Glucose Increases Intracellular Free Ca (2+) in Tanycytes Via ATP Released Through Connexin 43 Hemichannels

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Glia. Author manuscript; available in PMC 2013 January 01.
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Glia. 2012 January ; 60(1): 53–68. doi:10.1002/glia.21246.
Glucose increases intracellular free Ca2+ in tanycytes via ATP

released through connexin 43 hemichannels
Juan A. Orellana*, Pablo J. Sáez*, Christian Cortés-Campos‡, Roberto J. Elizondo‡, Kenji F.
Shoji*, Susana Contreras-Duarte*, Vania Figueroa*, Victoria Velarde*, Jean X. Jiang&,
Francisco Nualart‡, Juan C. Sáez*,£, and María A. García‡
*Departamento de Fisiología, Pontificia Universidad Católica de Chile, Santiago, Chile
‡Departamento de Biología Celular, Universidad de Concepción, Concepción, Chile

&Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX, USA
£Instituto Milenio, Centro Interdisciplinario de Neurociencias de Valparaíso, Valparaíso, Chile
Abstract
The ventromedial hypothalamus is involved in regulating feeding and satiety behavior, and its
neurons interact with specialized ependymal-glial cells, termed tanycytes. The latter express
glucose-sensing proteins, including glucose transporter 2, glucokinase and ATP-sensitive K+
(KATP) channels, suggesting their involvement in hypothalamic glucosensing. Here, the
transduction mechanism involved in the glucose-induced rise of intracellular free Ca2+
concentration ([Ca2+]i) in cultured β-tanycytes was examined. Fura-2AM time-lapse fluorescence
images revealed that glucose increases the intracellular Ca2+ signal in a concentration-dependent
manner. Glucose transportation, primarily via glucose transporters, and metabolism via anaerobic
glycolysis increased connexin43 (Cx43) hemichannel activity, evaluated by ethidium uptake and
whole cell patch clamp recordings, through a KATP channel-dependent pathway. Consequently,
ATP export to the extracellular milieu was enhanced, resulting in activation of purinergic P2Y1
receptors followed by inositol trisphosphate receptor activation and Ca2+ release from intracellular
stores. The present study identifies the mechanism by which glucose increases [Ca2+]i in
tanycytes. It also establishes that Cx43 hemichannels can be rapidly activated under physiological
conditions by the sequential activation of glucosensing proteins in normal tanycytes.
Keywords
glucosensing; hypothalamus; glucokinase; connexons
Introduction
The ventromedial hypothalamus (VMH) is involved in regulating feeding and satiety
behavior through their capacity to detect changes in glucose concentrations (Levin et al.,
2004). The arcuate nucleus (AN) and the ventromedial nucleus (VMN) form the VMH; their
neurons are in close contact with highly elongated ependymal-glial cells known as tanycytes
(Akmayev and Popov, 1977; Chauvet et al., 1995; Flament-Durand and Brion, 1985).
Tanycytes are the main glial cells present in the basal hypothalamus (García et al., 2001;
García et al., 2003; Millan et al., 2010) and are classified into four types, according to their
Address correspondence to: García MA, PhD, Departamento de Biología Celular, Universidad de Concepción, Concepción, Chile.
mgarcia@udec.cl Tel.: +5641 2203808, fax: +56412239687 or Juan A. Orellana, Ph.D. Departamento de Fisiología, Pontificia
Universidad Católica de Chile, Alameda 340, Santiago, Chile. jaorella@uc.cl, Tel.: +5626862860, fax: +562222551.
Orellana et al. Page 2
localization in the III–V ventricle and biochemical and molecular properties (Akmayev and
Fidelina, 1974): α1, α2, β1 and β2 (Akmayev and Popov, 1977). α1- and α2-tanycytes are
localized beside the VMN, while β1-tanycytes are localized within the lower lateral wall of
the third ventricle, contacting neurons through cell processes in the AN as well as capillaries
in the hypothalamus (García et al., 2001). β2-tanycytes are found in the floor of third
ventricle lining the median eminence (ME) (García et al., 2001).
Both α and β tanycytes express several glucose-sensing molecules, including glucose

transporter 2 (GLUT2), and ATP-sensitive K+ (KATP) channels, suggesting their possible
involvement in hypothalamus-mediated glucosensing (Alvarez et al., 1996; García et al.,
2003; Millan et al., 2010; Navarro et al., 1996). Notably, unlike α-tanycytes, high
expression of GLUT2 and glucokinase (GK) in the proximal pole has been observed in β1-
tanycytes in vivo (García et al. 2003; Millan et al., 2010). The specific localization of β1
tanycytes in direct contact with cerebral spinal fluid (CSF) and their prominent GLUT2/GK
expression strongly support the idea that these cells have a high glucose uptake capacity. In
fact, it has been proposed that β1-tanycytes could uptake and metabolize glucose to lactate
through the glycolytic pathway, and subsequently export lactate to neurons of the AN
through monocarboxylate transporters (MCTs) 1 and/or 4 (Cortés-Campos et al., 2011;
García et al., 2003; Millan et al., 2010).
In support of the putative brain glucosensor role of tanycytes, it was recently demonstrated
that extracellular glucose increases the intracellular free Ca2+ concentration ([Ca2+]i) in α1-
and α2-tanycytes (Frayling et al., 2011). Although expression of glucosensing proteins by
different tanycyte subtypes is well-established, the mechanism by which these cells exhibit
rises in [Ca2+]i upon exposure to extracellular glucose is not completely understood (Dale,
2011; Frayling et al., 2011). Recently, it was proposed that Ca2+ signaling could be
mediated by hemichannels (Schalper et al., 2010) and the hemichannel activity can
modulated by the intracellular free Ca2+ concentration ([Ca2+]i) (De Vuyst et al., 2009;
Schalper et al., 2008b). Hemichannels are formed by the oligomerization of six protein
subunits, termed connexins (Cxs), which are a highly conserved protein family encoded by
21 genes in humans. In addition, members of a recently described three-member
glycoprotein family unrelated to Cxs, termed pannexins (Panxs), can also form
hemichannels in the cell membrane of vertebrates (Orellana et al., 2009).
The use of exogenous expression systems has permitted the study of electrophysiological
(Sáez et al., 2005) and qualitative and quantitative permeability (Orellana et al. 2011a)
properties of some hemichannels as well as mechanisms that control their functional
activity. Under appropriate experimental conditions, physiologically relevant quantities of
signaling molecules (e.g., ATP, glutamate, NAD+, and PGE2) are released via hemichannels
to the extracellular milieu (Schalper et al., 2008a). Since hemichannels are believed to play
relevant roles in intercellular signaling, the aim of the present study was to address the
mechanism behind tanycyte glucosensing and determine whether it is associated with
hemichannel-dependent changes in [Ca2+]i. Here, the [Ca2+]i of cultured tanycytes increased
in response to glucose. This response was mediated by a transduction process involving the
canonical glucosensing pathway and rapid activation of Cx43 hemichannels.
Material and Methods

Reagents and antibodies
A chemiluminescence detection kit was purchased from GE Healthcare (Aulnaissous-Bois,
France). Anti-rabbit IgG antibodies-conjugated to horseradish peroxidase (HRP) was
purchased from Pierce (Rockford, IL, USA). Gap26 and 10panx1 mimetic peptides were
obtained from NeoMPS, SA. (Strasbourg, France). HEPES, minimal essential medium

