracted with an RNeasy kit (QIAGEN, Hilden, Germany) and reverse transcribed into cDNA with superscript II enzyme (Invitrogen Life Technologies). Amplification was by 35 cycles of PCR using primers designed to amplify a 300-bp fragment of GLUT8 cDNA (forward primer: 5'-GCATAATCCAGGTCCTGTTCACTGC; reverse primer: 5'-GGAAGATCTCTGACATGAGG). GAPDH was amplified using as forward primer 5'-GTCGGTGTGAACGGATTTGG and as reverse primer 5'-GACTCCACGACATACTCAGC.
Immunofluorescence microscopy
Detection of GLUT8myc on nonpermeabilized PC12 cells was performed after incubation of the cells in K5 solution [5 mM KCl, 127 mM NaCl, 10 mM D-glucose, 1 mM MgCl2, 20 mM HEPES, and 2.7 mM CaCl2 (pH 7.4)] for 10 min at 37 C or after treatment for 2 min with K80 [80 mM KCl, 53 mM NaCl, 10 mM D-glucose, 1 mM MgCl2, 20 mM HEPES, and 2.7 mM CaCl2 (pH 7.4)] at 37 C. Cells were then immediately incubated in PBS (10 mM Na2HPO4, 138 mM NaCl, 2.7 mM KCl, and 1.76 mM KH2PO4) containing 1% BSA at 4 C. Detection of GLUT8-myc on nonpermeabilized mouse hippocampal neurons was performed in Ringer buffer [145 mM NaCl, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, and 5 mM D-glucose (pH 7.61)] at room temperature. Both PC12 cells and hippocampal neurons were fixed at the end of the assay with 4% paraformaldehyde, followed by three washes with PBS.
For immunodetection of intracellular antigens, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS for 2 min. Detection of antigens was performed for 1 h at room temperature followed by a 1-h incubation with the secondary antibodies, which were CY3-conjugated goat antimouse Ig (Jackson ImmunoResearch, West Grove, PA) and fluorescein-conjugated goat antirabbit Ig (Calbiochem) antibodies. Images were taken with a Leica TCS NT microscope (Cellular Imaging Facility, Lausanne, Switzerland).
Antibody capture assay
PC12 cells and hippocampal neurons were incubated for 2 h at 37 C with 10 μg/ml antimyc 9E10 antibody (24). Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and then incubated for 1 h at room temperature with secondary goat antimouse antibodies coupled to Cy3.
Results
GLUT8myc for immunodetection studies
To study GLUT8 intracellular localization and cell surface translocation, we generated an epitope-tagged version of GLUT8 containing a myc sequence in the extracellular glycosylated loop. We first showed that the myc epitope could be used for immunodetection of GLUT8 when expressed in cell lines. We thus transfected PC12 cells with a cDNA for GLUT8myc (Fig. 1A) containing or not a mutation of the dileucine motif. Expression of GLUT8myc was detected in permeabilized cells using an antimyc antibody (Fig. 1, B and C) and showed a perinuclear localization as previously reported (16, 17). Cell surface expression of the GLUT8myc dileucine mutant on nonpermeabilized cells was also clearly observed using the same antimyc antibody (Fig. 1, D and E).
GLUT8myc subcellular localization in PC12 cells
To study GLUT8myc subcellular localization, we generated two recombinant adenoviruses for efficient expression of the transporter in PC12 cells or hippocampal neurons. The first was AdShuttle-GLUT8myc for the immunofluorescence microscopy colocalization studies and the second AdTrack-GLUT8myc that was use for all the other subcellular and translocation experiments (see below).
Subcellular localization of GLUT8myc was first evaluated by confocal immunofluorescence microscopy of AdShuttle-GLUT8myc-infected PC12 cells. GLUT8myc localized to a perinuclear area and no staining within cellular processes were observed (Fig. 2). A partial colocalization with TGN38 was observed (Fig. 2, A–C), suggesting that a fraction of GLUT8myc may reside in the trans-Golgi network. No colocalization with the early endosomal marker EEA1 was observed (Fig. 2, D–F), suggesting that GLUT8myc did not reside in a recycling compartment in PC12 cells. This was also supported by the lack of colocalization with NEEP21 (data not shown), a marker of neuronal early endosomes (25). Occasional colocalization with Lamp1, a marker of lysosomes, could be observed (Fig. 2, G–I). By staining cells with MitoTracker (Molecular Probes, Eugene, OR) and GLUT8, we could rule out a localization of GLUT8 within mitochondria (data not shown). Moreover, only a partial colocalization with calreticulin could be obtained, suggesting that GLUT8myc did not principally reside in the ER (Fig. 2, J–L).
To obtain complementary information on GLUT8myc intracellular localization, PC12 cells infected with AdTrack-GLUT8myc were homogenized, and the postnuclear supernatant was fractionated by equilibrium centrifugation on 10–50% sucrose gradients (Fig. 3). The gradient fractions were then analyzed by Western blot for the presence of GLUT8myc and markers of different intracellular compartments. GLUT8myc-containing fractions were clearly distinct from those containing Lamp1 and EEA1, indicating absence of GLUT8myc from endosomes or lysosomes. A partial overlap with calreticulin-containing fractions was observed, suggesting that a fraction of GLUT8myc may be present in the endoplasmic reticulum. The distribution of the synaptophysin and GLUT8myc-containing vesicles was clearly distinct, indicating that GLUT8myc was not present in synaptic-like vesicles.
GLUT8myc translocation in PC12 cells Membrane depolarization
Neuron activation requires energy production, which could come from increased glucose uptake and metabolism. Therefore, we tested whether short-term depolarization by high KCl treatment or glutamate could induce GLUT8myc translocation to the plasma membrane. Figure 4 shows that no GLUT8myc surface expression could be detected following either K+ (Fig. 4, A–C) or glutamate (Fig. 4, D–F)-induced depolarization of transduced cells identified by GFP expression. Figure 4, G–I, shows GLUT8myc expression in permeabilized GFP-positive cells.
