Hypothalamic ependymal-glial cells express the glucose transporter GLUT2, a protein involved in glucose sensing

Hypothalamic ependymal-glial cells express the glucose

transporter GLUT2, a protein involved in glucose sensing

Marıa de los Angeles Garcıa,* Carola Millan,� Carolina Balmaceda-Aguilera,� Tamara Castro,�Patricia Pastor,� Hernan Montecinos,� Karin Reinicke,� Felipe Zuniga,§ Juan Carlos Vera,§

Sergio A. Onate� and Francisco Nualart�

*Departamento de Biologıa Molecular, �Laboratorio de Neurobiologıa Celular, Departamento de Histologıa y Embriologıa,

§Departamento de Fisiopatologıa, Facultad de Ciencias Biologicas, Universidad de Concepcion, Chile

�Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Abstract

The GLUT2 glucose transporter and the K-ATP-sensitive

potassium channels have been implicated as an integral part

of the glucose-sensing mechanism in the pancreatic islet

b cells. The expression of GLUT2 and K-ATP channels in the

hypothalamic region suggest that they are also involved in a

sensing mechanism in this area. The hypothalamic glial cells,

known as tanycytes a and b, are specialized ependymal cells

that bridge the cerebrospinal fluid and the portal blood of the

median eminence. We used immunocytochemistry, in situ

hybridization and transport analyses to demonstrate the glu-

cose transporters expressed in tanycytes. Confocal micros-

copy using specific antibodies against GLUT1 and GLUT2

indicated that both transporters are expressed in a and b

tanycytes. In addition, primary cultures of mouse hypotha-

lamic tanycytes were found to express both GLUT1 and

GLUT2 transporters. Transport studies, including 2-deoxy-

glucose and fructose uptake in the presence or absence of

inhibitors, indicated that these transporters are functional in

cultured tanycytes. Finally, our analyses indicated that tany-

cytes express the K-ATP channel subunit Kir6.1 in vitro. As

the expression of GLUT2 and K-ATP channel is linked to

glucose-sensing mechanisms in pancreatic b cells, we pos-

tulate that tanycytes may be responsible, at least in part, for a

mechanism that allows the hypothalamus to detect changes in

glucose concentrations.

Keywords: glia, glucose sensing, glucose transporter 2,

hypothalamus, K-ATP-sensitive potassium channels, tany-

cytes.

J. Neurochem. (2003) 86, 709–724.

The hypothalamus is thought to be involved in modulating

feeding behavior and corporal growth through its ability to

detect changes in circulating glucose (Schwartz et al. 2000).

The hypothesis that the hypothalamus is able to detect

changes in glucose requires the identification of cells

involved in this process, as well as the expression and

secretion of the key molecules that participate in the

hypothalamic glucose-sensing mechanism (Oomura et al.

1969; Levin et al. 2001). The glucose-sensing mechanism by

pancreatic b cells involves a number of molecules, including

GLUT2, glucokinase, glucagon-like peptide-1 receptors and

the ATP-sensitive K+ channels (Guillam et al. 1997; Schuit

et al. 2001). The finding that ventricular hypothalamic glial

cells (tanycytes) express glucose sensor molecules suggests

that they may be responsible, at least in part, for glucose

sensing by the hypothalamus (Alvarez et al. 1996; Navarro

et al. 1996; Thomzig et al. 2001).

The hypothalamic peri-ventricular neurons are grouped in

nuclei (arcuate nucleus) that are in close contact with highly

elongated ependymal cells, namely tanycytes (Flament-

Durand and Brion 1985; Chauvet et al. 1995) (Fig. 1).

Received March 6, 2003; revised manuscript received April 29, 2003;

accepted April 29, 2003.

Address correspondence and reprint requests to Dr Francisco Nualart,

Laboratorio de Neurobiologıa Celular, Departamento de Histologıa y

Embriologıa, Facultad de Ciencias Biologicas, Universidad de Concep-

cion, casilla 160C, Chile. E-mail: [email protected]

Abbreviations used: 2-DOG, 2-deoxy-glucose; bNOS, nitric oxide

synthase-brain; CK-HMW, cytokeratin of high molecular weight; GFAP,

glial-fibrillary acidic protein; GLUT, glucose transporter; HVWF, von

Willebrand factor; LH-RH, luteinizing hormone-releasing hormone;

K-ATP, channels, K-ATP-sensitive potassium channels; MBP, myelin

basic protein; p75NGFr, neurotrophic growth factor receptor; TTR,

transthyretin.

Journal of Neurochemistry, 2003, 86, 709–724 doi:10.1046/j.1471-4159.2003.01892.x

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724 709

Different studies using the Golgi impregnation method, or

analysis by electron microscopy (Akmayev and Fidelina

1974), identified two main types of tanycytes, b1 and b2.The b1 tanycytes are present in the lateral lower part of the

third ventricle and they are capable of developing cell

processes that contact neurons of the arcuate nucleus as well

as the blood capillary vessels in the hypothalamus (Chauvet

et al. 1995; Peruzzo et al. 2000; Garcıa et al. 2001). The end

feet of the cells processes reach the lateral sulcus of the

infundibular region contacting luteinizing hormone-releasing

hormone (LH-RH) terminals, which are involved in hormone

release to the hypophyseal portal vessels.

The b2 tanycytes are located in the floor of third ventricle

lining the median eminence (Fig. 1). The proximal part of the

cells is in contact with the cerebrospinal fluid of the third

ventricle, while the dorsal part of the cells forms processes in

which the end feet reaches the pial surface of the brain or the

local capillary plexus in the median eminence (Chauvet et al.

1995; Peruzzo et al. 2000; Garcıa et al. 2001). These cells

develop tight-junctions that form the cerebrospinal fluid–

median eminence barrier. The existence of two additional

types of tanycytes, a1 and a2, has also been proposed

(Akmayev and Fidelina 1974) (Fig. 1). These cells line the

dorsal and lateral walls of the third ventricle mainly facing the

ventromedial nucleus of the hypothalamus. The functions of

tanycytes remain a matter of controversy and speculation.

Originally, it was suggested that they might represent a link

between the cerebrospinal fluid and the portal vessels, and that

they could be involved in regulating neuroendocrine function

(Rodrıguez et al. 1985). It has also been suggested that

tanycytes are involved in uptake and transport but the nature of

thematerial that might be transported remains to be discovered

(Flament-Durand and Brion 1985; Garcıa et al. 2001).

Differential gene expression of facilitative glucose trans-

porters (GLUT1–13) mediates the uptake of hexoses in

mammalian cells (Joost et al. 2002). Experiments designed

to identify the precise cellular localization of these transporters

indicate that GLUT1 and GLUT3 are the main isoforms

expressed in brain (Kalaria et al. 1988; Vannucci 1994;Gerhart

et al. 1995; Nualart et al. 1999). GLUT2 is a low-affinity

transporter for glucose and fructose expressed at low levels in

different regions of the brain (Brant et al. 1993). Because of

this low affinity for glucose, but high transport capacity,

GLUT2 is believed to play a major role in glucose-sensing

mechanisms (Guillam et al. 1997, 2000). In situ hybridization

data suggest that GLUT2 is present in hypothalamus (Navarro

et al. 1996). Immunohistochamical analysis indicates GLUT2

expression in astrocytes-like cells (Leloup et al. 1994), how-

ever, GLUT2 has also been detected in ependymal cells

(Ngarmukos et al. 2001).Maekawa et al. (2000) confirmed the

expression of GLUT2 in ependymal cells of the dorsal third

ventricle and cerebral aqueduct, but the hypothalamic ependy-

mal cells (tanycytes) were negative. Thus, the exact localiza-

tion of the specific glial cell types that express GLUT2 in the

hypothalamus remains to be determined.

Here, we report that tanycytes are the main glial cells in

the periventricular zone of the hypothalamus. Expression

analysis of the glucose transporters in mouse hypothalamic

tanycytes revealed that GLUT1 and GLUT2 are expressed in

both a and b tanycytes. The functional properties of GLUT1

and GLUT2 transporters were demonstrated in tanycytes

isolated from mouse hypothalamus. In addition to GLUT1

and GLUT2, we observed that tanycytes in culture express

the pore-forming subunit Kir6.1, an essential molecule in the

formation of K-ATP-sensitive potassium (K-ATP) channels.