(MEM), DNAse I, poly-L-lysine, water (W3500), La3+, ethidium (Etd) bromide,

thapsigargin (TG), xestospongin C (XeC), BAPTA AM, glibenclamide, xestospongin B
(XeB), 4,6,-O-ethylidene-D-glucose (ETDG), alloxan, diazoxide (diazox), cytochalisin B
(Cyto-B) and probenecid (Prob) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Fetal calf serum (FCS) and fetal bovine serum (FBS) were obtained from Hyclone (Logan,
UT, USA). Penicillin, streptomycin, 2-deoxyglucose (2-DOG), 2-(N-(7-nitrobenz-2-oxa-1,3-
diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), goat anti-mouse Alexa Fluor 488 and goat
anti-mouse Alexa Fluor 555 and TOPRO-3 were obtained from Invitrogen (Carlsbad, CA,
USA). 2-deoxy-D-[1,2-(N)3H]glucose (2-[H3]DOG) was obtained from DuPont–NEN
(Boston, MA, USA). Normal goat serum (NGS) was purchased from Zymed (San Francisco,
CA, USA). Cx43E2, a Cx43 hemichannel antibody (Siller-Jackson et al. 2008) was made
available by Dr. Jean X. Jiang (University of Texas Health Science Center).
Animals
All animals were handled in strict accordance with the Animal Welfare Assurance (permit
number 2010101A), and all animal work was approved by the appropriate Ethics and
Animal Care and Use Committee of the University of Concepción, Chile. Male adult
Sprague-Dawley rats were used for the experiments. Animals were kept in a 12-h light/12-h
dark cycle with food and water ad libitum. Experiments in cultured cells were performed
mainly at Departamento de Fisiología, Pontificia Universidad Católica de Chile (PUCC),
using protocols approved by the Ethic and Biosecurity Committee of PUCC.
Cell cultures
Cultures of hypothalamic glial cells from 1-day postnatal brains were prepared following the
method described previously (García et al., 2003). After decapitation, brains were removed
and the hypothalamic area was dissected to obtain a region close to the ependymal layer.
The dissection was carried out from tissues immersed in dissection buffer containing 10 mM
HEPES (pH 7.4, 340 mOsm/L). Samples were incubated with 0.25% trypsin-0.2% EDTA
(w/v) for 20 min at 37°C and then, it was transferred to planting medium containing MEM
(Invitrogen) with 10% (v/v) FBS (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 2
mg/mL DNAse I (Sigma-Aldrich). Cells were seeded at 1.2×105 cells/cm2 in culture dishes
coated with 0.2 mg/mL poly-L-lysine (Sigma-Aldrich). After 2 h, the culture medium was
changed to MEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin,
100 µg/mL streptomycin, and 2.5 µg/mL fungizone (Thermo Fisher Scientific Inc). Mixed
hypothalamic cultures were prepared using the same protocol described above; however,
they were cultured in Neurobasal medium supplemented with B27 (Invitrogen). Astroglial
cultures were prepared from rat brain cortex and cultured in MEM supplemented with 10%
FBS. Cells were cultured in the same dish for 3 weeks, and the medium was changed every
2 days. The purity of cell cultures was evaluated using molecular markers for several cell
types (Fig. 1). For all experiments, chemicals and saline solutions were prepared in ultra
pure H2O.
Intracellular calcium imaging

Cells plated on glass coverslips were loaded with 5 µM Fura-2-AM in DMEM without
serum for 45 min at 37°C and then, washed three times in Locke’s solution (154 mM NaCl,
5.4 mM KCl, 2.3 mM CaCl2, and 5 mM HEPES, pH 7.4) followed by a de-esterification
period of 15 min at 37°C. The experimental protocol for [Ca2+]i imaging involved data
acquisition every 5 s (emission at 510 nm) at 340- and 380-nm excitation wavelengths using
an Olympus BX 51W1I upright microscope with a 40x water immersion objective. Changes
were monitored using an imaging system equipped with a Retga 1300I fast-cooled
monochromatic digital camera (12-bit) (Qimaging, Burnaby, BC, Canada), monochromator
for fluorophore excitation, and METAFLUOR software (Universal Imaging, Downingtown,

PA) for image acquisition and analysis. Analysis involved determination of pixels assigned
to each cell. The average pixel value allocated to each cell was obtained with excitation at
each wavelength and corrected for background. Due to the low excitation intensity, no
bleaching was observed even when cells were illuminated for a few minutes. The ratio was
obtained after dividing the 340-nm by the 380-nm fluorescence image on a pixel-by-pixel
base (R=F340 nm/F380 nm).
Immunofluorescence and confocal microscopy

Cells grown on poly-L-lysine-coated glass coverslips in 24-well plates were fixed with 4%
paraformaldehyde in PBS for 30 min, washed with Tris-HCl buffer (pH 7.8), and incubated
in wash buffer containing 1% bovine serum albumin (BSA) and 0.2% Triton X-100 for 5
min at room temperature. Samples were then incubated with the following primary
antibodies overnight at room temperature: rabbit anti-Cx43 (1:200) and mouse anti-vimentin
(1:200, DAKO, Campintene, CA, USA), rabbit anti-GFAP (1:200, DAKO), mouse anti-
MAP2 (1:50, Chemicon Temecula, CA, USA), mouse anti- β3 tubulin (1:1000, Promega,
USA), rabbit anti-GLUT1 and GLUT2 (1:100, Alpha Diagnostic International, INC., San
Antonio, TX, USA), chicken anti-MCT1 (1:100, Millipore, Temecula, CA, USA), rabbit
anti-MCT4 (1:20, Millipore), rabbit anti-von Willebrand factor (VWF, 1:300, Sigma) or
anti-Kir1.6 (1:200, Santa Cruz Biotechnology California, USA). Samples were then
incubated with alexa fluor 488 or alexa fluor 555-labeled secondary antibodies and counter-
stained with the DNA stain, TOPRO-3 (1:1000, Invitrogen). Preparations were analyzed
using confocal laser microscopy (D-Eclipse C1 Nikon, Tokyo, Japan).
Dye uptake and time-lapse fluorescence imaging

For time-lapse fluorescence imaging, cells plated on glass coverslips were washed twice in
phosphate-buffered saline (PBS) solution, pH 7.4, and then bathed with Locke’s solution
containing 5 µM Etd. In some experiments, cells were bathed in a divalent cation-free
solution (DCFS) [in (mM): NaCl (148), KCl (5), MgCl2 (1), HEPES (5), EGTA (10), pH =
7.4; at 37°C] containing 5 µM Etd. Cells on glass coverslips were placed on the microscope
stage and data was acquired using the same microscope and camera described for
intracellular calcium imaging. Regions of interest were placed over random cells and
background was subtracted. Fluorescence was recorded every 30 s. To test for changes in
slope, regression lines were fitted to points before and after various treatments using the
Excel program and mean value of slopes were compared using GraphPad Prism software
(GraphPad Software Inc., San Diego CA).
Electrophysiology
Cells platted on glass coverslips were transferred to an experimental chamber mounted on
the stage of an inverted microscope (Olympus IX-51; Olympus Optical). For whole-cell
voltage clamp experiments, the bath solution contained 140 mM NaCl, 5.4 mM KCl, 1 mM
MgCl2, 1.8 mM CaCl2, 2 mM BaCl2, and 10 mM HEPES, pH 7.4. The pipette solution
contained 130 mM CsCl, 10 mM AspNa, 0.26 mM CaCl2, 1 mM MgCl2, 2 mM EGTA, 7
mM TEA-Cl, and 5 mM HEPES, pH 7.2. Patch pipettes were made from borosilicate glass
capillaries using a flaming/brown micropipette puller (P-87; Sutter Instruments, Union City,
CA). The tip resistance was 10–15 MΩ when filled with pipette solution. Currents were
filtered at 1 kHz and sampled at 5 kHz, and records were filtered with a digital low pass
filter of 0.5 kHz. Data acquisition and analysis were performed using pClamp 9 (Molecular
Devices, Novato, CA).

Western blot analysis

Cultures were rinsed twice with PBS (pH 7.4) and harvested by scraping with a rubber
policeman in ice-cold PBS containing protease and phosphatase inhibitors (1 mM
orthovanadate, 10 mM α-glycerophosphate) and a complete miniprotease inhibitor (Roche

Diagnostics, San Francisco, CA). Proteins were measured in aliquots of cell lysates with the
Bio-Rad protein assay (Bio- Rad, Richmond, CA). Pelleted cells were resuspended in 40 µL
of the protease and phosphatase inhibitor solution, placed on ice, and lysed by sonication
(Ultrasonic cell disrupter, Microson, Ultrasons, Annemasse, France) after which samples
were stored at −80°C or analyzed by immunoblotting. Aliquots of cell lysates or biotinylated
cell surface proteins were resuspended in 1 X Laemli’s sample buffer, boiled for 5 min,
separated on 8% SDS-PAGE and electro-transferred to nitrocellulose sheets.
Nonspecific protein binding was blocked by incubation of nitrocellulose sheets in PBS-

BLOTTO (5% nonfat milk in PBS). After 30 min blots were incubated with primary
antibody for 1 h at room temperature or overnight at 4°C, followed by four 15 min PBS
washes. Blots were incubated with goat anti-rabbit antibody conjugated to HRP.
Immunoreactivity was detected by enhanced chemiluminescence (ECL) detection using the
SuperSignal kit (Pierce, Rockford, IL) according to the manufacturer´s instructions.
Cell surface biotinylation and quantitation