Activation of intracellular signaling pathways
We next tested whether activation of protein kinase A (PKA) or C (PKC), which can stimulate exocytosis of synaptic vesicles, could induced GLUT8 appearance to the plasma membrane. Short-term treatment of transduced PC12 cells with 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cAMP (dbcAMP), glucagon-like peptide-1-(7–36)-amide-1 (a G protein-coupled receptor linked to the adenylyl cyclase pathway), or phorbol myristate acetate to activate PKC, however, had no effect on GLUT8myc localization (data not shown). Similarly, no translocation could be observed when the transduced cells were treated with the tyrosine kinase inhibitors genistein or erbstatin A or the phosphatase inhibitors okadaic acid or phenylarsine oxide. Insulin or IGF-I treatment also did not induce translocation (see Table 1).
Hyperosmotic shock
Because hyperosmotic shock can trigger GLUT4 translocation to the plasma membrane of 3T3L1 adipocytes (26, 27), we tested whether a similar treatment could induce GLUT8myc translocation. However, no translocation was observed after a 30-min challenge with hyperosmotic concentrations of sorbitol (600 mM, Table 1).
Glucose deprivation and AMP-activated protein kinase (AMPK) activation
Hippocampal neurons submitted to a glucoprivic stress in vivo or in vitro by 2-deoxy-D-glucose (2-DG) treatment display increased protection against subsequent excitotoxic and oxidative stress. This is, at least in part, controlled by the expression of the heat shock proteins GRP78 and HSP70 (28). Here we tested whether 2-DG exposure of PC12 cells for 10 min (and also for longer periods of time in hippocampal neurons; see below) could induce GLUT8myc surface expression as part of the protective effect. However, no surface appearance of GLUT8myc could be detected in these conditions (Table 1).
A decrease in glucose supply activates the metabolic sensor AMPK (29, 30), which may be part of the response to ischemia. In muscle, activation of this kinase can induce translocation of GLUT4 to the plasma membrane (31). We thus treated transduced PC12 cells with 5-amino-imidazole-4-carboxamide-1--D-ribofuranoside (AICAR), an activator of AMP kinase (see Table 1). This also failed to induce GLUT8myc surface appearance.
GLUT8myc does not constitutively cycle through the plasma membrane in PC12 cells
A previous report indicated that GLUT8 could recycle constitutively to the plasma membrane, through a dynamin-dependent pathway, when expressed in primary rat adipocytes (7). To test whether a similar recycling of GLUT8 to the plasma membrane also occurred when the transporter was expressed in PC12 cells, we coinfected these cells with AdTrack-GLUT8myc and an adenovirus coding for a HA-tagged mutant of dynamin (dynK44A). The dynamin mutant was overexpressed, compared with GLUT8myc (ratio of MOI of 10:1) to ensure that dynaminK44A was present in all GLUT8myc-transduced cells. This was verified by immunofluorescence detection of HA-tagged mutant dynamin. As shown in Fig. 5, dynamin K44A overexpression, in conditions shown previously to increase HMIT cell surface expression (23), did not lead to increased expression of GLUT8myc at the plasma membrane.
As a another test for the possible presence of a constitutive recycling to the plasma membrane, PC-12 cells were transfected with a plasmid containing the GLUT8myc cDNA. Two days later, the transfected cells were exposed for 2 h at 37 C to 10 μg/ml of antimyc antibodies. After washing, the cells were permeabilized and reacted at 4 C with an antimyc antibody. This failed to revealed internalization of the antimyc antibody. This therefore provided another indication that GLUT8myc did not recycle to the plasma membrane in the basal state.
GLUT8myc expression and translocation in hippocampal neurons
Inability to observe translocation of GLUT8myc to the plasma membrane in PC12 cells may be due to these cells failing to express the required translocation machinery. We thus chose to express GLUT8myc in hippocampal neurons, which normally express this transporter. Primary culture of hippocampal neurons were thus established and were first tested for the presence of endogenous GLUT8. RT-PCR analysis showed that the GLUT8 mRNA was expressed both in freshly isolated hippocampi and hippocampal neurons after a 7-d in vitro culture period (Fig. 6A). The cultured hippocampal neurons were then infected with the AdTrack-GLUT8myc, and the transporter was detected by immunofluorescence microscopy. As shown in Fig. 6B, GLUT8myc localizes to a perinuclear region similar to that found in differentiated PC12 cells (above) and hippocampal neurons, as detected by immunohistological methods in sections of hippocampus (16, 17, 32).
Neuronal activation
To evaluate GLUT8myc translocation to the plasma membrane in hippocampal cells, transduced neurons were treated with either 50 mM KCl, glutamate or other depolarizing agents (A23187 Ca2+ ionophore, N-methyl-D-aspartate (NMDA); see Table 1) directly added to the culture medium. As shown in Fig. 7 for K+ and glutamate activation of transduced neurons, no surface appearance of GLUT8 was observed.
PKA and PKC, insulin, and neurotrophin stimulation
cAMP can trigger synaptic vesicle exocytosis, and activation of PKC induces HMIT translocation to the plasma membrane in hippocampal neurons (23). We thus tested whether IBMX or phorbol-12-myristate-13-acetate (PMA) could induce GLUT8myc appearance on the plasma membrane. However, as for the PC12 cells, this was not the case (Table 1).
Results from other cellular systems indicate that GLUT8 may translocate to the plasma membrane after insulin stimulation (18, 33). Because hippocampal neurons express insulin receptors and insulin signaling transduction pathways components (34, 35), we tested whether insulin could induce GLUT8myc translocation in these cells. As shown in Fig. 8, insulin did not induce GLUT8myc translocation to the cell surface in hippocampal neurons after either short (30 min) or long (16 h) periods of stimulation.
Neurotrophins such as BDNF and nerve growth factor are well known for their neuroprotective effect in hippocampus in whole animals as well as cultured neurons. These factors act, at least partly, by enabling neurons to take up more glucose from the extracellular milieu, i.e. during development or during ischemic events, when extra glucose may be needed for neurons to avoid cell death. We thus tested whether BDNF could stimulate GLUT8myc to translocate to the plasma membrane. However, treatment with BDNF (2 or 16 h at 10 ng/ml) did not lead to GLUT8myc surface expression (see Table 1 and data not shown).
Glucoprivic stress
We next tested whether preconditioning of hippocampal neurons by 2-DG treatment for 20 min or 24 h (see above) or activation of AMP kinase by AICAR could induce GLUT8myc surface expression in hippocampal cells. No GLUT8myc could be observed at the plasma membrane (data not shown), indicating that hypoglycemia or decreased cellular energy levels did not induce transporter translocation.
