Chem. Senses 27: 7-11,
2002
© Oxford University Press 2002
The Glucose Transporter GLUT1 and the Tight Junction Protein Occludin in Nasal Olfactory Mucosa
1 Department of Histology, Institute of Anatomy, Tartu University, Tartu, Estonia 2 Laboratory of Molecular and Cellular Morphology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8512, Japan 3 Department of Anatomy and Cell Biology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan 4 Third Department of Medicine, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan 5 Department of Anatomy, School of Health Sciences, Kyorin University, Hachioji, Tokyo 192-8508, Japan
Correspondence to be sent to: Kuniaki Takata, Department of Anatomy and Cell Biology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan. e-mail: takata{at}med.gunma-u.ac.jp
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Abstract |
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The nervous cells in the brain and the peripheral nerves are isolated from the external environment by the blood-brain, blood—cerebrospinal fluid and blood—nerve barriers. The glucose transporter GLUT1 mediates the specific transfer of glucose across these barriers. The olfactory system is unique in that its sensory cells, olfactory receptor neurons, are embedded in the nasal olfactory epithelium and send their axons directly to the olfactory bulb of the brain. Only the apical parts of the olfactory receptor neurons are exposed to the lumen, and these serve as sensors for smell. Immunohistochemical examination showed that the tight junction protein occludin was present in the junctions of the olfactory epithelium. Endothelial cells in the blood vessels in the lamina propria of the olfactory mucosa were also positive for occludin. These observations suggest that the olfactory system is guarded from both the external environment and the blood. GLUT1 was abundant in these occludin-positive endothelial cells, suggesting that GLUT1 may serve in nourishing the cells of the olfactory system. Taken together, GLUT1 and occludin may serve as part of the machinery for the specific transfer of glucose in the olfactory system while preventing the non-specific entry of substances.
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Introduction |
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Epithelia or endothelia constitute the structural basis of the blood—tissue barriers such as the blood—brain, blood—nerve, blood—retina, blood—aqueous and placental barriers (Takata et al., 1997
). Tight junctions connecting the epithelial or endothelial
cells or syncytial cell layers prevent the free exchange of substances between
the blood and the compartments guarded by the barrier, and only a limited
number of substances are allowed to pass through it. Specific transporters
localized at plasma membranes of the cells of the barrier play pivotal roles
in the specific and effective transfer of selected substances transcellularly
across the barrier (Takata,
1996).
Glucose is one of the most important sources for ATP production as well as
for the synthesis of a variety of cellular molecules. In the small intestine,
dietary carbohydrates are hydrolyzed to monosaccharides, which are absorbed
and transferred to the blood stream through absorptive epithelial cells
(Takata et al., 1993;
Takata, 1996). Most of the
cells in the body utilize blood glucose, the level of which is strictly
controlled. Glucose transporters, also called sugar transporters, are membrane
proteins that serve in the transfer of sugars across the cellular membranes
(Baldwin, 1993;
Bell et al., 1993;
Takata et al., 1993;
Takata, 1996). Two types of
glucose transporters have been identified: the SGLT family and the GLUT
family. SGLT glucose transporters are sodium-dependent active transporters
serving in the concentrative transport of sugars in the small intestine and
the kidney. GLUT glucose transporters are passive facilitated-diffusion
transporters that transport sugars according to their concentration gradient.
We have shown previously that GLUT1, an isoform of the GLUT family, is
abundant in the cells of blood—tissue barriers (Takata et al.,
1990,
1997). GLUT1 is localized at
both the apical and basolateral domains of the cells of the barriers, and
appears to constitute a key molecule in the transcellular transfer of glucose
from the blood to the specialized compartments guarded by the barriers. The
importance of GLUT1 was made evident by a mutation of GLUT1 that was shown to
be responsible for seizures due to the decrease of the glucose level in the
cerebrospinal fluid caused by defective glucose transport across the
blood—brain barrier (Seidner et
al., 1998).
