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MISCELLANEOUS |
Institute of Pharmacology and Toxicology (M.I., M.U., B.T.) and Institute of Cell Biology and Morphology (B.M.R.), University of Lausanne, 1005 Lausanne, Switzerland; and Department of Pathology (B.G., J.R.), Division of Cell and Molecular Pathology, University of Zurich, 8091 Zurich, Switzerland
Address all correspondence and requests for reprints to: Bernard Thorens, Institute of Pharmacology and Toxicology, University of Lausanne, 27 rue du Bugnon, 1005 Lausanne, Switzerland. bernard. thorens{at}ipharm-unil.ch
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently we and others identified and functionally characterized a novel glucose transporter isoform referred to as GLUTX1 or GLUT8 (6, 7, 8). This transporter shows relatively little sequence homology with the previously characterized GLUT1–5 and is more closely related to hexose transporters in plants and bacteria.
The specific function of this novel glucose transporter is not yet known. Tissue distribution analysis by Northern blotting revealed its expression at high level in the testis, at intermediate levels in the brain, hypothalamus, and hippocampus, and at a low level in tissues such as brown and white adipose tissue, muscle, and adrenal glands (6). A specific expression was also reported in mouse oocytes in which it appears to be translocated to the cell surface on insulin action (8). The cellular localization of this transporter has not been formally elucidated. Nevertheless, its successful expression at the surface of Xenopus oocytes and HEK293T cells was found to be strictly dependent on the elimination, by mutagenesis, of a dileucine internalization motif present in the amino-terminal cytoplasmic tail (6).
Here, to gain further understanding of its possible physiological function, we mapped the sites of GLUTX1 expression in the testis and brain. The testis is the site of highest expression of GLUTX1 as deduced from Northern blot analysis (6). We therefore used this tissue to first demonstrate the specificity of antibody reactivity using Western blot analysis and immunofluorescence microscopy and by confirming these mapping results by in situ hybridization. Mapping of GLUTX1 in the brain, in which the level of expression is lower than in the testis, was then carried out by immunofluorescence and immunogold microscopy. Together the data show expression of GLUTX1 at specific stages of sperm development and in endocrine and nonendocrine brain neurons.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For Western blotting, GLUTX1 cDNA in a pcDNA3 vector was stably transfected in PC-12 cells (9), and cell membranes were prepared and analyzed using specific antibodies following previously published procedures (10). Testis membranes were prepared by Polytron homogeneization of testis and separation of the homogenate by centrifugation at 95,000 x g for 1 h on a 41% sucrose solution, as described (11). Endoglycosidase H and PNGaseF (New England Biolabs, Inc.) digestions were performed according to the manufacturer’s protocol.
In situ hybridization
For the in situ hybridization, rat testes were frozen
in liquid nitrogen, 12-µm thick cryostat sections were prepared,
postfixed with 4% paraformaldehyde in PBS, pH 7.4 for 10 min, and
rinsed with diethylpyrocarbonate in phosphate buffer. For the synthesis
of nonoverlapping probes, regions 1–500 and 935–145 of rat GLUTX1
were amplified by PCR with an inserted SP6 promoter sequence at either
5' extremities or 3' extremities for synthesis of sense or antisense
riboprobes, respectively. PCR products were then subjected to in
vitro transcription using SP6 polymerase (Promega Corp., Madison, WI) and DIG RNA labeling mix
(Roche, Basel, Switzerland). Hybridization was
carried out at 58 C in 5x standard sodium citrate (SSC) and
60% formamide for 16 h with a riboprobe concentration of 400
ng/ml, followed by several washes (60 min in 2x SSC at 65 C, 60 min in
0.1x SSC at 65 C) as previously described (12). The
staining with alkaline phosphatase was performed as described
(13). Sections were dehydrated and mounted with Eukitt (O.
Kindler, Freiburg, Germany). The specificity of hybridization
was ascertained by the use of sense GLUTX1 riboprobes with the same
length, guanine cytosine content, and specific activity as the
corresponding antisense riboprobes.