Materials and methods

Immunocytochemistry and confocal microscopy

Mice (C57BL/J6) brains were dissected and fixed immediately by

immersion in Bouin’s solution. Fixation in situ was performed by

vascular perfusion (Nualart et al. 1991). Samples were dehydrated in

graded alcohol solutions and embedded in paraffin. Frontal sections

Fig. 1 Schematic representation of mouse hypothalamus. The cells

that are directly identified are: 1, ciliated ependymocytes lining the

rostral wall of the third ventricle, 2, a tanycytes located in the dorsal

lateral wall of the third ventricle, 3, b1 tanycytes located in the lower

lateral wall of the third ventricle, 4, b2 tanycytes located in the median

eminence. The cells form the median eminence–cerebrospinal fluid

barrier (thick line). The projections of b1 and b2 tanycytes contact the

portal blood vessels of the median eminence and pars tuberalis that

are characterized by the absence of a blood–brain barrier. There is

experimental evidence that tanycytes and neurons of the lower lateral

wall of the third ventricle express glucokinase, glucagon-like peptide-1

receptor (GLP-1), and ATP-sensitive K+ channels; molecules involved

in the glucose sensing mechanism. V-, portal blood vessels without

blood–brain barrier.

710 M. A. Garcıa et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

(5 lm) of the hypothalamic area were mounted on poly L-lysine-

coated glass slides.

For immunohistochemical analyses, we used a panel of affinity-

purified antibodies raised against synthetic peptides encompassing the

last 10–13 carboxy terminal amino acid of each isoform of the human

facilitative hexose transporters (GLUT1 to GLUT5, Alpha Diagnos-

tic, San Antonio, TX, USA). Sections were incubated, overnight at

room temperature (22�C) in a humid chamber, with anti-GLUT

polyclonal antibodies (1 : 200–1 : 1000) diluted in a Tris-HCl buffer

(pH 7.8) containing 8.4 mM sodium phosphate, 3.5 mM potassium

phosphate, 120 mM NaCl and 1% bovine serum albumin. After

washing extensively, the sections were incubated for 2 h with Cy2-

conjugated affinity-purified donkey anti-rabbit IgG (1 : 200; Jackson

Immuno-Research, West Grove, PA, USA) at room temperature.

Alternatively, anti-mouse IgG (1 : 50; Dako, Carpinteria, CA, USA)

labeled with fluorescein isothiocyanate (1 : 30; Dako) was used as a

secondary antibody. For confocal laser microscopy analysis, the tissue

sections were incubated with propidium iodine in the absence of

RNAase for cellular staining. As negative controls for GLUT1 and

GLUT2, we utilized both primary antibodies pre-absorbed with the

respective peptides used to elicit them, and pre-immune serum. To

characterize glial cell distribution in the hypothalamic area, serial

tissue sections were immunostained using an anti-glial fibrillary acidic

protein (GFAP) polyclonal antibody (1 : 100; Dako).

In situ hybridization

A cDNA of approximately 2.2 kb subcloned in pGEM-4Z

(Clontech, Palo Alto, CA, USA) and encoding the human GLUT2

was used to generate sense and anti-sense digoxigenin-labeled

riboprobes. RNA probes were labeled with digoxigenin-UTP by

in vitro transcription with SP6 or T7 RNA polymerase following the

manufacturer’s procedure (Boehringer Mannheim, Mannheim,

Germany). In situ hybridization was performed on hypothalamic

frontal sections mounted on poly L-lysine-coated glass slides. The

sections were baked at 60�C for 1 h, deparaffinized in xylene, and

rehydrated in graded ethanol. Following proteinase K treatment

(5 min at 37�C in PBS, 1 lg/mL proteinase K), the tissue sections

were fixed with 4% p-formaldehyde for 5 min at 4�C, washed in

cold PBS and then acetylated in 0.1 M triethanol amine-HCl

(pH 8.0) at room temperature for 10 min. After a brief wash, the

sections were incubated in pre-hybridization solution for 15 min at

37�C, and then 25 lL of hybridization mix [50% formamide, 0.6 M

NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 · Denhart’s

solution, 10% PEG 8000, 10 mM DTT, 500 lg yeast tRNA/mL,

50 lg/mL heparin, 500 lg/mL DNA carrier, and 1 : 20–1 : 100

diluted riboprobe] were added to each slide. The slides were

covered with glass coverslips and placed in a humidified chamber at

42�C overnight. After removal of the coverslip, the slides were

rinsed in 4 · SSC and washed twice for 30 min at 42�C. The slideswere washed at 37�C for 30 min each in 2 · SSC, 1 · SSC and

0.3 · SSC. Visualization of digoxigenin was performed by incuba-

tion with a monoclonal antibody coupled to alkaline phosphatase

(anti-digoxigenin-alkaline phosphatase Fab fragments diluted

1 : 500; Boehringer Mannheim) for 2 h at room temperature.

Nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-

phosphate (Boehringer Mannheim) were used as substrates for the

alkaline phosphatase. Controls included use of the sense riboprobe

and omission of the probe.

Tissue culture

Ependymal cells

Tissue culture experiments used cells obtained from mice at 19 days

of gestation (C57BL/J6) (Gabrion et al. 1988; Chauvet et al. 1996).

The brain was removed, the hypothalamic area taken out, and

further dissected until obtaining a region close to the ependymal

layer. The dissection was done with the samples submerged in

10 mM Hepes (pH 7.3) containing 10 mM glucose, 44 mM sacarose,

135 mM NaCl, 5 mM KCl, 0.15 mM Na2HPO4 (340–350 mOsm/L).

Ventricular walls were incubated with 0.1% (w/v) trypsin for 15 min

at 37�C. Trypsinized tissue was transferred to a 15-mL culture tube

containing 10 mL of minimal essential medium (MEM, Gibco Co.,

Rockville, NY, USA) with 10% (v/v) of fetal bovine serum (FBS) to

stop trypsin action. The tissue was dissociated by trituration through

a siliconized Pasteur pipette, until a single cell suspension was

obtained. Cells were centrifuged for 5 min at 200 g, the supernatant

was aspirated off and the cells were resuspended in MEM and

seeded in culture dishes at a concentration of 0.5–1.0 · 106 cells by

dish. Culture medium was supplemented with 10% FBS, 4 mM

L-glutamine, 100 U/mL penicillin and 100 lg/mL streptomycin

(Nalgene, Rochester, NY, USA). Cells were cultured in the same

dish for 5 weeks and fed every 3 days. The dishes with the highest

concentration of epithelial confluent cells were expanded and used

for the uptake experiments.

Astrocytes

Cerebral hemispheres were removed and the meninges were excised

carefully and discarded. Cells were incubated in 0.1% trypsin for

15 min at 37�C and mechanically dissociated. The trypsinized

tissue was transferred to a 15-mL culture tube containing 10 mL of

MEM with 10% of FBS to stop trypsin action. Cells were

centrifuged for 5 min at 200 g, the supernatant aspirated off and the

cells resuspended in MEM and seeded in culture dishes at a

concentration of 0.5–1.0 · 106 cells/dish. The culture medium was

supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL

penicillin and 100 lg/mL streptomycin.

For immunocytochemistry, cells were grown on 8-well Laborat-

ory-Tek chamber microscopy slides (Nunc, Neperville, IL, USA),

fixed with 4% p-formaldehyde in PBS for 30 min at 4�C, washedwith PBS and incubated in PBS containing 1% bovine serum

albumin (BSA) and 0.2% Triton X-100 for 5 min at room

temperature. Cells were incubated with the different antibodies

overnight at room temperature: Anti-GLUT1-5 (1 : 250, Alpha

Diagnostic), anti-Kir6.1 (1 : 100, Santa Cruz Biotechnology, Santa

Cruz, CA, USA), anti-GFAP (1 : 200, Dako), anti-vimentin mono-

clonal antibody (1 : 10, Boehringer Mannheim), anti-cytokeratin of

high molecular weight (CK-HMW) monoclonal antibody (1 : 500,

Dako), anti-brain-S100a polyclonal antibody (1 : 400, Dako), anti-

myelin basic protein (MBP) monoclonal antibody (1 : 300, Boeh-

ringer Mannheim), anti-neurotrophic growth factor receptor

(p75NGFr) polyclonal antibody (1 : 5000; Chemicon, Temecula,

CA, USA), anti-transthyretin (TTR) polyclonal antibody (1 : 300,

Dako), anti-Tau monoclonal antibody (5 mg/mL, Boehringer

Mannheim), anti-MAP-2 monoclonal antibody (5 mg/mL, Boehrin-

ger Mannheim), anti-von Willebrand factor (HVWF) polyclonal

antibody (1 : 300, Sigma, St Louis, MO, USA), anti-endothelial cell

antigen (CD31) monoclonal antibody (1 : 50, Dako), anti–blood

GLUT2 expression in hypothalamic glial tanycytes 711

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

brain barrier monoclonal antibody (HT-7, 1 : 200, Sigma), and anti-

nitric oxide synthase-brain (bNOS) monoclonal antibody (1 : 250,

Sigma). Cells were then incubated with fluorescein isothiocyanate

conjugated with goat anti-rabbit IgG or rabbit anti-mouse IgG

(1 : 25, Boehringer Mannheim) for 2 h, mounted, and analyzed by

fluorescence microscopy. Similar analyses were done with cultured

astrocytes.