Cells cultured on 90-mm dishes were washed three times with ice-cold Hank’s saline
solution (pH 8.0), and 3 mL of sulfo-NHS-SS-biotin solution (0.5 mg/mL) was added
followed by incubation for 30 min at 4°C. Cells were washed three times with ice-cold
saline containing 15 mM glycine (pH 8.0) to block unreacted biotin. The cells were
harvested and incubated with an excess of immobilized NeutrAvidin (1 mL of NeutrAvidin
per 3 mg of biotinylated protein) for 1 h at 4°C after which 1 mL of wash buffer (saline
solution, pH 7.2 containing 0.1% SDS and 1% Nonidet P-40) was added. The mixture was
centrifuged for 2 min at 14.000 r.p.m. at 4°C. The supernatant was removed and discarded,
and the pellet was resuspended in 40 µl of saline solution, pH 2.8, and containing 0.1 M
glycine to release the proteins from the biotin. After the mixture was centrifuged at 14.000
r.p.m. for 2 min at 4°C, the supernatant was collected, and the pH was adjusted immediately
by adding 10 µL of 1 M Tris, pH 7.5. Relative protein levels were measured using Western
blot analysis as described above. Resulting immunoblot signals were scanned, and the
densitometric analysis was performed with the Scion Image software. Densitometric
arbitrary units were normalized to the signal obtained from total protein measured with
Ponceau red.
Glucose transport assay

For 2-[H3]DOG uptake assays, cells were grown in 12-well plates to a density of 1×105 cells
per well. Cultures were carefully selected under the microscope to ensure that only plates
showing uniformly grown cells were used. Cells were washed with incubation buffer (10
mM HEPES, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, and 0.8 mM MgCl2) and incubated
in this buffer for 30 min at room temperature. Uptake assays were performed in 500 µl of
incubation buffer containing 0.2 mM 2-DOG and 3 µCi 2-[H3]DOG (30.6 Ci/mmol). 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 and 0.2% SDS), and the incorporated radioactivity was
assayed by liquid scintillation counting. Where appropriate, competitors and inhibitors were
added to the uptake assays or pre-incubated with the cells.
For 2-NBDG uptake assays, cells were grown on glass coverslips. Tanycytes bathed in
Locke’s solution were observed with the same microscope and camera used for dye uptake
experiments (see above). 2-NBDG (100 µM) was exited at 488 nm, and the emission was

filtered at 505–550 nm (Porras et al. 2004). In each experiment, the resulting fluorescence
was measured with Metafluor software (Universal Imaging, Downingtown, PA), and for
each value, the background value was subtracted.
Measurement of extracellular ATP concentration

Tanycytes were grown (80% confluence) in 6-well plates for 15 days after which they were
incubated in Locke’s solution at 37°C for 30 min. Cells were then treated for 10 min with 10
mM glucose, and ATP concentration in the extracellular solution was measured using a
luciferin/luciferase bioluminescence assay kit (Sigma-Aldrich). Baseline measurements
were performed on separate cultures using standard Locke’s solution. The amount of ATP in
each sample was calculated from standard curves and normalized for the protein
concentration using the BCA assay obtained from Pierce (Rockford, IL, USA).
Data analysis and statistics

For each data group, results were expressed as mean ± standard error (SE), and n refers to
the number of independent experiments. For statistical analysis, each treatment was
compared with its respective control, and significance was determined using a one-way
ANOVA followed, in case of significance, by a Tukey post-hoc test. For multiple group
treatments, significance was determined using a two-way ANOVA followed in case of
significance by a Bonferroni post-hoc test.
Results
Glucose increases the intracellular free Ca2+ signal in cultured rat tanycytes via GLUTs,
glucokinase and KATP channels
As described previously (García et al., 2003), immunohistochemical analysis revealed that
cultures enriched in differentiated hypothalamic tanycytes exhibit an intense reactivity of
vimentin, Kir6.1 and GLUT2, but not GFAP, MAP2, βIII-tubulin and VWF, ruling out
contamination with other hypothalamic cell types (Fig. 1A–G). In contrast, mixed
hypothalamic cultures were highly immunopositive for MAP2 and vimentin (Fig. 1F),
whereas cortical astroglial cultures exhibited a strong GFAP, and low vimentin reactivity
(data not shown). Most tanycytes (>90%) were highly reactive to anti-GK, anti-MCT1 and -
MCT4 antibodies (Fig. 1G), suggesting a high enrichment in β1-tanycytes, since β1- but not
α-tanycytes express GK, MCT1 and MCT4 in vivo (Cortés-Campos et al., 2011; Millan et
al., 2010).
Tanycytes have been proposed to mediate, at least in part, the mechanism by which the
hypothalamus detects changes in extracellular glucose concentrations (Millan et al., 2010).
Supporting this idea, acute in situ application of glucose increases the [Ca2+]i in α-tanycytes
(Frayling et al., 2011). However, the mechanism underlying this phenomenon remains to be
elucidated. Thus, we investigated whether canonical glucosensing molecules used by
specialized cells, such as pancreatic β-cells, were involved in this process and if the
activation of hemichannels could contribute to the glucose-induced changes in [Ca2+]i. As
indicated by time-lapse measurements of Fura-2 ratio (340/380) (from now and on called
Ca2+ signal), cultured tanycytes maintained in saline containing 2 mM glucose showed a
low basal Fura-2 fluorescent ratio (Fig. 2A–E). However, exposure to 10 mM glucose
induced a rapid, strong and transient increase in Fura-2 fluorescent ratio; peaking at ~630%
as compared to basal levels (Fig. 2A, D and E). In addition, a delay (67.6 ± 7.8 s; n=9)
between glucose addition and increase in Ca2+ signal was evident (vertical gray line in Fig.
2A), suggesting that the glucose-induced rise in Ca2+ signal might require activation of a
signal transduction mechanism. Moreover, increasing glucose concentrations induced a
proportional rise in Ca2+ signal, approaching a plateau at about 20 mM glucose; peaking at

~815% as compared to basal levels (Fig. 2D). No changes in Ca2+ signal were observed
upon treatment with 10 mM sucrose or mannitol ruling out the possibility of an osmolarity-
induced effect (not shown).
Because glucosensing by specialized cells, such as pancreatic β-cells, involves GLUT2, GK

and KATP channels (Schuit et al., 2001), the effect of inhibitors of these proteins on glucose-
induced rise in Ca2+ signal of tanycytes was assessed. Two GLUT inhibitors, 100 µM
cytochalasin B (Cyto-B) or 100 mM 4,6,-O-ethylidene-D-glucose (ETDG) (Barros et al.,
2009), greatly reduced the glucose-induced increase in Ca2+ signal from ~630% to ~234%
and ~257%, respectively (Fig. 2E). Moreover, cytochalasin E did not affect the glucose-
induced rise in Ca2+ signal (not shown), ruling out unspecific effects of Cyto-B on actin
polymerization. These results suggest that the glucose-induced rise in Ca2+ signal is
triggered by glucose entry primarily via GLUTs; a small amount of glucose enters through
another pathway not yet identified (see below). In addition, pretreatment with alloxan (500
µM), a GK inhibitor, completely inhibited the glucose-induced increase in Ca2+ signal in
tanycytes from ~630% to ~109% (Fig. 2B and E). To investigate if the glucose-induced rise
in Ca2+ signal depends on ATP generated from anaerobic glycolysis and/or oxidative
phosphorylation, tanycytes were treated with iodoacetic acid (IA) or antimycin A (AA),
blockers of each metabolic pathway, respectively. Pretreatment with IA (1 mM) reduced the
glucose-induced rise in Ca2+ signal from ~630% to ~107%. However, no significant
reduction was observed using AA (200 nM; 612.1 ± 105.2%) (Fig. 2E), suggesting that
anaerobic glycolysis is the main source of ATP required for the glucose-induced rise in Ca2+
signal. In pancreatic β-cells, the rise in [Ca2+]i depends on closure of KATP channels
induced by an increase in intracellular ATP concentration (Schuit et al., 2001). Therefore,
diazoxide (1 µM, Diazox), an activator of KATP channels, was used to examine the possible
involvement of a similar mechanism in the glucose-induced rise in Ca2+ signal observed in
glucose-treated tanycytes. Pretreatment with Diazox for 10 min drastically reduced the
glucose-induced rise in Ca2+ signal from ~630% to ~98% (Fig. 2C and E).
Rat tanycytes exhibit functional hemichannels in their surface and express Cx43 in vitro
The rise in Ca2+ signal induced by glucose might result from opening of a Ca2+ permeable
cell membrane pathway and/or Ca2+ release from intracellular stores. In glial cells the main
connexin expressed is Cx43 (Giaume and Theis, 2010), which has been recently shown to be
Ca2+ permeable (Schalper et al. 2010). Therefore, we studied if rat tanycytes express
functional Cx43 hemichannels in their surface. Similar to cortical astrocytes (Orellana et al.,
2010), tanycytes cultured under control conditions exhibited a low Etd uptake rate (0.25 ±
0.02 AU/min; Fig. 3A, and C). The basal Etd uptake in tanycytes under control condition
was partially reduced to about 50% of the basal level by La3+ (200 µM), a general blocker of
connexin hemichannels, Gap26 (200 µM), and the Cx43E2 antibody (1:500) (Fig. 3D), two
Cx43 hemichnnel blockers, suggesting the presence of a basal Cx43 hemichannel activity.
However, the Panx1 hemichannel blockers, probenecid (Prob, 500 µM) or 10panx1 (200
µM), did not significantly alter the basal Etd uptake (97.3 ± 22.3% and 102.4 ± 6.2%,
respectively), suggesting that if tanycytes express Panx1 hemichannels, they are not active
under basal conditions (Fig. 3D). To address whether both Panx1 hemichannel blockers
effectively inhibit Panx1 hemichannels under our conditions, we treated neurons with
glutamate for 1h, a known condition that open Panx1 hemichannels in these cells (Orellana
et al., 2011b). Both blockers inhibited the increase in the glutamate-induced Etd uptake
mediated by Panx1 hemichannels (not shown).
The presence of connexin hemichannels in cultured tanycytes was supported by their