Tight junctions play important roles in the barrier function of the
epithelial and endothelial sheets (Anderson
et al., 1995). Recent studies have revealed that tight
junctions have a specialized membrane structure in which a number of specific
proteins assemble, such as zonula occludens-1 (ZO-1), ZO-2, ZO-3, 7H6 antigen,
cingulin, symplekin, occludin and claudins (Denkar and Nigam, 1998;
Tsukita and Furuse 1999;
Cereijido et al.,
2000; Tsukita et al.,
2001). We showed that occludin and GLUT1 were specifically
expressed in the cells of the blood—ocular
(Tserentsoodol et al.,
1998) and blood—nerve
(Tserentsoodol et al.,
1999) barriers. These two molecules may constitute the machinery
for the selective transfer of glucose across the barriers while preventing the
non-specific flow of blood constituents.
The olfactory system is a unique extension of the central nervous system
(Doucette, 1990). The sensory
cells of the olfactory system, olfactory receptor neurons (ORNs), are embedded
in the olfactory epithelium of the nasal mucosa, and protrude their dendrites
to the lumen (Graziadei, 1973, 1977;
Farbman, 1992). Freeze-fracture
replica examination has shown that ORNs and supporting cells are sealed by
rows of well-developed tight junction strands
(Kerjaschki and
Hörandner, 1976). The ORNs project
their axons directly to the olfactory bulb. In order to clarify whether these
specialized parts of the nervous system are surrounded by tight junctions and
whether glucose transporters are present in the barrier layer, we
immunolocalized occludin and GLUT1 in the rat olfactory mucosa. To identify
the olfactory mucosa in tissue sections, antisera to tubulin and protein gene
product 9.5 (PGP) were used. PGP is a useful marker for various types of
neurons (Thompson et al.,
1983; Doran et al., 1993) including mammalian ORNs
(Takami et al., 1993,
1995;
Taniguchi et al.,
1993).
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Materials and methods |
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Antibodies
Rabbit anti-GLUT1 and guinea pig anti-GLUT1 antibodies were raised using
synthetic partial peptides of human GLUT1 and characterized as described
previously (Takata et al.,
1990; Shin et al.,
1996,Shin et al.,
1996). Rabbit anti-chicken tubulin was from S.J. Singer
(University of California at San Diego)
(Rogalski and Singer, 1984).
Mouse anti-PGP was from Ultra Clone (Rossiters Farm House, Wellow, Isle of
Wight, UK) (Bonfanti et al.,
1992) and mouse anti-occludin was from Zymed (San Francisco, CA)
(Tserentsoodol et al.,
1999). Fluorescein isothiocyanate-labeled donkey antiguinea pig
immunoglobulin G (IgG), dichlorotriazinyl amino fluorescein-labeled and
rhodamine red X-labeled donkey anti-rabbit IgG, and Cy3-labeled donkey
anti-mouse IgG were products of Jackson Immunoresearch (West Grove, PA).
Immunofluorescence staining
Male Wistar rats, 4 weeks old (supplied from the Animal Breading Facility,
Gunma University), were anesthetized with an i.p. injection of sodium
pentobarbital. Mucous membranes of the olfactory region were taken under a
dissecting microscope. Specimens were fixed with 1-3% paraformaldehyde in
phosphate-buffered saline (PBS) at 4°C for 3-24 h. In some cases, rats
were perfused with the same fixative from the left ventricle under anesthesia,
and specimens removed were further fixed in the same way as in immersion
fixation. Specimens were washed with PBS, infused with 20% sucrose in 0.1 M
sodium phosphate buffer, pH 7.4, containing 0.02% sodium azide, frozen in
liquid nitrogen, and stored at -80°C until use. Cryostat sections, 4-8
µm thick, were cut and mounted on glass slides coated with poly-L-lysine.