Immunoblotting

For immunoblot analysis, mouse tanycytes, liver and whole brain cell

membraneproteinswereobtainedbyhomogenizing thecells in0.3 mM

sucrose, 3 mM DTT, 1 mM EDTA, 100 lg/mL PMSF, 1 lg/mL

Peptatin A and 2 lg/mL Aprotinin. Total membranes were collected

by high-speed centrifugation. Membrane protein (30 lg) was loadedin each lane, separated by polyacrylamide gel electrophoresis in the

presence of sodium dodecylsulfate, transferred to nitrocellulose

membranes, and probed with anti-GLUT, anti-Kir6.1 or pre-

absorbed antibodies (1 : 500–1 : 1500) (Zamora-Leon et al.

1996). The secondary antibodies were goat anti-rabbit IgG coupled

to peroxidase (1 : 5000) or rabbit anti-goat IgG coupled to

peroxidase (1 : 5000). The reaction was developed with enhanced

chemiluminiscence using the ECL western blotting analysis system

(Amersham Corporation, Arlington Heights, IL, USA).

Uptake analysis

For uptake assays, cells were grown in 6-well plates to a density of

2 · 105 cells per well. Cultures were carefully selected under the

microscope to ensure that only plates showing uniformly growing

cells were used. In each experiment, cells from six wells incubated

with buffer were removed and used to quantify the number of cells

present in each well. We did not observe a significant variation in

cell numbers between the wells after buffer incubation (Vera et al.

1993; Spielholz et al. 1997; Nualart et al. 2003). Cells were washed

with incubation buffer (15 mM HEPES, 135 mM NaCl, 5 mM KCl,

1.8 mM CaCl2, 0.8 mM MgCl2) and incubated in the same medium

for 30 min at room temperature. Uptake assays were performed in

1 mL of incubation buffer containing 0.2 mM deoxy-glucose and

3 lCi of 2-deoxy-D-[1,2-(N)3H]glucose (30.6 Ci/mmol; DuPont–

NEN, Boston, MA, USA). Uptake was stopped by washing the cells

with ice-cold PBS. Cells were lyzed in 0.5 mL of lysis buffer

(10 mM Tris-HCl, pH 8.0, 0.2% SDS), and the incorporated

radioactivity was assayed by liquid scintillation counting. Fructose

uptake assays were performed in incubation buffer containing 1 mM

fructose and 0.8 lCi of D-[U-14C]fructose/mL (285 mCi/mmol;

Amersham) (Zamora-Leon et al. 1996). Samples were processed as

indicated for deoxyglucose uptake. Where appropriate, competitors

and inhibitors were added to the uptake assays or pre-incubated with

the cells. Data represent means ± SD of three experiments done in

duplicate.

Results

Astrocytes and tanycytes localization in the

periventricular zone of the mouse hypothalamus

Astrocytes are stellated cells with multiple fine processes,

some of which are in close contact to capillary walls. Other

astrocyte processes are wrapped around synaptic contacts

which possess receptors for a variety of neurotransmitters.

In the periventricular area of the brain, the astrocytes are

located in the subventricular zone close to the ependymal

cells. Consistent with this characteristic, we found a normal

stellated astrocytes distribution in the dorsal ventricular wall

of the third ventricle using the classical astrocyte and

propidium iodine staining (Figs 2a and b, arrows). The

ependymal cells and neurons were negative for anti-GFAP

marker, indicating very-low-to-absent expression of GFAP

(Figs 2a and b). However, in the lower lateral walls of the

Fig. 2 Double-labeling studies to observe the astrocytes and tany-

cytes distribution in the hypothalamic area. Frontal sections of mouse

hypothalamus were stained with anti-GFAP (green) (a, c, e) and

propidium iodine (red) (b, d, f). High expression of GFAP was detected

in the dorsal subventricular zone of the third ventricle, where the

astrocytes are localized under the classical ependymal cells (a, b,

arrows). In the subventricular area of the hypothalamus, the processes

of the a tanycytes showed positive reaction with anti-GFAP (c, d). The

immunoreactive cell processes (c, d, arrows) contact ‘en passant’ the

neurons of the arcuate and ventromedial nucleus (d, insert). No

immunoreaction is detected in the lower lateral wall of the third vent-

ricle where the arcuate nucleus neurons and b1 tanycytes are located

(e, f). III-V, third ventricle; AN, arcuate nucleus; SVZ, subventricular

zone. Scale bar, 100 lm.

712 M. A. Garcıa et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

hypothalamus, stellated and GFAP positive cells were not

detected in the subventricular zone (SVZ) and the reaction

was observed in a tanycytes which presented long immuno-

reactive processes (Figs 2c and d, arrows). These processes

may come into contact, en passant, with the ventromedial

and arcuate nucleus neurons and also the blood vessels

(Fig. 2d, insert and data not shown). In the infundibular walls

of the hypothalamus, where b1 tanycytes contact the neurons

of the arcuate nucleus, we did not find positive reaction with

anti-GFAP (Figs 2e and f), suggesting that astrocytes were

totally replaced by tanycytes in this hypothalamic region.

Our results confirm that tanycytes are the main glial cells in

contact with the endocrine neurons localized in the arcuate

nucleus. This is one of the most important areas responsible

for the generations of signals involved in glucose sensing.

GLUT1 and GLUT2 are expressed in the hypothalamic

ependymal cells

Several studies suggest the expression of the facilitative

glucose transporters, GLUT1 and GLUT2, in ependymal

cells and neurons of the hypothalamic region (Leloup et al.

1994; Maekawa et al. 2000; Ngarmukos et al. 2001). To

confirm the expression of GLUT1 and to establish the precise

localization of GLUT2 in the hypothalamus of adult mice,

we used immunocytochemical detection with anti-GLUTs

polyclonal antibodies and confocal microscopy analysis.

Intense anti-GLUT1 immunoreactivity was observed in the

hypothalamic area, and the immunoreactive material was

associated with endothelial cells, neurophils and tanycytes

(Figs 3a–c). High-magnification analysis showed that

GLUT1 was localized mainly in the processes of the

tanycytes (Fig. 3d, arrows). The apical cytoplasm and the

blebs of the cells were also positives (Fig. 3e, arrow and

asterisks). The reaction was negative when the antibody was

pre-absorbed with the blocking peptide (Fig. 3f). As positive

controls, we observed the intense immunoreaction detected

in the ependymal cells of the third and lateral ventricle

(Figs 3g–i) and in the basolateral membrane of the choroids

plexus cells (Fig. 3h, arrows).

Additionally, we used immunofluorescent and confocal

microscopy to analyze, with higher sensitivity, the expression

of GLUT2 in the hypothalamic area. Our results showed

GLUT2 staining in a and b tanycytes (Figs 4a–c, arrows)

mainly localized in the apical cellular membranes and the

ventricular cytoplasmic regions of the cells (Figs 4d and e,

large arrows). The cell processes of both a and b tanycytes

presented a weak immunoreactivity (Figs 4d and e, short

arrows). In the lower infundibular region of the third

ventricle the b tanycytes presented a low positive reaction

in the blebs of the cells, similar to the reaction observed with

anti-GLUT1 (Fig. 4f, asterisks and short arrows). No immu-

nostaining was observed in the ependymal cells (Fig. 4g). An

intense immunoreaction was observed in the cellular mem-

brane of the islet b cells (Fig. 4i), demonstrating the high

specificity of the anti-GLUT2 antibody. The reaction was

always negative when we used the blocking peptide to

absorb the antibody (Fig. 4h).