activation in cells exposed to a divalent cation-free solution (DCFS), which increases the
open probability of connexin but not Panx1 hemichannels (Sáez et al., 2005). Under these
conditions, a high Etd uptake rate (0.56 ± 0.01 AU/min) was found compared to control

(224.3 ± 36.3%) that was almost completely blocked by Cx43E2 antibody (61.3 ± 12.1%)
(Fig. 3B, C and D), indicating that most if not all hemichannels are formed by Cx43.
The presence of functional Cx43 hemichannels was also in agreement with the
immunofluorescence detection of Cx43 in cultured tanycytes (Fig. 3E); Cx43 was primarily
localized at cell-cell interfaces, which may correspond with gap junctions (Fig. 3E).
Moreover, all cultures showed vesicular Cx43 reactivity that most likely corresponds to
intracellular Cx43 trafficking (Fig. 3E). Since a very small percent (~10%) of total Cx43 is
known to form hemichannels at the cell surface of cortical astrocytes (Retamal et al., 2006),
it is likely that Cx43 hemichannels also correspond to a small percent of the total Cx43
protein making difficult their detection by immunofluorescence. In spite, we detected Cx43
at the cellular surface using the Cx43E2 antibody in nonpermeabilized cells (Supplementary
Fig. 1A). In permeabilized cells Cx43 reactivity was also detected in vesicle-like structures
located at the cell interior in addition to the labeling detected at the cell periphery
(Supplementary Fig. 1B). We also detected Cx43 hemichannels at the cellular surface using
biotinylation of surface proteins and whole cell patch clamp (see below).
The glucose-induced increase in intracellular free Ca2+ signal is mediated mainly by Ca2+
release from intracellular stores and not by Ca2+ influx via hemichannels
The possible role of Cx43 hemichannels in the glucose-induced rise in Ca2+ signal was
studied using specific blockers of Cx43 hemichannels, the Cx43E2 antibody (Orellana et al.,
2011c; Siller-Jackson et al., 2008) and the mimetic peptide Gap26 with an amino acid
sequence identical to the second loop of Cx43 (Evans et al., 2006). Cx43E2 (1:500) and
Gap26 (200 µM) reduced almost completely the glucose-induced increase in Ca2+ signal in
tanycytes to ~113% and ~107%, respectively (Fig. 3G). However, pretreatment with the
preimmune antibodies (1:500) did not affect the glucose-induced increase in Ca2+ signal,
supporting the specificity of Cx43E2 (not shown). In contrast, 10panx1 (200 µM) and
probenecid (200 µM), both Panx1 hemichannel blockers (Pelegrin and Surprenant, 2006;
Silverman et al., 2008), did not reduce the glucose-induced increase in Ca2+ signal (Fig.
3G), ruling out the possible involvement of Panx1 hemichannels in the glucose-induced rise
in Ca2+ signal. In spite of the strong inhibition of the glucose-induced rise in Ca2+ signal
induced by Cx43E2 and Gap26, the absence of extracellular Ca2+ did not significantly
reduce this response (from ~615%, in the presence of extracellular Ca2+ to ~590% in Ca2+-
free) (Fig. 3F and G), suggesting that in tanicytes exposed to glucose, Cx43 hemichannels
do not allow a relevant Ca2+ inflow and thus, their role in the observed glucose-induced rise
in Ca2+ signal could be indirect. In addition, the lack of effect of extracellular Ca2+-free
solution on the glucose-induced rise in Ca2+ signal suggested strongly the involvement of
Ca2+ release from intracellular reservoirs instead of Ca2+ influx from the extracellular
medium.
Glucose enhances the Cx43 hemichannel activity via a glucokinase/KATP channel-

dependent pathway in rat tanycytes
To elucidate the involvement of Cx43 hemichannels in the glucose-induced rise in Ca2+
signal, the effect of glucose on the hemichannel activity was assessed. Under control
conditions (2 mM glucose), tanycytes exhibited low Etd uptake (Fig. 4A and D), which
prominently increased upon treatment with 10 mM glucose (from 0.25 ± 0.02 AU/min to
0.57 ± 0.02 AU/min, n=6) (Fig. 4B, D and F). An important difference with the glucose-
induced rise in Ca2+ signal, which showed a delay of about 1 min (Fig. 4A and B), was that
the delay of the glucose-induced rise in Etd uptake was <20 s (Fig. 4D). Moreover, as in the
case of the Ca2+ signal changes, the Etd uptake increase was glucose concentration-
dependent, approaching a plateau at 40 mM glucose (425.4 ± 182.3%, compared to basal)
(Fig. 4E). In these cells, no concurrent changes in intercellular communication mediated by

gap junctions, evaluated by the intercellular transfer of microinjected Lucifer yellow (LY),
was observed (Supplementary Fig. 2A–D).
To evaluate if the glucosensing proteins, GLUTs, glucokinase (GK) and KATP channels, are
involved in the glucose-induced Etd uptake, specific drugs against each of these molecular
components of the glucosensor transduction system were employed. As compared to control,
100 µM Cyto-B, 100 mM ETDG, 500 µM alloxan, 1 mM IA and 1 µM Diazox reduced the
glucose-induced Etd uptake from ~211% to ~138%, ~141%, ~103%, ~105% and ~109%,
respectively (Fig. 4F). However, 200 nM AA did not affect it (207.2 ± 19.4%, n=4) (Fig.
4F). Therefore, the glucose-induced Etd uptake requires the functional participation of
GLUTs, GK and glycolysis-derived ATP to block KATP channels. Relevant to this
interpretation is that inhibitors of these glucose-sensing proteins did not alter the DCFS-
induced Etd uptake (Supplementary Fig. 3), indicating that they act as glucosensing
modulators and not as hemichannel blockers.
Glucose increases the macroscopic Cx43 hemichannel current, but does not alter the
unitary conductance or the surface Cx43 levels in tanycytes
To identify the pathway through which glucose increases the membrane permeability of
tanycytes, whole-cell voltage-clamp studies were undertaken, and the macroscopic
membrane current (total current measured in the absence of other active membrane
channels) and the presence of hemichannel unitary events applying voltage ramp protocols
from −80 to +80 mV were assessed (Fig. 5A).
In control tanycytes, unitary current events were not detected in 17 cells (not shown);
however, 6 cells showed a few small unitary transitions at negative potentials (Fig. 5B). In
contrast, in tanycytes treated with 10 mM glucose for 3 min the total current was much
bigger than in control cells, and numerous unitary current events were detected at either
negative (at −60 mV from ~15 pA to ~197 pA, n=12 cells) (Fig. 5B and D) or positive
potentials (at +60 mV from ~14 pA to ~205 pA, n=12 cells) (Fig. 5B and D). In most cells
(n=18), unitary current events were recorded, and point-by-point conversion of current to
conductance values revealed single channels of 218 ± 6 pS (see inset in Fig. 5B). This value
is close to the expected unitary conductance (~220 pS) of Cx43 hemichannels (Contreras et
al., 2003). To elucidate the involvement of such hemichannels in the glucose-induced
currents, tanycytes were treated acutely with Cx43E2 (1:500). This Cx43 hemichannel
blocker reduced almost completely the rise in macroscopic current (at −60 mV from ~197
pA to ~27 pA and at +60 mV from ~205 pA to ~25 pA (n=12) and the appearance of
discrete unitary current transitions induced by glucose (Fig. 5B and D), indicating that
glucose induces activation of Cx43 hemichannels. Importantly, the reversal potential of all
the above mentioned currents was close to 0 mV. However, the glucose-induced currents
presented a reversal potential close +5 mV (Fig. 5B). Moreover, 10 mM glucose increased