Immunohistochemistry
For immunohistochemistry, rats were deeply anesthetized with
Nembutal and cardially perfused with 4% paraformaldehyde in PBS. The
brain was removed and postfixed in the same fixative for 7 h at 4
C. Brains were kept for 24 h in 30% sucrose and PBS at 4 C before
sectioning. Brains were coronally and sagittally cut (50 µm) with a
Microm congelation microtome. Sections were stored in series in a
freeze-protection solution (150 g sucrose, 300 ml ethylene glycol, 500
ml 50 mM PBS, pH 7.4, 200 mg sodium-azide). Sections were
kept at -20 C until use. Free-floating sections were rinsed with
Tris-buffered saline (TBS; 0.5 M Tris plus 121
mM NaCl, pH 7.2) at 4 C and then incubated for 30 min in
3% FCS in TBS at room temperature to block nonspecific
antibody-binding sites. The sections were incubated overnight at room
temperature with rabbit anti-GlutX1 antibody at a 1:50 or 1:100
dilution in TBS supplemented with 1% FCS. The sections were rinsed
several times with TBS and incubated for at least 2 h with 0.3%
peroxidase-conjugated antirabbit IgG (DAKO Corp.,
Carpinteria, CA) and revealed by using 4-chloro-1-naphthol as described
previously (14). Sections were mounted on glass slides and
covered with semisolid mounting fluid (15), and observed
with a digitized microscope (Carl Zeiss, Oberkochen,
Germany). Selected sections were used for immunodetection of
GlutX1 with rhodamine-labeled secondary antibodies. These sections were
examined with a confocal microscope (Leica Corp.,
Deerfield, IL) equipped with an Argon-Krypton laser set at
568-nm excitation corresponding filters. A 100x fluotar objective was
used, with additional digitalized zoom (2x). The resolution (100x) in
the xy plane is 139.4 nm and 235.8 nm in the xz plan. Picture size was
between 284 kb to 1 Mb. Optical sections at different planes of focus
was collected. Care was taken to use the full dynamic range of the
photomultipliers by using a special look-up table (glowover-glowunder,
Leica Corp.). Digitized images were processed with
image-editing software (Adobe Photoshop, Mountain View,
CA). Identification of stained structures was performed by comparison
with the rat brain atlas (16). All animal experiments were
approved by the local veterinary office.
Immunoelectron microscopy
Rats were anesthetized with Nembutal and perfused via the left
cardiac ventricle with oxygenated HBSS 10–20 mM HEPES (pH
7.4) containing 3% polyvinyl pyrrolidone (30 kDa; Fluka,
Buchs, Switzerland) and 70 mM NaNO2
(Merck, Darmstadt, Germany) for 2 min at 37 C followed by
the same solution additionally containing 3% formaldehyde (freshly
depolymerized from paraformaldehyde; Fluka) plus 0.1%
glutaraldehyde (vacuum distilled; Fluka) for 15 min at 37
C. Afterward, slices of brain and the pituitary gland were immersion
fixed in the same fixatives for 1 h 30 min at ambient temperature,
rinsed with PBS, immersed in PBS (10 mM phosphate buffer,
pH 7.4, 0.15 M NaCl) containing 50 mM
NH4Cl for 60 min, and stored in PBS at 4 C until
use.
For immunoelectron microscopy, small pieces of brain containing the nucleus supraopticus or the posterior pituitary lobe were immersed in 2 M sucrose containing 15% polyvinyl pyrrolidone (10 kDa), mounted on aluminum pins, and frozen and stored in liquid nitrogen. Frozen ultrathin sections were prepared according to Tokuyasu (17, 18) using an Ultracut S ultramicrotome equipped with an FCS cryochamber (Reichert) picked up on nickel grids, and stored overnight on gelatin at 4 C. Before immunolabeling, gelatin was liquefied at 37 C, nickels grids removed, and washed by floating them on droplets of PBS (pH 7.4).
For single immunolabeling, grids with the attached thin sections were conditioned on droplets of PBS containing 1% BSA, 0.01% Triton X-100, and 0.01% Tween 20 for 10 min at ambient temperature. Grids were then transferred to droplets of primary antibodies diluted in conditioning buffer for 2 h at ambient temperature, rinsed on droplets of PBS, and incubated with 8- or 10-nm labeled protein A-gold (19) or gold-labeled secondary antibodies (diluted to an absorbance of 0.06 and 0.1, respectively, in conditioning buffer containing 10% normal goat serum). Finally, grids with the attached thin sections were rinsed in PBS, fixed with 2% glutaraldehyde in PBS for 10–20 min, rinsed with PBS and distilled water, and embedded and stained with methylcellulose and uranyl acetate according to Tokuyasu (17, 18). For double immunolabeling (vasopressin/GLUTX1 and oxytocin/GLUTX1), the sequential protein A-gold method was applied.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that the affinity-purified
antibody recognizes a single band in cell membranes prepared from
stably transfected but not from control PC-12 cells. The size
corresponds to normally glycosylated GLUTX1, as previously
characterized (6). Using mouse or rat testis membranes,
the same antibody recognized a band migrating with a molecular
mass of approximately 35 kDa, which could be converted
completely to a faster migrating band of approximately 32 kDa on
deglycosylation by PNGaseF. This antibody was then initially used to
determine GLUTX1 localization on the testis. It was found to be present
in the seminiferous tubules (Fig. 2B) and
the GLUTX1 mRNA was shown by in situ hybridization to be
present in the same structures (Fig. 2A). The low-power magnification
of Fig. 2A shows that only a subset of tubule profiles was stained.