Isotopic in situ hybridization analyses have suggested the

expression of GLUT2 mRNA in hypothalamic neurons and

ependymal cells, but the low resolution of this technique has

prevented a clear identification of the specific cell types

expressing GLUT2. We analyzed the gene expression of

GLUT2 at the mRNA level by in situ hybridization using

digoxigenin-labeled cRNA probes specific for GLUT2. Both

hypothalamic tanycytes, a and b, showed a positive hybrid-

ization signal (Figs 5a and b, arrows) concentrated in the

region facing the third ventricle (Fig. 5b, insert). The neurons

showed a very low reaction similar to the staining observed

with the sense riboprobe (Figs 5b and c), however, the

reaction detected in tanycytes was completely abolished

when the sense probe was used (Fig. 5c, arrows). In

conclusion, these experiments confirmed GLUT1 expression

in hypothalamic tanycytes and demonstrated that these cells

also express the low affinity transporter GLUT2.

GLUT2 is highly expressed in primary tanycyte cultures

We seeded our cultures with cells obtained by thoroughly

dissecting the pre-natal mouse hypothalamic area. After

5 weeks in culture without passage, we selected flasks with

confluent cell growth having an elongated, epithelial aspect

(Fig. 6a). Most cells showed a polarized morphology that

consisted of a wide proximal cytoplasmic region containing

the nucleus and a long basal process (Fig. 6b). Immuno-

histochemical analysis revealed an intense positive reaction

with anti-vimentin and anti-p75 NGFr (Table 1, Figs 6c and

f, and 7a), and a low anti-S100a immunoreaction (Table 1,

Fig. 6e) in cultured tanycytes. Anti-GFAP, anti-CK-HMW

and anti-TTR produced a negative immunoreaction (Table 1,

Fig. 6d). The cells showed negative immunoreactivity for

antibodies against neurons (anti-Tau and anti-MAP2), oligo-

dendroglia (anti-MBP) and endothelial cell markers (anti-

HVWF, anti-BBB, anti-bNOS and anti-endothelial cells)

(Table 1). Astrocytes showed an intense immunostaining

with anti-GFAP (Table 1, Fig. 6g), however, they were

negative for anti-vimentin (Table 1 and Fig. 6h), anti-S100a,

anti-MBP and antip75 NGFr (Table 1).

Antibodies specific for facilitative glucose transporters

revealed expression of GLUT1 and GLUT2 in cultured

tanycytes (Table 1, Figs 7b–f). The anti-GLUT1 immuno-

reactivity showed some heterogeneity (Fig. 7b); in compar-

ison, anti-GLUT2 staining was consistently more intense and

was evenly distributed throughout the cell population

(Figs 7c and d). Moreover, the cell processes showed a

homogeneous and particularly intense reaction with anti-

GLUT2 (Fig. 7e, arrows). To control for the specificity of the

anti-GLUT1 and anti-GLUT2 immunoreactivity, we used

primary antibodies pre-absorbed with the blocking peptides

(Fig. 7f). To control for the expression of GLUT1 and

GLUT2 expression in hypothalamic glial tanycytes 713

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

GLUT2 in other glial cells in culture, we analyzed the

expression of these transporters in astrocytes. We detected a

clear immunoreaction for GLUT1 (Fig. 7g), however, anti-

GLUT2 was always negative (Fig. 7h). The astrocytes used

for these analyses presented intense staining for GFAP

(Fig. 7h, insert).

GLUT1 and GLUT2 expression was also evaluated

by western blotting using protein extracts isolated from

cultured tanycytes. Our analyses indicated that the tanycytes

expressed a protein band of approximately 55 kDa reactive

to anti-GLUT1 (Fig. 7i, lane 2) that co-migrated with a

similar protein band present in membranes prepared from

whole brain (Fig. 7i, lane 1). Parallel experiments in

tanycytes demonstrated the presence of a 60-kDa protein

band immunoreactive with anti-GLUT2 (Fig. 7j, lane 2)

which co-migrated with a similar protein band present in

Fig. 3 Double-labeling studies in the mouse hypothalamus to detect

GLUT1 expression. Frontal sections of mouse hypothalamus were

stained with propidium iodine (red) and anti-GLUT1 (green) (a–f). In

the arcuate nucleus zone of the hypothalamus, the b tanycytes show a

marked immunostaining with anti-GLUT1 (b, c). The reaction is pos-

itive in the proximal parts of b tanycytes which make contact with the

cerebrospinal fluid of the third ventricle and in the processes of the

cells (d, e, arrows). The cellular blebs of the tanycytes are also

immunopositives (e, asterisks). The ciliated ependymal cells of the

third and lateral ventricle, the choroids plexus cells and the endothelial

cells of the blood–brain barrier present an intense immunoreaction

(g–i). To control for the specificity of anti-GLUT1, the antibody was

pre-absorbed with the peptides used to elicit them (f). III-V, third

ventricle; AN, arcuate nucleus. Ep, ependymal cells; LV, lateral vent-

ricle; N, neurons. Scale bars in a–f, 10 lm; in g–i, 30 lm.

714 M. A. Garcıa et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

membranes prepared from mouse liver cells (Fig. 7j, lane 1).

No immunoreactive proteins were detected when anti-

GLUT1 and anti-GLUT2 antibodies were pre-absorbed with

the blocking peptides (Figs 7i and j, lanes 3 and 4).

Functional characterization of GLUT1 and GLUT2

in primary cultures of tanycytes

Hexose uptake studies revealed that the cultured tanycytes

incorporate 2-deoxy-glucose (2-DOG) (Fig. 8a). DOG is

transported by both GLUT1 and GLUT2. Short-term uptake

assays revealed that uptake proceeded in a linear fashion at a

rate of 3.3 nmol per millon cells per min for the first 90 s of

incubation (Fig. 8a). To demonstrate the presence of a

functional GLUT2, we determined the capacity of tanycytes

to transport fructose, a substrate that is transported by GLUT2,

but not GLUT1. As shown in Fig. 8(b), tanycytes transport

fructose in a time-dependent manner and competition studies

indicated that high concentrations of fructose decrease the

Fig. 4 Double-labeling studies in the mouse hypothalamus to detect

GLUT2 expression. Frontal sections of mouse hypothalamus were

stained with propidium iodine (red) (a–h) and anti-GLUT2 (green) (b–h).

In the hypothalamus, a and b tanycytes show a positive immuno-

staining with anti-GLUT2 (b, c). The reaction is positive in the proximal

part of a and b tanycytes, where the cells contact the cerebrospinal

fluid of the third ventricle (b–e, large arrows). The processes of the

cells presented lower immunoreaction (d–e, short arrows). The cellular

blebs of the tanycytes present low positive immunoreaction (f, aster-

isks and short arrows). The ciliated ependymal cells of the third

ventricle were negative (g). To control for the specificity of anti-GLUT2,

the antibody was pre-absorbed with the peptides used to elicit them

(h). The specificity of the anti-GLUT2 antibody was observed in pan-

creatic islet b cells, an intense immunoreaction was detected in the

cellular membranes of the cells. III-V, third ventricle; AN, arcuate

nucleus. Scale bars in a–h, 10 lm; in i, 15 lm.

GLUT2 expression in hypothalamic glial tanycytes 715

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

cellular uptake of 2-DOG (Fig. 8c), which is consistent with

the concept that tanycytes express a transporter with GLUT2-

like properties. Competition analysis demonstrates that

L-glucose is not transported by GLUT1 or GLUT2 (Fig. 8c).

To determine the kinetic properties of transporters

involved in 2-DOG uptake we used a short uptake period

of 1 min. As shown in Fig. 8(d), dose-dependence experi-

ments revealed that uptake of 2-DOG by tanycytes

approached saturation at substrate concentrations near

100 mM. Eadie-Hofstee analysis of the uptake rate data

revealed the existence of two functional components with

different affinities for the transport of 2-DOG in tanycytes

(Fig. 8e). The high-affinity component for glucose trans-

porter reveals an apparent KM of 2.8 mM, with a Vmax of

19.8 nmol/min per million cells (Fig. 8e). A second

component with a lower affinity reveals an apparent KM

of 41.6 mM and a Vmax of 115 nmol/min per million cells

for the transport of 2-DOG (Fig. 8e). Assuming that both

transporters transport glucose with an affinity similar to

that for 2-DOG, the apparent KM and Vmax values for the

transport of this sugar were used to estimate the relative

contribution of each transporter to the acquisition of

glucose under normal conditions. As shown in Fig. 8(f),

both transporters would make a similar contribution to the

uptake of glucose at a sugar concentration of 5 mM.