Osmotic shock
As mentioned above, hyperosmotic shock may generate a signal for surface expression of GLUT4 in adipocyte cell lines. The exposure of transduced hippocampal neurons to 600 mM sorbitol, however, did not induce expression of GLUT8myc at the cell surface.
Absence of constitutive recycling to the plasma membrane.
As for transfected PC-12 cells, we evaluated with an antibody capture assay whether GLUT8myc transduced in hippocampal neurons constitutively transited to the plasma membrane (24). The infected cells were exposed for 2 h at 37 C to 10 μg/ml of antimyc antibodies. After washing, the neurons were permeabilized and reacted at 4 C with an antimyc antibody. As for the PC-12 cells, this did not reveal internalization of the antimyc antibody, therefore failing to provide evidence for constitutive recycling to the plasma membrane.
Discussion
Our results show that GLUT8myc, when expressed in PC12 cells or hippocampal neurons, resides in an intracellular compartment distinct from endoplasmic reticulum, trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles, indicating association with a specific compartment. When expressed in either cell type, translocation to the cell surface could not be demonstrated in any conditions activating general intracellular signaling pathways, differentiation mechanisms, or by glucoprivic preconditioning or osmotic shock. Furthermore, no basal recycling of GLUT8myc from its intracellular location to the plasma membrane could be evidenced.
Our immunofluorescence studies showed that GLUT8myc did not significantly colocalize with markers for the endoplasmic reticulum, the trans-Golgi network complex, early endosomes, lysosomes, or synaptic vesicles. Subcellular fractionation studies of PC12 cells further showed that GLUT8myc was associated with gradient fractions clearly distinct from those containing Lamp1. This excluded a possible colocalization of the transporter with lysosomal structures, as the occasional costaining for Lamp1 and GLUT8myc in the immunofluorescence microscopy data could have suggested. The localization of GLUT8myc in a perinuclear region also excluded its presence in dense core granules, which are strongly enriched in cellular processes (23). Thus, GLUT8myc was localized to a specific compartment, the composition of which is not yet established.
GLUT8 is a high-affinity glucose transporter. Because there is normally no free glucose inside the cell, the need for a glucose transporter associated with an intracellular compartment is not clear. It has been reported that lysosomes have a glucose transport activity (36), which is required for export into the cytosol of glucose generated from degradation of glycoproteins. However, our data do not support an association of GLUT8 with this compartment. Furthermore, lysosomes are present in every cell type, whereas GLUT8 has a very restricted tissue distribution excluding a general housekeeping function. GLUT8 may thus play a specific role in the cells in which it is expressed.
An obvious hypothesis for GLUT8’s role is that it is conditionally expressed at the plasma membrane on specific cell stimulation to increase cellular glucose uptake. This has been shown to occur for GLUT8 expressed in mouse blastocysts, in which insulin appears to induce a redistribution of the transporter to the blastocyst surface (18). However, our present data failed to show induced translocation of GLUT8 to the plasma membrane of PC12 cells or hippocampal neurons, a cell type normally expressing GLUT8, under any of the stimulatory conditions tested. Also, in normally insulin sensitive cells such as adipocytes, no insulin-stimulated GLUT8 surface appearance could be observed. The observations made in blastocysts are thus unique.
In our attempts to identify conditions that could lead to cell surface expression of GLUT8, we tested stimuli that depolarize the plasma membrane, mimicking neuronal activation. These conditions were previously shown to induce translocation to the plasma membrane of the HMIT, a member of the glucose transporter family (Slc2A13) (23). Activation of PKA, which can stimulate fusion of synaptic vesicles with the plasma membrane or, in kidney-collecting duct cells, activate translocation to the apical membrane of the water channel aquaporin (37), had no effect. Treatment of the cells with PMA, which activates PKC but can also directly activate fusion of vesicles with the plasma membrane by its interaction with Munc 13 (38) and which stimulates translocation of HMIT to the cell surface, had no effect on GLUT8myc translocation.
Other signaling pathways dependent on tyrosine kinase activation (insulin, BDNF), inhibition of tyrosine kinase activity by genistein or erbstatin A, or inhibition of serine/threonine or tyrosine protein phosphatase by, respectively, okadaic acid and phenylarsine oxide did not lead to GLUT8myc translocation; whatever the periods of time, these different substances were applied to the cells. Thus, the above data suggest that classical intracellular signaling pathways were not involved in the control of GLUT8 translocation.
The absence of translocation after glucoprivic stress with 2-DG treatment or activation of the metabolic sensor AMP-activated kinase by AICAR suggests that appearance of GLUT8 to the plasma membrane may not be part of a response to stimuli mimicking food restriction or ischemic events.
Finally, our experiments with transduction of the dominant-negative form of dynamin indicated that GLUT8 did not constitutively recycle to the plasma in a dynamin-dependent manner. This is in contrast to previous studies in which GLUT8 was expressed in primary rat adipocytes and shown to accumulate at the plasma membrane when dynamin K44A was present (7). It must, however, be stressed that, here, we studied GLUT8 expression in neuronal cells that normally express this transporter. Thus, in its normal cellular context, GLUT8 probably remains within its specific intracellular compartment. As a further support of the absence of constitutive recycling to the plasma membrane, we showed that GLUT8myc-expressing PC12 cells or hippocampal neurons failed to capture antimyc antibodies from the extracellular medium after a 2-h incubation period. These experiments were designed according to previous ones demonstrating the recycling to the plasma membrane of epitope-tagged GLUT4. Together our present observations indicate the absence of constitutive recycling of GLUT8myc to the plasma membrane.
Finding proteins colocalized or interacting with GLUT8 in neuronal cells would probably help better define its localization and discover which signals, if any, lead to its translocation to the plasma membrane. Because GLUT8 is expressed during brain development, it may localize to the neuronal cell surface only during restricted developmental time periods and in defined areas of the brain when there are specific glucose needs. A formal possibility would be that GLUT8 is associated with a cellular compartment unique to the cells in which it is expressed (hippocampus, heart) involved in some unknown glucose handling mechanism.
Finally, an alternative hypothesis that cannot be ruled out is that glucose is not the primary substrate for GLUT8 and that within the cell, it transports other substrates.