This suggested that GLUTX1 was expressed in differentiating
spermatozoa. On the basis of these and complementary microscopic
analysis (not shown), GLUTX1 was determined to be expressed in type I
spermatocytes during a period approximately corresponding to the
preleptotene, leptotene, and zygotene stages of meiosis I but was no
longer expressed in mature spermatozoa.
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represents a selection of areas with
positive staining. In the hippocampus, granule cells of the dentate
gyrus were intensely stained. Of lesser intensity were the pyramidal
cells of the CA1 to CA4 regions. Among cortical neurons, those in the
piriform (primary olfactory) and entorhinal cortex and parts of the
amygdala area were strongly labeled. Among the amygdala nuclei
staining, the basomedial nucleus was strongest, the medial nucleus was
less intense, and the central and intercalated nuclei and anterior
amygdaloid area were weak or unstained. In the other areas of the
cortex (neocortex), staining of pyramidal and granule cells was weak
and sporadic. The most intense staining of all structures was found in
the median eminence of the hypothalamus. Several nuclei of the dorsal
hypothalamic area and ventral hypothalamus were stained, including the
supraoptic nucleus. In the cerebellum, only the perikarya of Purkinje
cells were weakly stained. In the brain stem, positive structures were
rare. The solitary nucleus revealed some staining.
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By immunofluorescence microscopy, GLUTX1 was found in intracellular
locations in cortical neurons (Fig. 3
, F and G). Mapping of GLUTX1 in
the brain is presented in Fig. 4. The
relative level of expression is indicated with shades of gray, darker
ones representing higher levels of expression. GLUTX1 was found in the
frontoparietal cortex, primary olfactive cortex, and amygdala.
Furthermore, staining was present in the dentate gyrus and the CA1–4
area of the hippocampus. Interestingly it was also present in the
dorsal and ventromedial hypothalamus. The solitary nucleus also
presented some GLUTX1 staining.
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, A through
D). Neurons of this supraoptico-paraventriculo-hypophyseal tract
synthesize and secrete vasopressin and oxytocin. To evaluate whether
GLUTX1 was associated with these neurons, double immunofluorescence and
double immunogold labeling were performed. Figure 5 (lower panels) and Fig. 6 show that GLUTX1 was colocalized
with vasopressin but not with oxytocin-containing cells.
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shows GLUTX1 localization in the
supraoptic nucleus. Immunogold labeling was associated with dense core
vesicles (Fig. 5A), which were also present close to a synaptic
junctions (Fig. 5B) and was absent from the perikarya of the
magnocellular cells. In the posterior pituitary, GLUTX1 was found
associated with the granules present in one subset of neurons (Fig. 7A). By double immunogold labeling, they were identified to correspond
to vasopressin cells (Fig. 6B) whereas the oxytocin cells were
unreactive for GLUTX1 (Fig. 6C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous Northern blot analysis had demonstrated a very high level of GLUTX1 expression in the testis. Thus, we initially evaluated GLUTX1 in this tissue to demonstrate the specificity of the antibody by Western blotting. These results were then extended by immunofluorescence and in situ hybridization to the cellular level. Together these data demonstrate that GLUTX1 was found in developing spermatozoa and was excluded from the supporting cells such as the Leydig cells. Expression of the mRNA and immunoreactivity for GLUTX1 was found in early stages of spermatocyte development coinciding with the first meiotic division and not in mature spermatozoa. The exact temporal expression pattern of GLUTX1 in the developing spermatocyte could not be determined from the present study and will require more detailed analysis. Nevertheless, our results support previous data of a role for GLUTX1 in the testis (6) and suggest that its temporal expression at both the RNA and protein level is tightly regulated and is confined to a specific stage in spermatogenesis.
The study in the testis allowed us to define the specificity of our affinity-purified antibodies and to ascertain that the immunolabeling correlated with the in situ hybridization signal. However, the most important aim of our study was to evaluate the expression of GLUTX1 in the brain. GLUTX1 was found in the hippocampus granule cells of the dentate gyrus, confirming a previous report (20). Its localization in other brain areas was discrete, and, interestingly, its cellular location appeared either clearly diffuse and intracellular in neurons of the supraoptic nucleus and dorsal hypothalamus whereas it appeared closely associated with the cell surface in the dentate gyrus and the cerebral cortex.