The transport data, combined with the results of the

immunohistochemistry, indicate that the two transporters

expressed in tanycytes are functionally active and corres-

pond to GLUT1 (the higher affinity component) and GLUT2

(the lower affinity component). To confirm our finding, we

examined the effect of cytochalasin B, a classical glucose

transporter inhibitor, on the transport of 2-DOG. At 0.2 mM

2-DOG, more than 70% of transport is carried out by the

higher affinity transporter, GLUT1 (Fig. 8f). In contrast, at

10 mM 2-DOG, more than 60% of transport is carried out by

the lower affinity component, GLUT2 (Fig. 8f). Cytochala-

sin B, a potent non-competitive inhibitor of glucose

transporters (Deves and Krupka 1978), shows a greater

affinity for GLUT1 than GLUT2, this characteristic can be

used to distinguish between these two isoforms. The

transport of 0.2 mM 2-DOG in the presence of cytochalasin

B was inhibited in a dose-dependent manner, with an IC50 of

0.1 lM. This is close to the inhibition constant of GLUT1 by

cytochalasin B and more than 80% inhibition was observed

at 1 lM cytochalasin B (Fig. 9a). Interestingly, inhibition of

the uptake of 10 mM 2-DOG by increasing concentrations of

cytochalasin B showed a biphasic behavior suggesting the

presence of two independent transporter activities (Fig. 9b).

The first component, with an IC50 for cytochalasin B of

0.1 lM accounted for 40% of the total transport activity, and

is consistent with the expected relative contribution and the

IC50 of GLUT1. The second component, with an IC50 of

2 lM accounted for the remaining 60% of transport, which

is consistent with the expected properties of GLUT2.

Consistently, cytochalasin E failed to affect the uptake of

0.2 and 10 mM 2-DOG by hypothalamic tanycytes,

confirming the specificity of the inhibition effect of

cytochalasin B.

Fig. 5 GLUT2 mRNA expression in hypothalamic cells. (a–c) Frontal

sections of the hypothalamus hybridized using digoxigenin-labeled

riboprobes specific for GLUT2. a and b tanycytes showed positive

hybridization signal (a, arrows). An intense hybridization was observed

at the proximal cytoplasm of b tanycytes (b, arrows and insert). The

arcuate nucleus neurons presented low or absent reaction similar to

the control (b). (c) Frontal sections of the hypothalamus hybridized

using digoxigenin-labeled sense probes, as a control. The reaction

observed in the tanycytes was completely abolished (arrows). III V,

third ventricle; AN, arcuate nucleus. Scale bar in a, 10 lm; in b, c,

30 lm.

716 M. A. Garcıa et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

Tanycytes in culture express the K-ATP channels

pore-forming subunit Kir6.1

Another component of the glucose-sensing mechanism is

the K-ATP channel. There is immunohistochemical data

showing the expression of the K-ATP channel pore-

forming subunit Kir6.1 in hypothalamic tanycytes in situ

(Thomzig et al. 2001). As the expression of this channel is

crucial to argument that tanycytes are potentially involved

in glucose sensing, we studied the expression of Kir6.1

subunit in cultured tanycytes using immunofluoresce and

immunoblot analysis.

Immunocytochemical analysis using specific antibodies

for Kir6.1 subunit indicates that Kir6.1 subunit is

expressed in tanycyte primary cultures (Table 1, Fig. 10a).

Positive immunoreactivity is detected in the cytoplasm and

processes of the tanycytes. Propidium iodine staining after

RNAase treatment confirms that most of the cells in

culture expressed Kir6.1 immunoreactivity (Fig. 10b).

Fig. 6 Immunocytochemistry analyses of cultured tanycytes and ast-

rocytes. The cells were obtained from mouse hypothalamus at

19 days of gestation. (a) Tanycytes after 5 weeks in culture. The cells

are organized in monolayers and show an elongated form. (b) High

magnification of a single cell using Nomarsky optics. The cell shows a

polarized aspect with an apical expanded area and a long single

process (labeled P). The position of the nucleus is also indicated

(labeled N). (c–f) Tanycytes immunostained with anti-vimentin (c),

anti-GFAP (d), anti-brain S100a (e) and anti-p75 NGFr (f). The tany-

cytes were anti-GFAP negative, however, the cells presented a strong

immunoreaction with anti-vimentin and anti-p75 NGFr, both markers

for tanycytes. (g, h) Astrocytes at 2 weeks in culture immunostained

with anti-GFAP (g) and anti-vimentin (h). Insert in h, Hoechst staining

indicating the cells present in the observed field. Scale bars in a,

100 lm; in b, 5 lm. Scale bars in c–h, 30 lm.

GLUT2 expression in hypothalamic glial tanycytes 717

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

Anti-Kir6.1 primary antibodies pre-absorbed with the

blocking peptide indicate that the immunoreaction was

specific (data not shown). Western immunoblotting analy-

sis using anti-Kir6.1 antibodies revealed a 47-kDa protein

band (Fig. 10d, lane 3). A similar protein band was found

in extract prepared from hypothalamus and liver (Fig. 10,

lanes 1 and 2).

Discussion

Initial biochemical studies indicate that the relative expres-

sion of GLUT2 in the brain is low (Brant et al. 1993; Leloup

et al. 1994). However, low relative levels of GLUT2 may be

due to restricted expression in a few cell types. Our detailed

immunohistochemical and functional analysis of the low

affinity glucose transporter, both in situ and in vitro,

consistently indicates that GLUT2 is primarily expressed in

a and b tanycytes of the mouse hypothalamus. These

experiments were performed using an anti-GLUT2 antibody

that gave a positive immunoblot reaction in membranes

isolated from mouse hepatocytes expressing high levels of

GLUT2 and in pancreatic b cells in situ. Interestingly, the

preferential GLUT2 expression observed in hypothalamic aand b tanycytes was localized in the proximal part of the cell

body, which corresponds to the region of the cell that is in

contact with the cerebrospinal fluid. Other cells of the

hypothalamus, such as neurons and endothelial cells, were

consistently negative for GLUT2 in our immunohistochem-

ical analysis.

Immunohistochemical data have indicated GLUT2 local-

ization in numerous punctate structures localized in the

hypothalamic area between the arcuate nucleus and ventro-

medial hypothalamus (Leloup et al. 1994). Further, immu-

nodetection using electron-microscopy suggests GLUT2

localization in astrocytes processes in close relationship with

nerve terminals or neuronal cell bodies (Leloup et al. 1994).

However, the antibodies used in this study were unable to

detect GLUT2 proteins by western blot analysis of samples

isolated from the hypothalamic arcuate nucleus. Ngarmukos

et al. (2001) were unable to detect GLUT2 expression in

ventromedial hypothalamic cells; however, GLUT2 immu-

noreactivity was detected in the ependymal cells of the dorsal

third ventricle and in scattered cells in the arcuate and

periventricular nuclei. Further, Maekawa et al. (2000) detec-

ted a clear expression of GLUT2 in ependymal cells of the

dorsal third ventricle and cerebral aqueduct, but the

hypothalamic glial cells were negative. In detail, GLUT2

was localized in ciliated ependymal cells of the cerebral

aqueduct, specifically in the cell membrane of the cilia

(Maekawa et al. 2000). Thus, the localized expression of

GLUT2 in a few ventricular ependymal cells may explain the

low relative expression of this transporter when the analysis

includes the whole brain. In situ hybridization analyses have

revealed GLUT2 expression in a region of the hypothalamus

containing neurons and ependymal cells (Navarro et al.

1996). However, the lower resolution capacity of autoradi-

ographic detection of the GLUT2 probe prevented the

identification of the specific cell types expressing GLUT2.

Our in situ non-isotopic hybridization clearly identifies the

a- and b-tanycytes as the hypothalamic cells expressing

GLUT2. We observed a reduced hybridization in neuronal

soma, however, the negative controls using the sense probe

showed a similar reaction, indicating low-to-absent expres-

sion of GLUT2 mRNA in arcuate nucleus neurons and

astrocytes-like cells.