the frequency of unitary currents of Cx43 hemichannels with conductance around ~220 pS
(Supplementary Fig. 4A–D), whereas did not significantly alter the decay constant (τ) of
open time compared to control (3.7 ± 0.7 ms and 5.2 ± 0.6 ms, respectively) (Supplementary
Fig. 4E and F). Furthermore, 10 mM glucose increased the mean open time compared to
controls conditions (0.7 ± 0.1 ms and 0.20 ± 0.03 ms, respectively).
The increase in Cx43 hemichannel activity might be due to an increase in open probability
per hemichannel or/and increase in the number of hemichannels present in the cell
membrane. Previous studies have associated hemichannel-mediated dye uptake with
increased surface levels of hemichannels (Orellana et al., 2010). Therefore, the effect of
glucose on the total and surface levels of Cx43 in tanycytes was evaluated. Neither total nor
surface levels of Cx43 were affected by treatment with 10 mM glucose for 5 min (Fig. 5C
and E, respectively, n=3). Also, neither 500 µM alloxan nor 1 µM diazox in combination

with glucose affected total Cx43 or surface levels in tanycytes (Fig. 5C and E, respectively,
n=3). Importantly, 10 mM glucose might decrease the total non-phosphorylated Cx43 and
increase the total phosphorylated form of Cx43 (Fig. 5C), however total levels of Cx43 were
not affected (Fig. 5E).
Tanycytes are permeable to glucose via GLUTs and Cx43 hemichannels

Since neither the rise in Ca2+ signal nor the increase in Etd uptake induced by glucose were
completely inhibited with GLUT blockers (Fig. 2F and 4F) and because other glial cells also
present glucose up take though Cx43 hemichannels (Retamal et al. 2007), the role of these
channels as an alternative pathway for glucose entry into tanycytes was explored.
Specifically, uptake of 2-DOG and 2-NBDG in tanycytes under control conditions or bathed
in DCFS to enhance the open probability of Cx43 hemichannels (Sáez et al., 2005) was
measured. After one-minute, 2-DOG uptake close to 20–30 nmoles per million of cells (22.1
± 8 nmol/106 cells) (Fig. 6A) and low 2-NBDG uptake (37.4 ± 7.3 A.U., n=6) was observed
under control conditions (Fig. 6B). Uptake of 2-DOG and 2-NBDG was drastically reduced
by 100 mM ETDG (~6 nmol/106 cells and ~9 AU, respectively, n=5) or 100 µM Cyto-B (~7
nmol/106 cells and ~7 AU, respectively, n=5), whereas Cx43E2 (1:500) only partially
inhibited 2-DOG and 2-NBDG uptake (~15 nmol/106 cells and ~22 AU, respectively, n=3)
(Fig. 6). Notably, co-application of Cyto-B and Cx43E2 almost completely blocked the
uptake of 2-DOG and 2-NBDG (~0.8 nmol/106 cells and 0.8 AU, respectively, n=3) (Fig.
6B).
As predicted, tanycytes bathed in DCFS exhibited higher 2-DOG (~38 nmol/106 cells, n=5)
and 2-NBDG (~66 AU, n=5) uptake than tanycytes under control conditions (Fig. 6A and
B). Under these conditions, uptake of 2-DOG or 2-NBDG was more prominently inhibited
by Cx43E2 (~14 nmol/106 cells and ~24 AU, respectively, n=3), than by ETDG (~22 nmol/
106 cells and ~31 AU, respectively, n=5) or Cyto-B (~24 nmol/106 cells and ~27 AU,
respectively, n=5) (Fig. 6). Furthermore, in cells treated with Cyto-B plus Cx43E2, the
uptake of 2-DOG and 2-NBDG was almost completely blocked (~1 nmol/106 cells and ~0.7
AU, respectively, n=3).
The glucose-induced increase in intracellular free Ca2+ signal occurs via ATP released
through Cx43 hemichannels
Because in parallel experiments the onset of glucose-induced Etd uptake occurred before
(Fig. 4D) than the rise in Ca2+ signal (Fig. 2A), simultaneous measurements of Etd uptake
and Ca2+ signal in tanycytes exposed to glucose were performed to better elucidate the onset
of these sequential changes. In these studies, the Etd uptake induced by 10 mM glucose
occurred at ~12 s (Fig. 7A–H and I, n=8), whereas the glucose-induced rise in Ca2+ signal
was observed at ~67 s (Fig. 7A–H and I, n=9). Because the glucose-induced changes did not
depend on the extracellular [Ca2+] (Fig. 2B and F), the above results suggest that opening of
Cx43 hemichannels results in the extracellular release of a molecule that reaches a
concentration sufficient to trigger Ca2+ release from intracellular stores.
Previous reports have demonstrated that Cx43 hemichannels can mediate the release of ATP
(Kang et al., 2008). Because ATP can induce Ca2+ release from intracellular stores via P2Y
receptors, ATP release by tanycytes in response to glucose was examined. The increase in
Ca2+ signal induced by glucose was prevented by 10 U/mL apyrase, a phosphatase that
degrades ATP, and 200 µM suramine, an inhibitor of both P2Y and P2X receptors, peaking
at ~135% or ~126%, respectively (Fig. 7J). However, no differences were observed using
200 µM oATP, a general P2X receptor blocker, or 10 µM brilliant blue G (BBG), a P2X7
receptor blocker (~610% or ~624%, respectively) (Fig. 7J). These results indicated that ATP
and P2Y receptors are crucial for glucose-induced increases in Ca2+ signal. In support of

this interpretation, MRS2179, a P2Y1 receptor blocker, inhibited the glucose-induced rise in
Ca2+ signal (129.2 ± 17.3%) (Fig. 7J). Moreover, 2 µM thapsigargin, a compound that
depletes the intracellular Ca2+ stores, or 5 µM xestospongin C (XeC) and 5 µM xestospongin
B (XeB), both IP3 receptor blockers, inhibited the rise in Ca2+ signal induced by glucose
(~158%, ~125% and ~120% respectively) (Fig. 7J), suggesting the involvement of IP3-
mediated Ca2+ release.
Then, the effect of glucose on ATP release via Cx43 hemichannels and glucosensing
signaling was examined. The extracellular medium of tanycytes treated with 10 mM glucose
exhibited higher ATP levels than that of control tanycytes (from ~1 pmol/106 cells to ~45
pmol/x106 cells, n=6) (Fig. 7K). The increase in extracellular ATP concentration induced by
glucose was prevented by 100 µM Cyto-B, 500 µM alloxan, 1 mM IA, 1 µM Diazox, and
Cx43E2 (~7 pmol/106, ~1 pmol/106, ~0.9 pmol/106, ~0.2 pmol/106 and ~0.2 pmol/106,
respectively, n=6) (Fig. 7K).
The effect of ATP itself on the Ca2+ signal in tanycytes was also examined; 100 µM ATP
induced a rise in Ca2+ signal similar to that observed with 10 mM glucose (~682%, n=3)
(Fig. 8A and B). Moreover, the ATP-induced rise in Ca2+ signal was inhibited by 200 µM
suramine (~133%, n=3), 2 µM thapsigargin (~147%, n=3), 5 µM XeC (~156%, n=3) and 5
µM XeB (~130%, n=3), but not by 200 µM oATP or 10 µM BBG (~671%, and ~656%,
respectively, n=3) (Fig. 8B). Thus, the results support the hypothesis that proposes
extracellular ATP as mediator of the glucose-induced rise in [Ca2+]i.
Due to the complexity behind the activation of Cx43 hemichannels of this system, we
investigated whether an IP3 receptor-mediated Ca2+ signal is needed to promote Cx43
hemichannel opening. XeC and XeB did not modify the glucose-induced hemichannel
activity (Supplementary Fig. 5). Similar results were obtained in tanycytes loaded with
BAPTA (Supplementary Figure 5), indicating that cytoplasmic Ca2+ does not mediate the
activation of Cx43 hemichannel opening. On the other hand, as it was shown before, KATP
channel inhibition was necessary for the glucose-induced hemichannel activity. However,
glibenclamide by itself, an inhibitor of KATP channels, did not induce hemichannel opening
(Supplementary Figure 5), suggesting that an additional mechanism activated almost
simultaneously and not yet identified is necessary for Cx43 hemichannel activation.
Discussion
The present study demonstrates that tanycytes possess a rapid, glucose-activated signal
transduction pathway, which includes GLUTs, GK, anaerobic glycolysis-derived ATP,
KATP channels, Cx43 hemichannels, extracellular ATP, P2Y receptors, and IP3 activated
intracellular Ca2+ stores.
Recently, glucose and non-metabolizable analogues of glucose were demonstrated to