In conclusion, our present study shows that in PC12 cells and hippocampal neurons, cells that normally express GLUT8, the transporter is associated with a specific intracellular compartment distinct from the endoplasmic reticulum, the trans-Golgi network, early endosomes, lysosomes, and synaptic vesicles. No constitutive, dynamin-dependent, or independent recycling could be observed in these cells. Surface expression of GLUT8myc could not be induced by stimuli-inducing membrane depolarization; various intracellular signaling events activating the adenylyl cyclase, the phospholipase C, or different kinase/phosphatase pathways; osmotic shock; or ischemic events. These data therefore suggest that surface expression is induced by a still uncharacterized stimulus or that GLUT8 is an intracellular glucose transporter performing a function that is not known or that its primary physiological substrate is not glucose. Further studies with GLUT8 knockout mice may help shed light on the physiological function of this transporter.
Acknowledgments
The authors thank Dr. Marie-Christine Broillet for precious help in isolating hippocampal neurons and Dr. Karin Pierre for extensive help for confocal microscopy.
Footnotes
This work was supported by Grant 3100-065219-01 from the Swiss National Science Foundation (to B.T.).
Abbreviations: AICAR, 5-Amino-imidazole-4-carboxamide-1--D-ribofuranoside; AMPK, AMP-activated protein kinase; BDNF, brain-derived neurotrophic factor; dbcAMP, dibutyryl cAMP; 2-DG, 2-deoxy-D-glucose; EEA, early endosome autoantigen; GFP, green fluorescent protein; HA, hemagglutinin; HMIT, H(+)-myo-inositol cotransporter; IBMX, 3-isobutyl-1-methylxanthine; Lamp, lysosome-associated membrane protein; MOI, multiplicity of infection; NMDA, N-methyl-D-aspartate; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate.
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Abstract
GLUT8 is a high-affinity glucose transporter present mostly in testes and a subset of brain neurons. At the cellular level, it is found in a poorly defined intracellular compartment in which it is retained by an N-terminal dileucine motif. Here we assessed GLUT8 colocalization with markers for different cellular compartments and searched for signals, which could trigger its cell surface expression. We showed that when expressed in PC12 cells, GLUT8 was located in a perinuclear compartment in which it showed partial colocalization with markers for the endoplasmic reticulum but not with markers for the trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles. To evaluate its presence at the plasma membrane, we generated a recombinant adenovirus for the expression of GLUT8 containing an extracellular myc epitope. Cell surface expression was evaluated by immunofluorescence microscopy of transduced PC12 cells or primary hippocampal neurons exposed to different stimuli. Those included substances inducing depolarization, activation of protein kinase A and C, activation or inhibition of tyrosine kinase-linked signaling pathways, glucose deprivation, AMP-activated protein kinase stimulation, and osmotic shock. None of these stimuli-induced GLUT8 cell surface translocation. Furthermore, when GLUT8myc was cotransduced with a dominant-negative form of dynamin or GLUT8myc-expressing PC-12 cells or neurons were incubated with an anti-myc antibody, no evidence for constitutive recycling of the transporter through the cell surface could be obtained. Thus, in cells normally expressing it, GLUT8 was associated with a specific intracellular compartment in which it may play an as-yet-uncharacterized role.
Introduction
THE TRANSPORT OF glucose through biological membranes is facilitated by several glucose transporter isoforms. GLUT1–5 have been studied in detail, and their physiological roles in various aspects of the control of glucose homeostasis have been well established (1, 2, 3, 4, 5). More recently screening gene banks for GLUT homologs revealed the existence of additional GLUT-like molecules in the rodent and human genomes. These sequences [GLUT6–12, HMIT, and GLUT14] have been cloned and studied in various experimental settings to determine their transport characteristics (6, 7, 8, 9, 10, 11, 12, 13). GLUT8, formerly named GLUTX1, was the first member of this extended GLUT family to be identified. It has a large extracellular, glycosylated loop between transmembrane domains 9 and 10, a characteristic of the type III glucose transporters, and it transports glucose with a relatively high affinity (Km = 2 mM) (14, 15).
GLUT8 expression is mostly restricted to the testis and some brain regions, primarily the hippocampus and hypothalamus (16, 17). It is also found at a lower level in insulin-sensitive tissues such as the liver, heart, white and brown fat, and adrenal glands. Interestingly, GLUT8 was shown to be present in nerve terminals in the supraoptic nucleus and to colocalize with vasopressin granules in neurons forming the posterior pituitary (16). In all these tissues as well as in transfected cells, GLUT8 was present in intracellular compartments and not at the plasma membrane. The retention in an intracellular compartment depends on the presence of an N-terminal dileucine motif; mutating this motif into dialanine results in the localization of GLUT8 to the cell surface (14).
The only instance in which GLUT8 was reported to appear to the cell surface in a regulated manner is in murine blastocysts after insulin stimulation (18). However, in primary rat adipocytes, insulin did not stimulate GLUT8 translocation to the cell surface (7).
Thus, at present, the identity of the intracellular compartment harboring GLUT8 is not well defined, and it is not known whether in physiological situations GLUT8 translocates to the cell surface to take up glucose and which signals could control this translocation. The aim of this study was thus to assess the subcellular localization of GLUT8 in PC12 cells and primary culture of hippocampal neurons, which normally express this transporter, and investigate whether translocation to the cell surface could be triggered by a variety of stimuli.
Materials and Methods
Cell culture and antibodies
PC12ES cells were grown in DMEM (Invitrogen Life Technologies, Inc., Paisley, Scotland, UK) containing 25 mM glucose and supplemented with 6% horse serum (Amimed, BioConcept, Allschwil, Switzerland), 6% fetal bovine serum (Life Technologies), 10 mM HEPES, 1 mM sodium pyruvate, and 2 mM glutamine at 37 C with 5% CO2. 293AD cells were grown in DMEM containing 10% fetal bovine serum. PC12 differentiation was obtained by changing the high-serum-containing medium to DMEM containing 1% horse serum and 50 ng/ml nerve growth factor 7S (Alomone Labs, Jerusalem, Israel). All experiments were done on differentiated PC12 cells.