The cellular and subcellular localization of GLUTX1 was more precisely defined in the supraoptico-paraventriculo-hypophyseal tract in which this transporter is expressed at highest level. Our immunofluorescence microscopy observations indicated that GLUTX1 was associated only with the vasopressin neurons. By immunogold labeling of ultrathin cryosections, GLUTX1 was found in two types of vesicles. In the supraoptic nucleus, it was detected in synaptic dense core vesicles of nerve terminals ending close to the perikarya of the magnocellular cells. In the vasopressin neurons of the supraoptico-paraventriculo-hypophyseal tract, it was clearly localized to the secretory granules. No evidence for plasma membrane localization was obtained under these steady-state conditions.
GLUTX1 has a clearly established glucose transporter function. Its expression in intracellular vesicles was previously demonstrated to rely on the presence of a dileucine internalization motif present in its amino-terminal, cytoplasmic tail. These data were obtained in transfected HEK293T cells and in Xenopus laevis oocytes. Our present study demonstrates that at least in the subset of neurons that we studied by immunoelectron microscopy, GLUTX1 was present in synaptic vesicles and vasopressin secretory granules. These intracellular vesicles can undergo stimulus-dependent exocytosis. This may subsequently lead to GLUTX1 expression at the plasma membrane and to an increase in glucose uptake. It has been well established that stimulation of nerve activity induces an increase in glucose metabolism that is mostly restricted to the sites of exocytotic activity rather than to the perikarya (21). This has been specifically described in the supraoptico-paraventriculo-hypophyseal tract in which stimulation of vasopressin secretion by salt-loading (21) or water deprivation (22) of rats induces a marked increase in glucose utilization in the posterior pituitary and, upon water deprivation, also in the supraoptic nucleus. It was reported that increased glucose utilization in the neurohypophysis on water deprivation for 72 h was accompanied with a 40–50% increase in GLUT1 and GLUT3 transporter expression (23).
Stimulation of vasopressin secretion in response to hypotension
induced by 
-adrenergic blockade also induced glucose utilization in
the supraoptic nucleus. In this situation however, stimulation of the
magnocellular cells is through brain stem neurons projecting to the
supraoptic nucleus. Increased glucose utilization in the supraoptic
nucleus is thus owing to the activated nerve terminals controlling the
function of the magnocellular cells (21). Why GLUTX1 was
not found in the oxytocin cells is not known. One possibility is that
these endocrine neurons express another isotype of glucose transporter
such as GLUT1 or GLUT3, which are known to be present in the
hypothalamus and neurohypophysis and could compensate for the lack of
GLUTX1 in these neurons (5, 23). It is also noteworthy
that the vasopressin neurons of the paraventricular hypothalamus were
not positive for GLUTX1 immunoreactivity. This emphasizes that the
expression of this transporter in specific neurons and subcellular
compartment plays a unique, although not yet elucidated, role.
Our results suggest a difference in GLUTX1 function between endocrine and cortical neurons. In the endocrine system, GLUTX1 is associated with granules and may be involved in transport functions at the vesicular membrane in neurons of the hypothalamus. Localization in cortical neurons was more discrete but specific and localized to the plasma membrane of cell bodies. The stained neurons mainly belong to archaic brain areas, which in animals have essential functions in survival and behavior. The difference in the subcellular localization of GLUTX1 may therefore suggest a difference in function that may relate more to glucose transport at the plasma membrane level.
Taken together our data indicate that GLUTX1 is localized to specific brain neurons and that its subcellular location may vary depending on the cells in which it is expressed. Whereas confirmation of plasma membrane expression of GLUTX1 will need further ultrastructural studies, its localization to synaptic vesicles and hormone granules has been established in the present study. The presence of GLUTX1 in vesicles that can fuse with the plasma membrane on nerve stimulation is a mechanism by which increased cell surface expression of this transporter could be achieved and a subsequent increase in glucose utilization. Whether GLUTX1 surface translocation can indeed be observed will require further study and may involve the development of a cellular assay using primary cultures of neurons.
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Acknowledgments |
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Footnotes |
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Abbreviations: GLUTX1, A newly characterized glucose transporter isoform; SSC, standard sodium citrate; TBS, Tris-buffered saline.
Received June 27, 2001.
Accepted for publication September 17, 2001.
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