The expression of GLUT2 in a- and b-tanycytes suggest

that tanycytes contacting ventromedial hypothalamic neu-

rons (a) and arcuate nucleus neurons (b) are involved in

glucose uptake using the same low-affinity transporter. In

both types of cells, the localization of GLUT2 is observed

in the proximal pole of the tanycyte which contains the

cerebrospinal fluid, and thus tanycytes may be primarily

involved in detecting glucose concentration in the cere-

brospinal fluid of the ventricular system. Similar expres-

sion of GLUT2 transporter has been detected in ependymal

Table 1 Comparative immunohistochemical analysis of cultured

tanycytes and astrocytes

Tanycytes Astrocytes

Antibodies Reaction

Glial and ependymal cell markers

Anti-vimentin +++ –

Anti-b-S100a + –

Anti-GFAP – +++

Anti-CK-HMW – nd

Anti-TTR – nd

Anti-MBP – –

Anti-p75 NGFr +++ –

Neuronal markers

Anti-Tau – nd

Anti-MAP-2 – nd

Endothelial cell markers

Anti-HVWF – nd

Anti-endothelial cells – nd

Anti-BBB – nd

Anti-bNOS – nd

Glucose transporters markers

Anti-GLUT1 +++ +++

Anti-GLUT2 +++ –

Anti-GLUT3 – –

Anti-GLUT4 – –

Anti-GLUT5 – –

K-ATP channels subunit

Anti-Kir6.1 +++ nd

nd, not determined; –, negative; +, weak reaction; +++, strong reac-

tion.

718 M. A. Garcıa et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

Fig. 7 Cultured tanycytes express GLUT1 and GLUT2. Immunocyto-

chemical and immunoblot analysis. (a–f) Tanycyctes at 5 weeks in

culture analyzed with different antibodies. (a) Anti-vimentin. (b) Anti-

GLUT1. (c) and (e, f) anti-GLUT2. GLUT2 is detected in the proximal

part of the tanycytes (c) and in the long processes of the cells (e). (d)

Hoechst staining indicating the total cells present in the observed field.

The numbers 1–4 represent the same cells shown in c with anti-

GLUT2. (f) Control of the immunostaining using anti-GLUT2 pre-

absorbed with the peptide. (g, h) Cultured astrocytes analysed with

anti-GLUT1 (g) and anti-GLUT2 (h). The astrocytes were positives to

anti-GFAP (h, insert). (i) Immunoblotting of total cellular membranes of

mouse brain (lane 1) and tanycytes (lane 2) incubated with anti-

GLUT1. The tanycytes showed a 55-kDa band (lane 2) similar to the

band located in cellular membranes isolated from whole mouse brains

(lane 1). (j) Immunoblotting of total cellular membranes of mouse liver

(1) and tanycytes (2) incubated with anti-GLUT2. A 60-kDa band that

corresponds to GLUT2 was detected in both samples. A total of 30 lg

of membrane protein was loaded in each lane. Controls were per-

formed incubating the membranes with antibodies pre-absorbed with

the peptides (i, j, lanes 3–4). Scale bars in a–h, 30 lm.

GLUT2 expression in hypothalamic glial tanycytes 719

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

cells of the cerebral aqueduct and dorsal third ventricle

(Maekawa et al. 2000), indicating that different cells

facing the ventricular system may be involved in glu-

cose-sensing mechanisms.

GLUT2 expression was also observed in primary cultures

of tanycytes dissected from the mouse hypothalamic area.

In vitro, cells presented a highly elongated and polarized

form reminiscent of tanycytes observed in vivo. Immuno-

staining with antibodies specific for markers found in

different brain cells strongly supports the concept that the

cells in culture corresponded to differentiated tanycytes.

Thus, the cells were immuno-positive for two tanycytes

markers, vimentin and p75 NGFr (Gudino-Cabrera and

Nieto-Sampedro 2000), and were immunonegative for anti-

bodies against markers specific for astrocytes (anti-GFAP),

neurons (anti-Tau), endothelial cells (anti-HVWF, anti-BBB),

and oligodendroglia (anti-MBP). Immunohistochemical ana-

lysis of glucose transporter expression showed the presence

of GLUT1 and GLUT2 in the cultured cells, this is analogous

to the in vivo situation. Immunoblot analyses confirmed the

expression of GLUT1 and GLUT2 transporters in cultured

tanycytes, and molecular weight analyses revealed that the

tanycytes expressed a 55-kDa GLUT1 isoform. A similar

analysis combining molecular weight determination with

anti-GLUT2 immunoblotting revealed that the size of

GLUT2 (60 kDa) in tanycytes was identical to that in

hepatocyte GLUT2. Overall, the immunolocalization and

immunoblotting data strongly support the concept that the

cells grown in vitro, after dissection from the hypothalamic

area, corresponded to hypothalamic tanycytes.

A combination of transport and competition assays

revealed that the tanycytes expressed two functionally active

transporters directly involved in the uptake of hexose by

these cells. The kinetic data confirmed that these transporters

correspond to the isoforms GLUT1 and GLUT2 as detected

by immunocytochemical techniques. The higher affinity

transport component had the expected properties (kinetic and

sensitivity to inhibitors) of GLUT1, with a transport Km of

2.8 mM. In contrast, kinetic analysis of the lower affinity

transporter revealed an apparent Km of 41 mM, which fits the

description of GLUT2. The capacity of the cultured tanycytes

to take up fructose, a substrate specific for GLUT2 (Gould

and Holman 1993), confirmed that GLUT2 is fully func-

tional.

The high-affinity glucose transporter GLUT3 plays a

central role in the metabolism of glucose in the brain because

Fig. 8 Kinetic analysis of 2-deoxy-D-glucose (2-DOG) and fructose

uptake in cultured tanycytes. (a) Time-course of 0.2 mM 2-DOG

uptake. Experiments were performed at room temperature. (b) Time-

course of 1 mM fructose uptake. Hexose uptake analyses demon-

strated that cultured tanycytes were able to transport 2-DOG and

fructose. (c) Semi-log plot of the concentration dependence for inhi-

bition of deoxyglucose transport by fructose and L-glucose. Measure-

ments were performed at 0.2 mM deoxyglucose using 60-s uptake

assays. (d) Substrate dose-dependence for the uptake of 2-DOG

using 1 min assays. (e) Eadie-Hofstee analysis of the data presented

in panel d. Two components with different affinities for the transport of

2-DOG are observed. (f) Substrate dose-dependency for each of the

high- and low-affinity hexose transporters present in cultured tany-

cytes. The data indicate that mouse hypothalamic tanycytes express

functional GLUT1 and GLUT2. Data represent the mean ± SD of three

samples.

720 M. A. Garcıa et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

it is the main glucose transporter expressed by neurons

(Vannucci 1994). The results of our functional studies

indicated absence of expression of a GLUT3-like high

affinity glucose transporter (Km < 1 mM) in the cultured

tanycytes. We confirmed the absence of expression of

GLUT3 in the tanycytes in vitro and in situ by immunocyto-

chemistry and immunobloting with anti-GLUT3 antibodies

(Table 1 and data not shown). Thus, the data indicate that

GLUT3 plays no role in glucose uptake by the tanycytes.

The expression of GLUT2 in hypothalamic tanycytes may

have important physiological consequences. An elevated Km

for glucose transport implies that tanycytes expressing

GLUT2 will increase their glucose uptake rate in direct

proportion to extracellular changes in glucose concentration.

This property of GLUT2 determines its participation in the

glucose-sensing mechanism of the pancreatic b cell (Guillam

et al. 1997; Yang et al. 1999; Guillam et al. 2000; Schuit

et al. 2001). Therefore, it is possible that GLUT2 might play

a similar role in the brain (Wan et al. 1998), which opens up

the possibility for the involvement of tanycytes in a glucose-

sensing mechanism in the hypothalamus. In this context,

there is data indicating that proteins of the type involved in

the b cell glucose-sensing mechanism are expressed in the

hypothalamus (Alvarez et al. 1996; Navarro et al. 1996;

Miki et al. 2001) (Fig. 1).

In order to obtain more evidence to reinforce our theory

that tanycytes are involved in glucose sensing, we analyzed

the expression of ATP-sensitive potassium channels in

cultured tanycytes. These channels are essential to couple

the energy state of a cell to its excitability, participating in

glucose-sensing mechanism in pancreatic islet b cells (Schuit

et al. 2001). Recently, the presence of functional K-ATP

channels in glial cells has been suggested. This study

detected the presence of the Kir6.1 subunit, the main pore-

forming protein detected in hippocampal, cortical and

cerebellar astrocytes, tanycytes and Bergmann glial cells

(Thomzig et al. 2001). We confirmed the expression of the

K-ATP channel subunit Kir6.1 in cultured tanycytes, indica-

ting that these cells maintain in vitro some of the molecular

and functional properties observed in situ.