For occludin staining, unfixed fresh tissue specimens were directly embedded
in OCT compound and rapidly frozen in liquid nitrogen. Cryostat sections were
cut, mounted on glass slides, and fixed in acetone and ethanol
(Tserentsoodol et al.,
1999). Immunofluorescence staining was carried out basically as
described previously (Tserentsoodol et
al., 1999). In short, sections were first covered with 5%
normal goat serum, then sequentially incubated with the primary antibody and
the fluorescence-labeled secondary antibody. For double-immunofluorescence
labeling, specimens were sequentially incubated with a mixture of the primary
antibodies raised in different animal species, then with a mixture of
fluorescence-labeled species-specific secondary antibodies. Immunolabeled
samples were mounted in 22% polyvinylalcohol in 56 mM Tris—HCl buffer,
pH 9.0, 11% glycerol and 5% 1,4-diazabicyclo[2,2, 2]octane
(Shin et al.,
1996,Shin et al.,
1996), and examined with an AX-70 epifluorescence microscope
(Olympus, Tokyo, Japan). Images were captured with a PXL1400 cooled-CCD camera
(Photometrics, Tucson, AZ) using an IPLab Spectrum software (Signal Analytics,
Vienna, VA). Confocal observation was carried out with an Olympus BX-50
epifluorescence microscope equipped with an MRC-1024 laser confocal system
(Bio-Rad, Hercules, CA). Images were captured and processed with Laser Sharp
software (Bio-Rad).
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Results |
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Anti-tubulin antibody strongly stained the apices of the olfactory epithelial cells where numerous cilia are present (Figures 1 and 2). Nerve fiber bundles emanating from the epithelium and thicker fibers formed by the further bundling thereof were also strongly positive for tubulin (Figure 1). Anti-PGP antibody stained ORNs in the olfactory epithelium and the nerve fibers running underneath (Figure 3). These results show that PGP serves as a marker for the olfactory epithelium and nerve fibers emanating thereof, and that tubulin acts as a marker for the nerve fibers.
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The tight junction membrane protein occludin was concentrated in the tight junctions connecting adjacent epithelial cells (Figures 4 and 5). Observation of tangential sections of the epithelia revealed that occludin encircles the apical portions of the ORNs (Figure 6). Occludin was also found in the cells of the ducts of Bowman's glands (data not shown), and was seen to be concentrated in the endothelial cells of blood vessels running in the connective tissue of the subepithelial lamina propria (Figures 2, 4 and 7). Nerve fiber bundles were negative for occludin (Figure 2).
The glucose transporter GLUT1 is abundant in the blood vessels in the olfactory mucosa (Figures 1, 3 and 4). Double labeling for GLUT1 and occludin revealed that GLUT1 is abundant in the endothelial cells connected by tight junctions of occludin (Figures 4 and 7). Blood vessels in the non-olfactory regions were negative for GLUT1 (data not shown). Axons of ORNs leave the epithelium as thin nerve fiber bundles, which then bundle together into thicker ones, forming olfactory nerves, which enter the olfactory bulb of the brain by passing through pores of the ethmoid bone. Double-immunofluorescence labeling for GLUT1 and tubulin, or for GLUT1 and PGP, revealed that thin nerve fiber bundles inside the epithelium and nerve fiber bundles in the underlying lamina propria were negative for GLUT1 (Figures 1 and 3). Some of thick nerve fiber bundles were weakly positive for GLUT1 (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The ORNs are unique bipolar neurons whose cell bodies are located in the olfactory epithelium with dendrites protruding to the nasal cavity and axons directly extending all the way to the olfactory bulb in the central nervous system (Doucette, 1990
). The
central nervous system and peripheral nerves are guarded against free access
from the outside by the blood—brain, blood—cerebrospinal fluid and
blood—nerve barriers (Takata et al.,
1993,
1997). Eyes are specialized
extensions of the central nervous system and are guarded by the
blood—ocular barrier, composed of retinal pigment epithelium, the
epithelium of the ciliary body and the endothelial cells of the intraocular
blood vessels (Raviola, 1977;
Takata et al., 1997).