GLUT8myc was detected with anti-myc antibodies (9E10 clone; Roche, Basel, Switzerland). Polyclonal antibodies used for double-labeling studies were the following: anti-TGN38 (gift of Professor Grenningloh, Lausanne, Switzerland), anti-early endosome autoantigen (EEA)-1 (Affinity BioReagents, Golden, CO), anti-NEEP21 (gift of Professor Hirling, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland), anti-lysosome-associated membrane protein (Lamp)1 (Santa Cruz Biotechnology, Santa Cruz, CA), anticalregulin (Santa Cruz), antisynaptophysin (Santa Cruz). Monoclonal antibodies against hemagglutinin (HA) were from Boehringer (Mannheim, Germany). All other reagents were from Sigma (St. Louis, MO) except A23187 ionophore (Calbiochem, La Jolla, CA), insulin (Novo Nordisk, Copenhagen, Denmark), and brain-derived neurotrophic factor (BDNF) (Alomone Labs).
Preparation and infection of hippocampal neurons
Hippocampi were dissected from E17 mouse brains of the C57BL6/J strain (Iffa-Credo, Lyon, France) (19, 20). Neurons were isolated by gentle trituration as described and then immediately plated on 10-mm round poly-L-lysine-coated (30–70 kDa; Sigma) glass coverslips in Neurobasal medium (Invitrogen Life Technologies) containing 2% B-27 supplement (Invitrogen Life Technologies), 0.5 mM glutamine, and 25 μM glutamate. Cell viability was assessed before plating by trypan blue staining. After 7–12 d of culture at 37 C and 5% CO2, neurons were infected during 48 h with high doses [multiplicity of infection (MOI) > 100] of adenovirus.
Expression of GLUT8myc in PC12 cells and hippocampal neurons
A human GLUT4 cDNA-containing seven c-myc epitope (GLUT4myc) in the first exofacial loop was kindly provided by Jonathan S. Bogan (Yale University, New Haven, CT) and Harvey F. Lodish (Whitehead Institute, Cambridge, MA). A fragment containing the c-myc epitopes was amplified from this plasmid by using specific primers containing additional 5' and 3' extensions corresponding to positions 1076–1095 and 1096–1114 of the rat GLUT8 cDNA sequence. A second PCR fragment (position 272-1095 of rat GLUT8 cDNA) was amplified by PCR from a rat GLUT8ss cDNA with specific primers containing additional 3' extension corresponding to position 193–208 of GLUT4myc cDNA. Finally, a third fragment (position 1096–1590) of GLUT8 cDNA containing a 5' extension corresponding to position 345–360 of GLUT4myc cDNA was obtained by PCR. A mix of 10 ng of each fragment was used as a template for a fourth PCR to generate a fragment of 1318 bp corresponding to position 272-1590 of GLUT8 cDNA with one c-myc epitope inserted into the exofacial loop between transmembrane domains 9 and 10. This PCR product digested by SacII and BglII enzymes was subcloned in pcDNA3 (Invitrogen Life Technologies) vector containing the wild-type and mutant forms of GLUT8 digested with the same enzymes. The cDNA for rat GLUT8myc was then subcloned into adenoviral vectors pAdTrack-CMV for determination of cell surface GLUT8 and pAdShuttle-CMV for colocalization studies (referred to as AdTrack-GLUT8myc and AdShuttle-GLUT8myc). High-titer stocks of adenovirus were prepared by infecting 293 cells as described in details elsewhere (21). Viral titers were evaluated by counting green fluorescent protein (GFP)-positive cells for AdTrack-GLUT8myc adenovirus and positive cells after anti-myc and anti-HA immunofluorescence for AdShuttle-GLUT8myc and AdShuttle-HA-dyn1K44A, respectively. PC12 cells were infected at an MOI of 5. Plasmid coding for mutant GLUT8 cDNA was transiently transfected into PC12 cells using Lipofectamine 2000 (Invitrogen Life Technologies) following the manufacturer’s instructions. Adenoviruses coding for HA-tagged mutant dynamin1 K44A were a kind gift of J. Pessin (State University of New York, Stony Brook, NY) (22).
Subcellular fractionation
PC12 cells infected with AdTrack-GLUT8myc were homogenized by 200 strokes in a dounce homogenizer in 2 ml of a solution consisting of 0.25 M sucrose, 20 mM HEPES (pH 7.4), 10 μg/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride (homogenization buffer). The homogenate was then centrifuged at 1500 rpm (270 x g), the pellet was discarded, and the supernatant loaded on top of a 10–50% continuous sucrose gradient containing 5 mM HEPES (pH 7.4). The homogenate was centrifuged at 4 C for 18 h at 30,000 rpm (160,000 x g) in an SW40Ti rotor (Beckman, Palo Alto, CA). Fractions (700 μl) were collected and the protein and sucrose concentrations were measured. An equal volume of 80 μl of each fraction was then separated by electrophoresis of 10% sodium dodecyl sulfate-polyacrylamide gels and the proteins were transferred onto nitrocellulose membranes for immunodetection using different antibodies as described. These procedures were as described previously (23).
Endogenous expression of GLUT8 in hippocampal neurons
The endogenous expression of GLUT8 in hippocampal neurons was assessed by RT-PCR. Total RNA was extracted with an RNeasy kit (QIAGEN, Hilden, Germany) and reverse transcribed into cDNA with superscript II enzyme (Invitrogen Life Technologies). Amplification was by 35 cycles of PCR using primers designed to amplify a 300-bp fragment of GLUT8 cDNA (forward primer: 5'-GCATAATCCAGGTCCTGTTCACTGC; reverse primer: 5'-GGAAGATCTCTGACATGAGG). GAPDH was amplified using as forward primer 5'-GTCGGTGTGAACGGATTTGG and as reverse primer 5'-GACTCCACGACATACTCAGC.
Immunofluorescence microscopy
Detection of GLUT8myc on nonpermeabilized PC12 cells was performed after incubation of the cells in K5 solution [5 mM KCl, 127 mM NaCl, 10 mM D-glucose, 1 mM MgCl2, 20 mM HEPES, and 2.7 mM CaCl2 (pH 7.4)] for 10 min at 37 C or after treatment for 2 min with K80 [80 mM KCl, 53 mM NaCl, 10 mM D-glucose, 1 mM MgCl2, 20 mM HEPES, and 2.7 mM CaCl2 (pH 7.4)] at 37 C. Cells were then immediately incubated in PBS (10 mM Na2HPO4, 138 mM NaCl, 2.7 mM KCl, and 1.76 mM KH2PO4) containing 1% BSA at 4 C. Detection of GLUT8-myc on nonpermeabilized mouse hippocampal neurons was performed in Ringer buffer [145 mM NaCl, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, and 5 mM D-glucose (pH 7.61)] at room temperature. Both PC12 cells and hippocampal neurons were fixed at the end of the assay with 4% paraformaldehyde, followed by three washes with PBS.