increase the [Ca2+]i in hypothalamic slices, specifically in α tanycytes located in the most
dorsal region of the hypothalamus (Frayling et al., 2011), suggesting that the pancreatic β-
cell paradigm does not apply to these cells. However, in the present study, glucose increased
the [Ca2+]i in a concentration-dependent manner in cultured β-tanycytes by a process in
which glucose uptake mainly via GLUT is critical as Cyto-B and ETDG strongly inhibited
this response. This is in agreement with the reported expression of GLUT1 and the
glucosensing transporter, GLUT2, by β1-tanycytes (García et al., 2003). Because the
glucose-induced rise in Ca2+ signal was prevented by a GK blocker as well as a KATP
channel activator, these two proteins may play an essential role in tanycyte glucosensing,
which is also consistent with the high in vivo expression of these proteins by these cells
(Marty et al., 2007; Thomzig et al., 2001). In addition, in vivo pharmacological and

molecular inhibition of GK or KATP channels impairs feeding behavior in rodents (Miki et

al., 2001; Sanders et al., 2004). The difference between the results of the present study and
those of Frayling et al (2011) could be explained by the type of tanycyte population used
and the model (ex vivo versus in vitro). Frayling et al (2011) used α-tanycytes from brain
slices, whereas our cultures were highly enriched in β1-tanycytes, which express the same
enzymes and transporters detected in situ (e.g., GLUT1, GLUT2, MCT1, MCT4, GK)
(Cortés-Campos et al., 2011; García et al. 2001; 2003; Millan et al., 2010). In this regard,
tanycytes may sense glucose by more than one mechanism, which is determined by the
predominant type of tanycyte.
In the present study, glucose-induced Etd uptake was not completely inhibited when GLUTs
were blocked, suggesting the contribution of another source of glucose transport/diffusion
through the cell membrane. Accordingly, the glucose uptake was completely inhibited when
both GLUTs and connexin hemichannels were blocked, suggesting that under control
conditions, Cx43 hemichannels participate in glucose diffusion toward the cytoplasm of
tanycytes to a minor extent despite the increase in Cx43 hemichannel activity, while GLUTs
are the main protagonist in this transport.
Intracellular ATP generated by anaerobic glycolysis but not produced by oxidative

phosphorylation was required for the glucose-induced rise in Ca2+ signal since the effect of
glucose was prevented by IA and not by AA. This finding is similar to that reported for
pancreatic β-cells (Mertz et al., 1996). The high glycolytic activity of tanycytes has been
previously demonstrated; they release lactate at physiological concentrations of glucose,
which is inhibited by classical MCT inhibitors (Cortés-Campos et al., 2011). In pancreatic
β-cells, the Ca2+ influx proceeds through voltage-dependent Ca2+ channels and is critical for
glucosensing (Schuit et al., 2001). However, data from the present study indicate that
extracellular Ca2+ influx is not involved in this response in tanycytes, since the glucose-
induced rise in Ca2+ signal was observed even in the absence of extracellular Ca2+,
indicating the involvement of intracellular Ca2+ stores. Nevertheless, the glucose-induced
rise in Ca2+ signal was not detected after Cx43 hemichannel blockade, implying that they
are involved in changing the [Ca2+]i by allowing release of a paracrine factor likely to be
ATP. In agreement with this interpretation, an increase in Cx43 hemichannel activity was
observed in tanycytes treated with glucose and the increase in Ca2+ signal and Etd uptake
were completely inhibited by several Cx43 hemichannel blockers (e.g., La3+, Gap26 and
Cx43E2). In contrast, Panx1 hemichannel blockers were without effect, indicating for the
first time that tanycytes exhibit functional Cx43 hemichannels, which is consistent with the
high Cx43 immunoreactivity detected in these cells. In support of the idea that Cx43
hemichannels are involved in glucosensing, patch clamp experiments revealed an increase in
macroscopic membrane current induced by glucose. This is most likely due to opening of
Cx43 hemichannels already present in the cell surface due to the following: 1) it was
completely inhibited by Cx43E2, a specific blocker of Cx43 hemichannels (Orellana et al.,
2011c; Siller-Jackson et al., 2008), 2) the total surface levels of Cx43 hemichannels were not
affected by glucose, and 3) the unitary events were of ~ 220 pS, characteristics which
correspond to that of Cx43 hemichannel (Contreras et al., 2003). Thus, it is likely that the
glucose-induced increase in total current was due to an increase in open probability of each
Cx43 hemichannel already present at the cell surface and not to recruitment of more
hemichannels at the cell surface or increased unitary hemichannel conductance. Unlike
cortical astrocytes that express hemichannels formed by Panx1 (Iglesias et al., 2009;
Iwabuchi and Kawahara, 2011) or Cx43 (Retamal et al., 2007; Orellana et al., 2011c),
cultured tanycytes seems to express mainly hemichannels formed by Cx43. The slightly
positive reversal potential observed in tanicytes treated with glucose might reflect either a
change Cx43 hemichannel selectivity or the presence of a more selective membrane channel
simultaneously activated by glucose and co-recorded with Cx43 hemichannels.

Since the glucose-induced hemichannel activity was inhibited by blockers of the canonical
glucosensing pathway (e.g., GLUTs, GK and KATP channels), activation of Cx43
hemichannels likely occurs downstream of the glucosensing molecules (Fig. 9). A puzzling
aspect is how KATP channel inhibition leads to increased Cx43 hemichannel activity. One
possibility is that it might result from membrane depolarization as consequence of KATP
channel inhibition by intracellular ATP. In agreement with this possibility, Cx43
hemichannels expressed in HeLa cells are activated by positive potential (Contreras et al.,
2003). However, in glucose-treated tanycytes, the activity of Cx43 hemichannels was also
high at negative potentials, suggesting that membrane depolarization is not the only possible
mechanism involved in enhancing Cx43 hemichannel activity. In support to this notion,
membrane depolarization under control conditions did not enhance the membrane current
mediated by Cx43 hemichannels. Moreover, glibenclamide by itself did not induce
hemichannel opening, suggesting that both glucose or its derivates (e.g., ATP) and inhibition
of KATP channels are necessary for Cx43 hemichannel activation. Among the putative
mechanism it is possible that glucose or its derivates activate kinases/phosphatases
regulating the phosphorylation state of Cx43 hemichannels, which are known to affect the
gating mechanism of these channels (Sáez et al., 2010). However, further studies are
required to examine these possible and other alternative possibilities to identify the
mechanism by which Cx43 hemichannels are activated by glucose in tanycytes.
Acute glucose increases the release of ATP in tanycytes (Frayling et al., 2011). Accordingly,
the present study shows that the extracellular ATP concentration is enhanced in tanycyte
cultures treated with glucose and most likely is the consequence of more ATP released via
Cx43 hemichannels since inhibitors of Cx43 hemichannels and not of Panx1 hemichannels
prevented the glucose-induced increase in extracellular ATP levels. This increase in
extracellular ATP concentration was also prevented by MRS2179, thapsigargin and
xestospongin C, revealing the involvement of the metabotropic P2Y1 receptors, IP3
receptors and intracellular Ca2+ stores, respectively. Supporting the possible regulatory role
of ATP in feeding behavior, intracerebroventricular infusion of this molecule stimulates
neurons localized in the lateral hypothalamic area (Wollmann et al. 2005), dorsomedial
hypothalamic nucleus (Matsumoto et al., 2004) and VMN (Sorimachi et al., 2001).
Moreover, extracellular ATP may regulate food intake via activation of P2Y1 receptors
(Kittner et al., 2006). ATP release by tanycytes may modulate neuronal activity in
hypothalamic areas associated with feeding behavior, including the AN and VMN, which
are in close contact with these cells. If so, the activation of purinergic P2Y receptors might
be turned off in part by diffusion of ATP to distal regions as well as by desensitization of
P2Y1 receptors (Choi et al., 2008) and degradation of extracellular ATP by extracellular
phosphatases in the extracellular compartment of tanycytes (Firth and Bock, 1976).
Future in vivo studies will be required to determine whether tanycytes could sense
extracellular changes in glucose concentration and transmit them to neurons via Ca2+ waves
and/or the release of paracrine factors (e.g., ATP). The present work shows the involvement
of several glucosensing proteins in the glucose-induced rise in [Ca2+]i on β1-ltanycytes (Fig.
9), which might contribute to the knowledge on the glucosensing mechanism in the brain
and may open novel pharmacological strategies for therapeutic treatment of feeding
behavior disorders.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
This work was partially supported by the following grants: CONICYT 24080055 (to JAO); FONDECYT 1100705
(to MAG, FN); FONDECYT 1111033 (to JCS); FONDECYT 1100396 (to FN, MAG), FONDEF DO7I1086 (to
JCS); Anillo ACT-71 (to JCS); NIH AR46798 and Welch Foundation Grant AQ-1507 (to JXJ).
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Figure 1. Immunocytochemistry characterization of cultured tanycytes