Glucose is a ubiquitous nutrient used in the cells of the body. GLUT1 is
abundant in the cells of the barrier, serving as the specific transfer
machinery of glucose across these barriers
(Harik et al., 1990;
Takata et al., 1990,
1997). It was an open question
whether GLUT1 and tight junctions are present in the olfactory system as well.
We have shown in this work that GLUT1 is abundant in the endothelial cells of
blood vessels in the lamina propria of the olfactory mucosa. The tight
junction membrane protein occludin was present in these endothelial cells.
ZO-1 was reported to be present in the blood vessels in the olfactory mucosa
(Miragall et al.,
1994). These observations suggest that the endothelia in the blood
vessels in the lamina propria serve as the barrier layer. In
blood—tissue barriers, such as in the brain, the retina, the iris and
the peripheral nerve fiber bundles, abundance of GLUT1 in endothelial cells
has been shown to be restricted to the cells of the barrier
(Takata et al., 1997;
Tserentsoodol et al.,
1999). The presence of both occludin and GLUT1 in the endothelial
cells of blood vessels in the lamina propria of the olfactory mucosa suggests
that these molecules constitute a part of the machinery for the prevention of
the non-specific flow of substances while allowing specific transfer of
glucose in this region.
Tight junctions with well-developed strands of intramembranous particles in
the apicolateral portion of the olfactory epithelium have been reported in the
mouse (Kerjaschki and
Hörandner, 1976) and the rat
(Menco, 1988). ZO-1 was
localized between the apical dendritic portions of the olfactory neurons and
the supporting cells (Miragall et
al., 1994). We show here that occludin is also present in
these regions. These observations indicate that, like other epithelia,
olfactory epithelium serves as a barrier layer against the luminal
constituents, thereby maintaining the environment of the epithelium and lamina
propria underneath.
ORNs are bipolar neurons that extend their axons up to the olfactory bulb.
The axons leave the epithelium as thin fiber bundles, which are further
bundled to form thicker olfactory nerves prior to penetration through the
ethmoid bone (Farbman, 1992).
Nerve fiber bundles were basically negative for occludin or GLUT1. Only weak
labeling for GLUT1 was seen in the ensheathing cells in thick nerve fiber
bundles. Although the presence of another tight junction protein ZO-1 was
reported in the glial fibrillar acidic protein-positive ensheathing cells
(Miragall et al.,
1994), absence of occludin indicates that these cells do not
constitute the barrier property. These observations suggest that nerve fibers
in the olfactory mucosa are exposed to the environment of the lamina propria.
Endothelial cells positive for both GLUT1 and occludin cells may serve as the
primary site of the barrier, and glucose passes through this barrier via
abundant GLUT1. This makes a marked contrast to the blood—nerve barrier
in typical peripheral nerves such as the sciatic nerve
(Tserentsoodol et al.,
1999), where perineurial cells positive for occludin and GLUT1
serve as the barrier, and the surrounding blood vessels are negative for
occludin and GLUT1.
In summary, we suggest that the environment of the olfactory system may be maintained as follows. The olfactory epithelium containing ORNs and supporting cells is protected from the harsh environment of the nasal cavity by well-developed tight junctions, as commonly seen in other types of epithelia. The axons of the olfactory nerves running in the lamina propria underneath are isolated from the blood constituents by the impermeable blood vessels lined with occludin-positive endothelial cells. Abundant GLUT1 in these endothelial cells may serve for the selective supply of glucose to the olfactory system, including the olfactory epithelium, as well as to the bundles of axons.
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Acknowledgments |
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We wish to thank S. Matsuzaki and F. Miyata for secretarial assistance. This work was supported in part by Grants-in-Aids for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan. P.H. was the recipient of a Japanese Government (Monbusho) scholarship. T.M. and N.T. are JSPS research fellows.
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Accepted September 3, 2001
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