For immunodetection of intracellular antigens, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS for 2 min. Detection of antigens was performed for 1 h at room temperature followed by a 1-h incubation with the secondary antibodies, which were CY3-conjugated goat antimouse Ig (Jackson ImmunoResearch, West Grove, PA) and fluorescein-conjugated goat antirabbit Ig (Calbiochem) antibodies. Images were taken with a Leica TCS NT microscope (Cellular Imaging Facility, Lausanne, Switzerland).
Antibody capture assay
PC12 cells and hippocampal neurons were incubated for 2 h at 37 C with 10 μg/ml antimyc 9E10 antibody (24). Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and then incubated for 1 h at room temperature with secondary goat antimouse antibodies coupled to Cy3.
Results
GLUT8myc for immunodetection studies
To study GLUT8 intracellular localization and cell surface translocation, we generated an epitope-tagged version of GLUT8 containing a myc sequence in the extracellular glycosylated loop. We first showed that the myc epitope could be used for immunodetection of GLUT8 when expressed in cell lines. We thus transfected PC12 cells with a cDNA for GLUT8myc (Fig. 1A) containing or not a mutation of the dileucine motif. Expression of GLUT8myc was detected in permeabilized cells using an antimyc antibody (Fig. 1, B and C) and showed a perinuclear localization as previously reported (16, 17). Cell surface expression of the GLUT8myc dileucine mutant on nonpermeabilized cells was also clearly observed using the same antimyc antibody (Fig. 1, D and E).
GLUT8myc subcellular localization in PC12 cells
To study GLUT8myc subcellular localization, we generated two recombinant adenoviruses for efficient expression of the transporter in PC12 cells or hippocampal neurons. The first was AdShuttle-GLUT8myc for the immunofluorescence microscopy colocalization studies and the second AdTrack-GLUT8myc that was use for all the other subcellular and translocation experiments (see below).
Subcellular localization of GLUT8myc was first evaluated by confocal immunofluorescence microscopy of AdShuttle-GLUT8myc-infected PC12 cells. GLUT8myc localized to a perinuclear area and no staining within cellular processes were observed (Fig. 2). A partial colocalization with TGN38 was observed (Fig. 2, A–C), suggesting that a fraction of GLUT8myc may reside in the trans-Golgi network. No colocalization with the early endosomal marker EEA1 was observed (Fig. 2, D–F), suggesting that GLUT8myc did not reside in a recycling compartment in PC12 cells. This was also supported by the lack of colocalization with NEEP21 (data not shown), a marker of neuronal early endosomes (25). Occasional colocalization with Lamp1, a marker of lysosomes, could be observed (Fig. 2, G–I). By staining cells with MitoTracker (Molecular Probes, Eugene, OR) and GLUT8, we could rule out a localization of GLUT8 within mitochondria (data not shown). Moreover, only a partial colocalization with calreticulin could be obtained, suggesting that GLUT8myc did not principally reside in the ER (Fig. 2, J–L).
To obtain complementary information on GLUT8myc intracellular localization, PC12 cells infected with AdTrack-GLUT8myc were homogenized, and the postnuclear supernatant was fractionated by equilibrium centrifugation on 10–50% sucrose gradients (Fig. 3). The gradient fractions were then analyzed by Western blot for the presence of GLUT8myc and markers of different intracellular compartments. GLUT8myc-containing fractions were clearly distinct from those containing Lamp1 and EEA1, indicating absence of GLUT8myc from endosomes or lysosomes. A partial overlap with calreticulin-containing fractions was observed, suggesting that a fraction of GLUT8myc may be present in the endoplasmic reticulum. The distribution of the synaptophysin and GLUT8myc-containing vesicles was clearly distinct, indicating that GLUT8myc was not present in synaptic-like vesicles.
GLUT8myc translocation in PC12 cells Membrane depolarization
Neuron activation requires energy production, which could come from increased glucose uptake and metabolism. Therefore, we tested whether short-term depolarization by high KCl treatment or glutamate could induce GLUT8myc translocation to the plasma membrane. Figure 4 shows that no GLUT8myc surface expression could be detected following either K+ (Fig. 4, A–C) or glutamate (Fig. 4, D–F)-induced depolarization of transduced cells identified by GFP expression. Figure 4, G–I, shows GLUT8myc expression in permeabilized GFP-positive cells.
Activation of intracellular signaling pathways
We next tested whether activation of protein kinase A (PKA) or C (PKC), which can stimulate exocytosis of synaptic vesicles, could induced GLUT8 appearance to the plasma membrane. Short-term treatment of transduced PC12 cells with 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cAMP (dbcAMP), glucagon-like peptide-1-(7–36)-amide-1 (a G protein-coupled receptor linked to the adenylyl cyclase pathway), or phorbol myristate acetate to activate PKC, however, had no effect on GLUT8myc localization (data not shown). Similarly, no translocation could be observed when the transduced cells were treated with the tyrosine kinase inhibitors genistein or erbstatin A or the phosphatase inhibitors okadaic acid or phenylarsine oxide. Insulin or IGF-I treatment also did not induce translocation (see Table 1).
Hyperosmotic shock
Because hyperosmotic shock can trigger GLUT4 translocation to the plasma membrane of 3T3L1 adipocytes (26, 27), we tested whether a similar treatment could induce GLUT8myc translocation. However, no translocation was observed after a 30-min challenge with hyperosmotic concentrations of sorbitol (600 mM, Table 1).
Glucose deprivation and AMP-activated protein kinase (AMPK) activation
Hippocampal neurons submitted to a glucoprivic stress in vivo or in vitro by 2-deoxy-D-glucose (2-DG) treatment display increased protection against subsequent excitotoxic and oxidative stress. This is, at least in part, controlled by the expression of the heat shock proteins GRP78 and HSP70 (28). Here we tested whether 2-DG exposure of PC12 cells for 10 min (and also for longer periods of time in hippocampal neurons; see below) could induce GLUT8myc surface expression as part of the protective effect. However, no surface appearance of GLUT8myc could be detected in these conditions (Table 1).