Tanycytes obtained from rat hypothalamus at 1 day postnatal were cultured for 3 weeks. (A–
E) Representative confocal images depicting vimentin (A, green), GFAP (B, green), MAP2
(C, green), βIII-tubulin (D, green), Kir6.1 (E, green) in rat tanycytes under control
conditions. (F) MAP2 (red) and vimentin (green) staining in mixed hypothalamic cultures of
tanycytes and neurons. In blue are shown nuclei stained with TOPRO-3. (G) Quantification
of immunopositive expression normalized to total cells of vimentin, Kir6.1, GLUT2, GK,
MCT1, MCT4, GFAP, βIII-tubulin, MAP2 and Von Willebrand factor (VWF) in tanycytes
under control conditions. Scale bar = 80 µm.

Figure 2. Glucose-induced increase in intracellular free Ca2+ signal occurs by a glucokinase- and
KATP channel-dependent pathway in rat tancytes
(A–C) Representative plots of relative changes in Ca2+ signal (340/380 nm) over time in
cultured rat tanycytes subjected to changes in glucose concentrations (horizontal bars, from
2 to 10 mM glucose) under control conditions (A), in presence of 500 µM alloxan, a
glucokinase inhibitor (B) or in presence of 1 µM diazoxide (C), a KATP channel activator. In
each panel, three photomicrographs of time-lapse images show changes in Fura-2 ratio
(pseudo-colored scale). The delay between the addition of glucose and the increase in Fura-2
ratio is represented by the vertical gray line. (D) Averaged data normalized to control
(dashed line) of maximal Fura-2 fluorescence intensity during the peak in tanycytes exposed

to increasing glucose concentration. (E) Averaged data normalized to control (dashed line)
of maximal Fura-2 fluorescence intensity during the peak in tanycytes exposed to 10 mM
glucose alone or in combination with the following blockers: 100 µM Cyto-B, 100 mM 4,6,-
O-ethylidene-D-glucose (ETDG), 500 µM alloxan, 1 mM iodoacetate (IA), 200 nM AA

(AA) and 1 µM diazoxide (Diazox). *** p < 0.001, effect of 10 mM glucose compared to
control; # p < 0.05, ## p < 0.005, effect of blockers compared to glucose treatment. Averaged
data were obtained from at least five independent experiments.

Figure 3. Rat tanycytes exhibit functional Cx43 hemichannels that are involved in the glucose-
induced increase in intracellular Ca2+ signal
(A–B) Fluorescence micrographs of Etd uptake (10 min exposure) in tanycytes under
control conditions (A) and then exposed for 10 min to a divalent cation (Ca2+/Mg2+)-free
solution (DCFS) (B). (C) Time-lapse measurement of Etd uptake in rat tanycytes under
control conditions (basal) or exposed to divalent cation free solution (DCFS). La3+ (200
µM), a connexin hemichannel blocker, applied at ~7 min of Etd uptake measurements
reduced the Etd uptake to a value even lower than that recorded under basal condition. (D)
Averaged data normalized to control (dashed line) of Etd uptake rate by tanycytes treated
with the following Cx43 hemichannel blockers co-applied during dye uptake recording: 200
µM La3+, 200 µM Gap26 and 1:500 Cx43E2; or with the following Panx1 hemichannel
blockers: 200 µM 10panx1 or 500 µM probenecid (Prob). Also, it is shown the Etd uptake
rate induced by DCFS conditions alone or plus the anti-Cx43 hemichannel blocker,
Cx43E2. *** p < 0.001, ** p < 0.005, treatments compared to control; # p < 0.05, effect of
DCFS conditions compared to blockers. Averaged data were obtained from at least 5
independent experiments. Scale bar = 30 µm. (E) Representative confocal image depicting
vimentin (red) and Cx43 (green) immunolabeling of cultured rat tanycytes under control
conditions. In blue are shown nuclei stained with TOPRO-3. Scale bar = 15 µm. (F)
Representative plot of relative changes in Ca2+ signal over time in cultured tanycytes

subjected to changes in glucose concentrations (horizontal bars, from 2 to 10 mM glucose)

in absence of extracellular Ca2+ (Ca2+-free) (G) Averaged data normalized to control
(dashed line) of maximal Fura-2 fluorescence intensity during the peak in tanycytes exposed
to 10 mM glucose alone or in combination with the following blockers: 200 µM 10panx1,

500 µM probenecid (Prob), 200 µM Gap26, 1:500 Cx43E2 antibody and in absence of
extracellular Ca2+. All blockers were applied 10 min before treatment with glucose. # p <
0.005, effect of blockers compared to glucose treatment. Averaged data were obtained from
at least five independent experiments.

Figure 4. Increased Etd uptake induced by glucose is mediated by a glucokinase/KATP channel-

dependent pathway in cultured rat tanycytes
(A–C) Representative immunofluorescence images depicting vimentin (white) labeling and
Edt (red) nuclei-staining from dye uptake experiments (10 min exposure to dye) in cultures
of tanycytes under control conditions (A) or treated with 10 mM glucose for 5 min alone (B)
or glucose plus 1:500 Cx43E2, an anti-Cx43 antibody that blocks Cx43 hemichannel (C). (D)
Time-lapse measurement of Etd uptake in rat tanycytes treated with 2, 10 or 20 mM glucose.
La3+ (200 µM) applied at ~10 min of Etd uptake measurement inhibited dye uptake. (E)
Average of Etd uptake rate normalized to control (dashed line) in tanycytes exposed to
increasing concentrations of glucose. * p<0.001, treatments compared to control. (F)

Averaged Etd uptake rate normalized to control (dashed line) by tanycytes treated with 10
mM glucose alone or in combination with the following blockers: 100 µM cytochalasin B
(Cyto-B), 100 mM ETDG, 500 µM alloxan, 1 mM iodocetic acid (IA), 200 nM antimycin A
(AA), 1 µM diazoxide (Diazox), 200 µM La3+, 200 µM Gap26, 1:500 Cx43E2, 200
µM 10panx1, or 500 µM Prob. ** p < 0.005, 10 mM glucose treatment compared to control; #
p < 0.05, ## p < 0.005, effect of 10 mM glucose treatment compared to blockers. Averaged
data were obtained from at least four independent experiments. Scale bar = 15 µm.

Figure 5. Glucose increases Cx43 hemichannel currents, but does not affect the surface Cx43
levels in cultured rat tanycytes
(A) Voltage ramps from −80 to +80 mV, 5 s in duration, were applied. Each ramp was
initiated and finished by a transition from 0 to −80 and +80 to 0 mV, respectively.
Membrane currents were measured in a whole-cell voltage-clamp configuration using low-
density cultures of rat tanycytes. (B) Voltage ramp from −80 to +80 mV, 5 s duration in
tanycytes under control conditions (black line) or treated for 3 min with 10 mM glucose
alone (magenta line) or 10 mM glucose plus 1:500 Cx43E2 (green line). A record section at
aboy +60 mV the current trace was point-by-point converted in conductance values showing
unitary events of about 220 pS. (C) Cultured tanycytes were treated for 5 min with 10 mM

glucose. Total (left panel) and surface (right panel) levels of Cx43 in tanycytes under control
conditions (lane 1) or treated for 5 min with 10 mM glucose alone (lane 2), 10 mM glucose
plus 500 µM alloxan (lane 3) or plus 1 µM diazoxide (Diazox) (lane 4). The Cx43
phosphorylated (P1-P2) and nonphosphorylated (NP) forms are indicated in the left. Total
levels of each analyzed protein were normalized according to the levels of α-tubulin
detected in each lane. Surface levels of each analyzed protein were normalized according to
the total protein loaded as revealed by staining with Ponceau red (PR) in each lane. Similar
observations were made in two other independent experiments. (D) Averaged data on
current events at +60 (white bars) or −60 mV (black bars) in tanycytes under control
conditions, exposed to 10 mM glucose alone or 10 mM glucose plus Cx43E2. * p < 0.001,
glucose treatment compared to control; # p < 0.001, inhibitory effect of blocker on the
glucose-induced response. Averaged data were obtained from three independent
experiments in which at least 8 cells were analyzed per treatment. (E) Quantification of total
(white bars) and surface (black bars) levels of Cx43 normalized to control (dashed line) in
tanycytes treated for 5 min with 10 mM glucose alone, plus 500 µM alloxan or 1 µM
Diazox. Averaged data were obtained from three independent experiments.