In addition to GLUT2 and K-ATP channels, cells

involved in glucose sensing express a high Km glucokin-

ase, an enzyme that shows low affinity for glucose and is

not inhibited by glucose-6-phosphate. Glucokinase is

expressed in the hypothalamus, specifically in ependymal

cells (tanycytes) and some neurons (Roncero et al. 2000).

The high Km glucokinase has also been detected in the

lower brain stem (Lynch et al. 2000; Maekawa et al.

2000). Other areas within the brain, specifically the area

post rema, the medulla oblongata and the tractus solitarius

nucleus have also been postulated to have glucose sensor

mechanisms that modulate feeding and reproduction

(Leloup et al. 1994; Schwartz et al. 2000). Additionally,

a sensor mechanism triggered by low glucose concentra-

tions in the hind brain controls the secretion of GnRH that

inhibits the pulsatile LH secretion in rats (Murahashi et al.

1996). It might be possible that the effects associated with

a glucose-sensing mechanism in the lower brain stem may

be functionally co-ordinated with a hypothalamic glucose-

sensing mechanism (Grill and Kaplan 2002). GnRH is

secreted in the basal and lateral part of the median

eminence, an area in which the axonal terminals of the

neurons that release GnRH are in contact with the end feet

of the tanycyte processes (Meister et al. 1988). The end

feet of the tanycyte processes wrap around and support the

portal blood vessels of the median eminence, thereby

blocking the nerve endings, containing hypothalamic

factors, from reaching the peri-capillary spaces in the

Fig. 9 Effect of cytochalasin B on 2-deoxy-D-glucose uptake by cul-

tured tanycytes. (a) Dose-dependent effect of cytochalasin B and cy-

tochalasin E using a concentration of 0.2 mM 2-DOG to test the higher

affinity transporter. (b) Dose-dependent effect of cytochalasin B and

cytochalasin E using a concentration of 10 mM 2-DOG to test the lower

affinity transporter. Data represent the mean ± SD of three samples.

DOG, deoxyglucose; Cyt, cytochalasin.

GLUT2 expression in hypothalamic glial tanycytes 721

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

median eminence (Kozlowski and Coates 1985; Hokfelt

et al. 1988). It has been proposed that dopamine regulates

the contact between the tanycyte ending and the blood

vessels (Bergland and Page 1979; Hokfelt et al. 1988).

When dopamine is released, it induces the retraction of the

tanycyte endings, allowing the access of the neuropeptide-

containing nerve terminal to the portal blood vessels. This

property of tanycytes is regulated by dopamine, but its

function may also be induced by intracellular signals

generated directly in the tanycytes. Tanycytes are in

simultaneous contact with the cerebrospinal fluid and the

blood vessels, and it is reasonable to think that they may

be able to detect changes in glucose concentration

occurring in both fluids. Thus, variations in glucose

concentration may induce the first changes in tanycytes,

and dopamine (whose release may be regulated by axons

afferent from the lower brain stem) could modulate the

structural change. These various systems provide a selec-

tion of routes by which glucose may reach the brain and

affect phenomena as diverse as sleep, orgasm, reproduction

cycle and feeding behavior (Bergland and Page 1979).

The level of GLUT2 expression in tanycytes may be

similar to the expression of GLUT3 and GLUT1 in

neurons and astrocytes. Thus, these transporters need high-

sensitivity immunohistochemical methods (Leloup et al.

1994). Additionally, an up-regulation in the expression of

GLUT1 and GLUT3 has been observed in cultured cells

(Gould and Holdman 1993). A similar up-regulation of

GLUT2 occurred in cultured tanycytes, allowing us to

detect the transporter with conventional immunofluores-

cence. We did not detect changes in GLUT2 expression in

cells after prolonged culture, suggesting that the regulation

of GLUT2 expression in tanycytes is different to that in

hepatocytes, where cells lost the expression of GLUT2 in

short time cultures with a simultaneous induction of

GLUT1 expression (Zheng et al. 1995).

Acknowledgements

The authors thank Ximena Campos, Universidad de Concepcion for

her technical support and Dr Simon Watkins, Department of Cell

Biology and Physiology, University of Pittsburgh, for his support in

confocal microscopy. We also thank Dr Rodolfo Medina for the

critical reading of the manuscript. This work was in part supported

by grants 1010843 from FONDECYT, Chile, DIUC-GIA

201.034.006–1.4, DIUC 202.031.089–1 and DIUC 201.035.002–

1.0, Universidad de Concepcion, Chile.

Fig. 10 Tanycytes in vitro express the K-ATP channel subunit Kir6.1.

The cells were obtained from mouse hypothalamus at 19 days of

gestation and used to perform immunocytochemical (a, b) or immu-

noblot (c) analysis. (a, b) Tanycytes after 5 weeks in culture stained

with anti-Kir6.1 subunit antibody (a) and propidium iodine (b). The

merge image shows that most of the cells in culture present Kir6.1

immunoreaction (b). (c) Immunoblotting of total cellular membranes of

liver (lane 1), hypothalamus (lane 2) and cultured tanycytes (lane 3)

incubated with anti-Kir6.1. The tanycytes show a 47-kDa band similar

to the band located in cellular membranes isolated from liver and

hypothalamus. Scale bar in c, 15 lm.

722 M. A. Garcıa et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

References

Akmayev I. G. and Fidelina O. V. (1974) Morphological aspects of the

hypothalamic-hypophyseal system. V. The tanycytes: their relation

to the hypophyseal adrenocorticotrophic function. An enzyme-

histochemical study. Cell Tissue Res. 152, 403–410.

Alvarez E., Roncero I., Chowen J. A., Thorens B. and Blazquez E.

(1996) Expression of the glucagon-like peptide-1 receptor gene in

rat brain. J. Neurochem. 66, 920–927.

Bergland R. M. and Page R. B. (1979) Pituitary-brain vascular relations:

a new paradigm. Science 204, 18–24.

Brant A. M., Jess T. J., Milligan G., Brown C. M. and Gould G. W.

(1993) Immunological analysis of glucose transporters expressed

in different regions of the rat brain and central nervous system.

Biochem. Biophys. Res. Commun. 192, 1297–1302.

Chauvet N., Parmentier M. L. and Alonso G. (1995) Transected axons of

adult hypothalamo-neurohypophysial neurons regenerate along

tanycytic processes. J. Neurosci. Res. 41, 129–144.

Chauvet N., Privat A. and Alonso G. (1996) Aged median eminence

glial cell cultures promote survival and neurite outgrowth of

cocultured neurons. Glia 18, 211–223.

Deves R. and Krupka R. M. (1978) Cytochalasin B and the kinetics of

inhibition of biological transport: a case of asymmetric binding to

the glucose carrier. Biochim. Biophys. Acta 510, 339–348.

Flament-Durand J. and Brion J. (1985) Tanycytes: morphology and

functions: a review. Int. Rev. Cytol. 96, 121–155.

Gabrion J., Peraldi S., Faivre-Bauman A., Klotz C., Ghandour M. S.,

Paulin D., Assenmacher I. and Tixier-Vidal A. (1988) Characteri-

zation of ependymal cells in hypothalamic and choroidal primary

cultures. Neuroscience 24, 993–1007.

Garcıa M. A., Carrasco M., Godoy A., Reinicke K., Montecinos V. P.,

Aguayo L. G., Tapia J. C., Vera J. C. and Nualart F. (2001)

Elevated expression of glucose transporter-1 in hypothalamic

ependymal cells not involved in the formation of the brain–

cerebrospinal fluid barrier. J. Cell Biochem. 80, 491–503.

Gerhart D. Z., Leino R. L., Borson N. D., Taylor W. E., Gronlund K. M.,

McCall A. L. and Drewes L. R. (1995) Localization of glucose

transporter GLUT3 in brain: comparison of rodent and dog using

species-specific carboxy-terminal antisera. Neuroscience 66,

237–246.

Gould G. W. and Holdman G. D. (1993) The glucose transporter family:

structures, function and tissue-specific expression. Biochem. J. 295,

329–341.

Grill H. and Kaplan J. (2002) The neuroanatomical axis for control of

energy balance. Front. Neuroendocrinol. 23, 2–40.

Gudino-Cabrera G. and Nieto-Sampedro M. (2000) Schwann-like

macroglia in adult rat brain. Glia 30, 49–63.

Guillam M. T., Hummler E., Schaerer E., Yeh J. Y., Birnbaum M. J.,

Beermann F., Schmidt A., Deriaz N. and Thorens B. (1997) Early

diabetes and abnormal pancreatic islet development in mice lack-

ing GLUT2. Nat. Genet. 17, 327–330.