A decrease in glucose supply activates the metabolic sensor AMPK (29, 30), which may be part of the response to ischemia. In muscle, activation of this kinase can induce translocation of GLUT4 to the plasma membrane (31). We thus treated transduced PC12 cells with 5-amino-imidazole-4-carboxamide-1--D-ribofuranoside (AICAR), an activator of AMP kinase (see Table 1). This also failed to induce GLUT8myc surface appearance.
GLUT8myc does not constitutively cycle through the plasma membrane in PC12 cells
A previous report indicated that GLUT8 could recycle constitutively to the plasma membrane, through a dynamin-dependent pathway, when expressed in primary rat adipocytes (7). To test whether a similar recycling of GLUT8 to the plasma membrane also occurred when the transporter was expressed in PC12 cells, we coinfected these cells with AdTrack-GLUT8myc and an adenovirus coding for a HA-tagged mutant of dynamin (dynK44A). The dynamin mutant was overexpressed, compared with GLUT8myc (ratio of MOI of 10:1) to ensure that dynaminK44A was present in all GLUT8myc-transduced cells. This was verified by immunofluorescence detection of HA-tagged mutant dynamin. As shown in Fig. 5, dynamin K44A overexpression, in conditions shown previously to increase HMIT cell surface expression (23), did not lead to increased expression of GLUT8myc at the plasma membrane.
As a another test for the possible presence of a constitutive recycling to the plasma membrane, PC-12 cells were transfected with a plasmid containing the GLUT8myc cDNA. Two days later, the transfected cells were exposed for 2 h at 37 C to 10 μg/ml of antimyc antibodies. After washing, the cells were permeabilized and reacted at 4 C with an antimyc antibody. This failed to revealed internalization of the antimyc antibody. This therefore provided another indication that GLUT8myc did not recycle to the plasma membrane in the basal state.
GLUT8myc expression and translocation in hippocampal neurons
Inability to observe translocation of GLUT8myc to the plasma membrane in PC12 cells may be due to these cells failing to express the required translocation machinery. We thus chose to express GLUT8myc in hippocampal neurons, which normally express this transporter. Primary culture of hippocampal neurons were thus established and were first tested for the presence of endogenous GLUT8. RT-PCR analysis showed that the GLUT8 mRNA was expressed both in freshly isolated hippocampi and hippocampal neurons after a 7-d in vitro culture period (Fig. 6A). The cultured hippocampal neurons were then infected with the AdTrack-GLUT8myc, and the transporter was detected by immunofluorescence microscopy. As shown in Fig. 6B, GLUT8myc localizes to a perinuclear region similar to that found in differentiated PC12 cells (above) and hippocampal neurons, as detected by immunohistological methods in sections of hippocampus (16, 17, 32).
Neuronal activation
To evaluate GLUT8myc translocation to the plasma membrane in hippocampal cells, transduced neurons were treated with either 50 mM KCl, glutamate or other depolarizing agents (A23187 Ca2+ ionophore, N-methyl-D-aspartate (NMDA); see Table 1) directly added to the culture medium. As shown in Fig. 7 for K+ and glutamate activation of transduced neurons, no surface appearance of GLUT8 was observed.
PKA and PKC, insulin, and neurotrophin stimulation
cAMP can trigger synaptic vesicle exocytosis, and activation of PKC induces HMIT translocation to the plasma membrane in hippocampal neurons (23). We thus tested whether IBMX or phorbol-12-myristate-13-acetate (PMA) could induce GLUT8myc appearance on the plasma membrane. However, as for the PC12 cells, this was not the case (Table 1).
Results from other cellular systems indicate that GLUT8 may translocate to the plasma membrane after insulin stimulation (18, 33). Because hippocampal neurons express insulin receptors and insulin signaling transduction pathways components (34, 35), we tested whether insulin could induce GLUT8myc translocation in these cells. As shown in Fig. 8, insulin did not induce GLUT8myc translocation to the cell surface in hippocampal neurons after either short (30 min) or long (16 h) periods of stimulation.
Neurotrophins such as BDNF and nerve growth factor are well known for their neuroprotective effect in hippocampus in whole animals as well as cultured neurons. These factors act, at least partly, by enabling neurons to take up more glucose from the extracellular milieu, i.e. during development or during ischemic events, when extra glucose may be needed for neurons to avoid cell death. We thus tested whether BDNF could stimulate GLUT8myc to translocate to the plasma membrane. However, treatment with BDNF (2 or 16 h at 10 ng/ml) did not lead to GLUT8myc surface expression (see Table 1 and data not shown).
Glucoprivic stress
We next tested whether preconditioning of hippocampal neurons by 2-DG treatment for 20 min or 24 h (see above) or activation of AMP kinase by AICAR could induce GLUT8myc surface expression in hippocampal cells. No GLUT8myc could be observed at the plasma membrane (data not shown), indicating that hypoglycemia or decreased cellular energy levels did not induce transporter translocation.
Osmotic shock
As mentioned above, hyperosmotic shock may generate a signal for surface expression of GLUT4 in adipocyte cell lines. The exposure of transduced hippocampal neurons to 600 mM sorbitol, however, did not induce expression of GLUT8myc at the cell surface.
Absence of constitutive recycling to the plasma membrane.
As for transfected PC-12 cells, we evaluated with an antibody capture assay whether GLUT8myc transduced in hippocampal neurons constitutively transited to the plasma membrane (24). The infected cells were exposed for 2 h at 37 C to 10 μg/ml of antimyc antibodies. After washing, the neurons were permeabilized and reacted at 4 C with an antimyc antibody. As for the PC-12 cells, this did not reveal internalization of the antimyc antibody, therefore failing to provide evidence for constitutive recycling to the plasma membrane.
Discussion
Our results show that GLUT8myc, when expressed in PC12 cells or hippocampal neurons, resides in an intracellular compartment distinct from endoplasmic reticulum, trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles, indicating association with a specific compartment. When expressed in either cell type, translocation to the cell surface could not be demonstrated in any conditions activating general intracellular signaling pathways, differentiation mechanisms, or by glucoprivic preconditioning or osmotic shock. Furthermore, no basal recycling of GLUT8myc from its intracellular location to the plasma membrane could be evidenced.
Our immunofluorescence studies showed that GLUT8myc did not significantly colocalize with markers for the endoplasmic reticulum, the trans-Golgi network complex, early endosomes, lysosomes, or synaptic vesicles. Subcellular fractionation studies of PC12 cells further showed that GLUT8myc was associated with gradient fractions clearly distinct from those containing Lamp1. This excluded a possible colocalization of the transporter with lysosomal structures, as the occasional costaining for Lamp1 and GLUT8myc in the immunofluorescence microscopy data could have suggested. The localization of GLUT8myc in a perinuclear region also excluded its presence in dense core granules, which are strongly enriched in cellular processes (23). Thus, GLUT8myc was localized to a specific compartment, the composition of which is not yet established.
GLUT8 is a high-affinity glucose transporter. Because there is normally no free glucose inside the cell, the need for a glucose transporter associated with an intracellular compartment is not clear. It has been reported that lysosomes have a glucose transport activity (36), which is required for export into the cytosol of glucose generated from degradation of glycoproteins. However, our data do not support an association of GLUT8 with this compartment. Furthermore, lysosomes are present in every cell type, whereas GLUT8 has a very restricted tissue distribution excluding a general housekeeping function. GLUT8 may thus play a specific role in the cells in which it is expressed.
An obvious hypothesis for GLUT8’s role is that it is conditionally expressed at the plasma membrane on specific cell stimulation to increase cellular glucose uptake. This has been shown to occur for GLUT8 expressed in mouse blastocysts, in which insulin appears to induce a redistribution of the transporter to the blastocyst surface (18). However, our present data failed to show induced translocation of GLUT8 to the plasma membrane of PC12 cells or hippocampal neurons, a cell type normally expressing GLUT8, under any of the stimulatory conditions tested. Also, in normally insulin sensitive cells such as adipocytes, no insulin-stimulated GLUT8 surface appearance could be observed. The observations made in blastocysts are thus unique.
In our attempts to identify conditions that could lead to cell surface expression of GLUT8, we tested stimuli that depolarize the plasma membrane, mimicking neuronal activation. These conditions were previously shown to induce translocation to the plasma membrane of the HMIT, a member of the glucose transporter family (Slc2A13) (23). Activation of PKA, which can stimulate fusion of synaptic vesicles with the plasma membrane or, in kidney-collecting duct cells, activate translocation to the apical membrane of the water channel aquaporin (37), had no effect. Treatment of the cells with PMA, which activates PKC but can also directly activate fusion of vesicles with the plasma membrane by its interaction with Munc 13 (38) and which stimulates translocation of HMIT to the cell surface, had no effect on GLUT8myc translocation.
Other signaling pathways dependent on tyrosine kinase activation (insulin, BDNF), inhibition of tyrosine kinase activity by genistein or erbstatin A, or inhibition of serine/threonine or tyrosine protein phosphatase by, respectively, okadaic acid and phenylarsine oxide did not lead to GLUT8myc translocation; whatever the periods of time, these different substances were applied to the cells. Thus, the above data suggest that classical intracellular signaling pathways were not involved in the control of GLUT8 translocation.
The absence of translocation after glucoprivic stress with 2-DG treatment or activation of the metabolic sensor AMP-activated kinase by AICAR suggests that appearance of GLUT8 to the plasma membrane may not be part of a response to stimuli mimicking food restriction or ischemic events.
Finally, our experiments with transduction of the dominant-negative form of dynamin indicated that GLUT8 did not constitutively recycle to the plasma in a dynamin-dependent manner. This is in contrast to previous studies in which GLUT8 was expressed in primary rat adipocytes and shown to accumulate at the plasma membrane when dynamin K44A was present (7). It must, however, be stressed that, here, we studied GLUT8 expression in neuronal cells that normally express this transporter. Thus, in its normal cellular context, GLUT8 probably remains within its specific intracellular compartment. As a further support of the absence of constitutive recycling to the plasma membrane, we showed that GLUT8myc-expressing PC12 cells or hippocampal neurons failed to capture antimyc antibodies from the extracellular medium after a 2-h incubation period. These experiments were designed according to previous ones demonstrating the recycling to the plasma membrane of epitope-tagged GLUT4. Together our present observations indicate the absence of constitutive recycling of GLUT8myc to the plasma membrane.
Finding proteins colocalized or interacting with GLUT8 in neuronal cells would probably help better define its localization and discover which signals, if any, lead to its translocation to the plasma membrane. Because GLUT8 is expressed during brain development, it may localize to the neuronal cell surface only during restricted developmental time periods and in defined areas of the brain when there are specific glucose needs. A formal possibility would be that GLUT8 is associated with a cellular compartment unique to the cells in which it is expressed (hippocampus, heart) involved in some unknown glucose handling mechanism.
Finally, an alternative hypothesis that cannot be ruled out is that glucose is not the primary substrate for GLUT8 and that within the cell, it transports other substrates.
In conclusion, our present study shows that in PC12 cells and hippocampal neurons, cells that normally express GLUT8, the transporter is associated with a specific intracellular compartment distinct from the endoplasmic reticulum, the trans-Golgi network, early endosomes, lysosomes, and synaptic vesicles. No constitutive, dynamin-dependent, or independent recycling could be observed in these cells. Surface expression of GLUT8myc could not be induced by stimuli-inducing membrane depolarization; various intracellular signaling events activating the adenylyl cyclase, the phospholipase C, or different kinase/phosphatase pathways; osmotic shock; or ischemic events. These data therefore suggest that surface expression is induced by a still uncharacterized stimulus or that GLUT8 is an intracellular glucose transporter performing a function that is not known or that its primary physiological substrate is not glucose. Further studies with GLUT8 knockout mice may help shed light on the physiological function of this transporter.
Acknowledgments
The authors thank Dr. Marie-Christine Broillet for precious help in isolating hippocampal neurons and Dr. Karin Pierre for extensive help for confocal microscopy.
Footnotes
This work was supported by Grant 3100-065219-01 from the Swiss National Science Foundation (to B.T.).
Abbreviations: AICAR, 5-Amino-imidazole-4-carboxamide-1--D-ribofuranoside; AMPK, AMP-activated protein kinase; BDNF, brain-derived neurotrophic factor; dbcAMP, dibutyryl cAMP; 2-DG, 2-deoxy-D-glucose; EEA, early endosome autoantigen; GFP, green fluorescent protein; HA, hemagglutinin; HMIT, H(+)-myo-inositol cotransporter; IBMX, 3-isobutyl-1-methylxanthine; Lamp, lysosome-associated membrane protein; MOI, multiplicity of infection; NMDA, N-methyl-D-aspartate; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate.
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