Guillam M. T., Dupraz P. and Thorens B. (2000) Glucose uptake,

utilization, and signaling in Glut2-Null islets. Diabetes 49,

1485–1491.

Hokfelt T., Foster G., SchultzbergM., Meister B., SchallingM., Goldstein

M., Hemmings H. C. Jr, Ouimet C. and Greengard P. (1988)

DARPP-32 as a marker for D-1 dopaminoceptive cells in the rat

brain: prenatal development and presence in glial elements (tany-

cytes) in the basal hypothalamus. Adv. Exp. Med. Biol. 235, 65–82.

Joost H. G., Bell G. I., Best J. D., Birnbaum M. J., Charron M. J., Chen

Y. T., Doege H., James D. E., Lodish H. F., Moley J. F., Mueckler

M., Rogers S., Schurmann A., Seino S. and Thorens B. (2002)

Nomenclature of the GLUT/SLC2A family of sugar/polyol trans-

port facilitators. Am. J. Physiol. Endocrinol. Metab. 282, 974–976.

Kalaria R. N., Gravina S. A., Schmidley J. W., Perry G. and Harik S. I.

(1988) The glucose transporter of the human brain and blood–brain

barrier. Ann. Neurol. 24, 757–764.

Kozlowski G. P. and Coates P. W. (1985) Ependymoneuronal speciali-

zations between LHRH fibers and cells of the cerebroventricular

system. Cell Tissue Res. 242, 301–311.

Leloup C., Arluison M., Lepetit N., Cartier N., Marfaing-Jallat P., Ferre

P. and Penicaud L. (1994) Glucose transporter 2 (GLUT2):

expression in specific brain nuclei. Brain Res. 638, 221–226.

Levin B. E., Dunn-Meynell A. A. and Routh V. H. (2001) Brain

glucosensing and the KATP channel. Nat. Neurosci. 4, 459–460.

Lynch R. M., Tompkins L. S., Brooks H. L., Dunn-Meynell A. A. and

Levin B. E. (2000) Localization of glucokinase gene expression in

the rat brain. Diabetes 49, 693–700.

Maekawa F., Toyoda Y., Torii N., Miwa I., Thompson R. C., Foster D.,

Tsukahara S., Tsukamura H. and Maeda K. (2000) Localization of

glucokinase-like immunoreactivity in the rat lower brain stem: for

possible location of brain glucose-sensing mechanisms. Endo-

crinology 141, 375–384.

Meister B., Hokfelt T., Tsuruo Y., Hemmings H., Ouimet C., Greengard

P. and Glodstein M. (1988) DARPP-32, a dopamine-and cyclic

AMP-regulated phosphoprotein in tanycytes of the mediobasal

hypothalamus: distribution and relation to dopamine and lutein-

izing hormone-releasing hormone neurons and other glial elements.

Neuroscience 27, 607–622.

Miki T., Liss B., Minami K., Shiuchi T., Saraya A., Kashima Y., Hor-

iuchi M., Ashcroft F., Minokoshi Y., Roeper J. and Seino S. (2001)

ATP-sensitive K+ channels in the hypothalamus are essential for

the maintenance of glucose homeostasis. Nat. Neurosci. 4, 507–

512.

Murahashi K., Bucholtz D. C., Nagatani S., Tsukahara S., Tsukamura H.,

Foster D. L. and Maeda K. I. (1996) Supression of luteinizing

hormone pulses by restriction of glucose availability is mediated by

sensors in the brain stem. Endocrinology 137, 1171–1176.

Navarro M., Rodrıguez de Fonseca F., Alvarez E., Chowen J. A., Zueco

J. A., Gomez R., Eng J. and Blazquez E. (1996) Colocalization of

glucagon-like peptide-1 (GLP-1) receptors, glucose transporter

GLUT-2, and glucokinase mRNAs in rat hypothalamic cells: evi-

dence for a role of GLP-1 receptor agonists as an inhibitory signal

for food and water intake. J. Neurochem. 67, 1982–1991.

Ngarmukos C., Baur E. and Kumagai A. (2001) Co-localization of

GLUT1 and GLUT4 in the blood–brain barrier of the rat ventro-

medial hypothalamus. Brain Res. 900, 1–8.

Nualart F., Hein S., Rodrıguez E. and Oksche A. (1991) Identification

and partial characterization of the secretory glycoproteins of the

bovine subcommissural organ-Reissner’s fiber complex. Evidence

for the existence of two precursor forms. Brain Res. Mol. Brain.

Res. 11, 227–238.

Nualart F., Godoy A. and Reinicke K. (1999) Expression of the hexose

transporters GLUT1 and GLUT2 during the early development of

the human brain. Brain Res. 824, 97–104.

Nualart F., Rivas C., Montecinos V., Godoy A., Guaiquil V., Golde D.

and Vera J. C. (2003) Recycling of vitamin C by a bystander effect.

J. Biol. Chem. 278, 10128–10133.

Oomura Y., Ono T., Ooyama H. and Wayner M. J. (1969) Glucose and

osmosensitive neurons of the rat hypothalamus. Nature 222, 282–

284.

Peruzzo B., Pastor F., Blazquez J., Schoebitz K., Pelaez B., Amat P. and

Rodrıguez E. (2000) A second look at the barriers of the medial

basal hypothalamus. Exp. Brain Res. 132, 10–26.

Rodrıguez E. M., Pena P., Aguado L. and Schoebitz K. (1985)

Involvement of tanycytes in the control of gonadotropin secretion,

in Proceedings of the International Symposium Comparative

Endocrinology. Current Trends in Comparative Endocrinology

GLUT2 expression in hypothalamic glial tanycytes 723

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724

(Lofts B. and Holmes W. N., eds), pp. 113–116. Hong Kong

University Press, Hong Kong.

Roncero I., Alvarez E., Vasquez P. and Blasquez E. (2000) Functional

glucokinase isoforms are expressed in rat brain. J. Neurochem. 74,

1848–1857.

Schuit F. C., Huypens P., Heimberg H. and Pipeleers D. G. (2001)

Glucose sensing in pancreatic b-cells. A model for study of other

glucose-regulated cells in gut, pancreas, and hypothalamus. Dia-

betes 50, 1–11.

Schwartz M. W., Woods S. C., Porte D. Jr, Seeley R. J. and Beskin D. G.

(2000) Central nervous system control of food intake. Nature 404,

661–671.

Spielholz C., Golde D. W., Houghton A. N., Nualart F. and Vera J. C.

(1997) Increased facilitated transport of dehydroascorbic acid

without changes in sodium-dependent ascorbate transport in human

melanoma cells. Cancer Res. 57, 2529–2537.

Thomzig A., Wenzel M., Karschin Ch, Eaton M., Skatchkov S. N.,

Karschin A. and Veh R. W. (2001) Kir6.1 is the principal pore-

forming subunit of astrocyte but not neuronal plasma membrane

K-ATP channels. Mol. Cell Neurosci. 18, 671–690.

Vannucci S. J. (1994) Developmental expression of GLUT1 and GLUT3

glucose transporters in rat brain. J. Neurochem. 62, 240–246.

Vera J. C., Rivas C., Fischbarg J. and Golde D. (1993) Mammalian

facilitative hexose transporters mediate the transport of dehydro-

ascorbic acid. Nature 364, 79–82.

Wan H. Z., Hulsey M. G. and Martin R. J. (1998) Intracerebroven-

tricular administration of antisense oligodeoxynucleotide against

GLUT2 glucose transporter mRNA reduces food intake, body

weight change and glucoprivic response in rats. J. Nutr. 128,

287–329.

Yang X. J., Kow L. M., Funabashi T. and Mobbs C. V. (1999)

Hypothalamic glucose sensor: similarities to and differences from

pancreatic b-cells mechanisms. Diabetes 48, 1763–1772.

Zamora-Leon S. P., Golde D. W., Concha I. I., Rivas C. I., Delgado-

Lopez F., Baselga J., Nualart F. and Vera J. C. (1996) Expression of

the fructose transporter GLUT5 in human breast cancer. Proc. Natl

Acad. Sci. USA 93, 1847–1852.

Zheng Q., Levitsky L. L., Mink K. and Rhoads D. B. (1995) Glucose

regulation of glucose transporters in cultured adult and fetal

hepatocytes. Metabolism 44, 1553–1558.

724 M. A. Garcıa et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 709–724