Figure 6. Glucose uptake occurs via GLUTs and Cx43 hemichannels in rat tanycytes
Averaged data at 1 min of 2-DOG (A) and 2-NBDG (B) uptake in tanycytes under control
conditions or treated with 100 mM ETDG, 100 µM cytochalasin B (Cyto-B), 1:500 Cx43E2
or 1:500 Cx43E2 plus 100 µM Cyto-B. In addition, 2-DOG and 2-NBDG uptake by
tanycytes exposed to DCFS conditions for 1 min alone or plus 100 mM (ETDG), 100 µM
Cyto-B, 1:500 Cx43E2 or 1:500 Cx43E2 plus 100 µM Cyto-B was analyzed. *** p < 0.001, **
p < 0.005, * p < 0.05; treatments compared to control; # p < 0.05, treatments compared to
DCFS conditions. Averaged data were obtained from at least four independent experiments.

Figure 7. Glucose increases the intracellular free Ca2+ signal in tanycytes by ATP released via
Cx43 hemichannels
(A–H) Representative fluorescence micrographs of simultaneous time-lapse imaging
showing changes in Fura-2 ratio (A–D, pseudo-colored scale) and Etd uptake (E–H, red) in
tanycytes treated with 10 mM glucose for 0 (A and E), 48 (B and F), 50 (C and G) and 200 s
(D and H). (I) Simultaneous plots of relative changes in [Ca2+]i and Etd uptake over time of
cells 1 (red), 2 (green), 3 (yellow) and 4 (cyan) depicted in panel A. The delay between the
increase in Etd uptake and Fura-2 ratio is indicated by the vertical dashed line. (J) Averaged
data normalized to control (dashed line) of maximal Fura-2 fluorescence intensity during the
peak in tanycytes exposed to 10 mM glucose alone or in combination with the followed

blockers: 10 U/mL apyrase (Apyr), 200 µM oxidized ATP (oATP), 10 µM (BBG), 200 µM
suramin (Sur), 10 µM MRS2179, 2 µM thapsigargin (TG), 5 µM xestospongin C (XeC) and
5 µM xestospongin (XeB). ** p < 0.005, 10 mM glucose treatment compared to blockers.
(K) Averaged values of ATP released by tanycytes under control conditions or treated with
10 mM glucose alone or in combination with the following blockers: 100 µM cytochalisin B
(Cyto-B), 500 µM alloxan, 1 mM IA, 1 µM diazoxide (Diazox), 200 µM 10panx1, 500 µM
probenecid (Prob), 200 µM La3+, 200 µM Gap26 or 1:500 Cx43E2. *** p < 0.001, 10 mM
glucose treatment compared to control; ## p < 0.005, effect of 10 mM glucose treatment
compared to blockers. Averaged data were obtained from at least four independent
experiments. Scale bar = 35 µm.

Figure 8. ATP increases the intracellular free Ca2+ signal in tanycytes via P2Y receptors
(A) Representative plots of relative changes in Ca2+ signal over time in cultured rat
tanycytes subjected to 100 µM ATP under control conditions or pretreated with MRS21. (B)
Averaged data normalized to control (dashed line) of maximal Fura-2 fluorescence intensity
during the peak in tanycytes exposed to 100 µM ATP alone or in combination with the
followed blockers: 200 µM oxidized ATP (oATP), 10 µM brilliant blue G (BBG), 200 µM
suramine (Sur), 10 µM MRS2179, 2 µM thapsigargin (TG) 5 µM xestospongin (XeC) and 5
µM xestospongin (XeB). * p < 0.001, 100 µM ATP treatment compared to control; # p <
0.001, 100 µM ATP treatment compared to blockers.

Figure 9. Model of glucosensing mechanism in tanycytes

Upon a rise in extracellular glucose concentration, glucose enters (1) mainly through GLUTs
and to a minor extent via Cx43 hemichannels, leading to glucokinase-dependent
phosphorylation of glucose (2). The ATP generated during glycolysis (3) via processing of
glucose-derived substrates causes closure of KATP channels (4). This event promotes by an
unknown mechanism the opening of Cx43 hemichannels (5). The ATP released via Cx43
hemichannels (6) activates P2Y receptors (7) and induces formation of cytoplasmic inositol
(1,4,5)-trisphosphate (IP3), which promotes the release of Ca2+ stored in the endoplasmic
reticulum (8), raising the [Ca2+]i (9).

Glucose Increases Intracellular Free Ca (2+) in Tanycytes Via ATP Released Through Connexin 43 Hemichannels

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Glia. 2012 January ; 60(1): 53–68. doi:10.1002/glia.21246.

Glucose increases intracellular free Ca2+ in tanycytes via ATP

‡Departamento de Biología Celular, Universidad de Concepción, Concepción, Chile

Both α and β tanycytes express several glucose-sensing molecules, including glucose

Material and Methods

Glia. Author manuscript; available in PMC 2013 January 01.

(MEM), DNAse I, poly-L-lysine, water (W3500), La3+, ethidium (Etd) bromide,

Intracellular calcium imaging

Glia. Author manuscript; available in PMC 2013 January 01.

Immunofluorescence and confocal microscopy

using confocal laser microscopy (D-Eclipse C1 Nikon, Tokyo, Japan).

Dye uptake and time-lapse fluorescence imaging

Glia. Author manuscript; available in PMC 2013 January 01.

Western blot analysis

orthovanadate, 10 mM α-glycerophosphate) and a complete miniprotease inhibitor (Roche

Nonspecific protein binding was blocked by incubation of nitrocellulose sheets in PBS-

Cell surface biotinylation and quantitation

Glucose transport assay

Glia. Author manuscript; available in PMC 2013 January 01.

Measurement of extracellular ATP concentration

Data analysis and statistics

Glia. Author manuscript; available in PMC 2013 January 01.

Because glucosensing by specialized cells, such as pancreatic β-cells, involves GLUT2, GK

The presence of connexin hemichannels in cultured tanycytes was supported by their

Glia. Author manuscript; available in PMC 2013 January 01.

Glucose enhances the Cx43 hemichannel activity via a glucokinase/KATP channel-

Glia. Author manuscript; available in PMC 2013 January 01.

from −80 to +80 mV were assessed (Fig. 5A).

presented a reversal potential close +5 mV (Fig. 5B). Moreover, 10 mM glucose increased

Glia. Author manuscript; available in PMC 2013 January 01.

not affected (Fig. 5E).

Tanycytes are permeable to glucose via GLUTs and Cx43 hemichannels

Glia. Author manuscript; available in PMC 2013 January 01.

extracellular ATP as mediator of the glucose-induced rise in [Ca2+]i.

Recently, glucose and non-metabolizable analogues of glucose were demonstrated to

Glia. Author manuscript; available in PMC 2013 January 01.

molecular inhibition of GK or KATP channels impairs feeding behavior in rodents (Miki et

Intracellular ATP generated by anaerobic glycolysis but not produced by oxidative

Glia. Author manuscript; available in PMC 2013 January 01.

Glia. Author manuscript; available in PMC 2013 January 01.

Influx/Efflux in Tanycytes Involved in Glia-Neuron Metabolic. Plos one. 2011; 6(1):e16411.

96:121–155. [PubMed: 2416706]

Glia. Author manuscript; available in PMC 2013 January 01.

after stimulation of hypothalamic P2Y1 receptors in rats: modulation of feeding behaviour by

Colocalization of glucagon-like peptide-1 (GLP-1) receptors, glucose transporter GLUT-2, and

Glia. Author manuscript; available in PMC 2013 January 01.

Physiol Cell Physiol. 2008; 295(3):C761–C767. [PubMed: 18596212]

Glia. Author manuscript; available in PMC 2013 January 01.

Figure 1. Immunocytochemistry characterization of cultured tanycytes

Glia. Author manuscript; available in PMC 2013 January 01.

Glia. Author manuscript; available in PMC 2013 January 01.

O-ethylidene-D-glucose (ETDG), 500 µM alloxan, 1 mM iodoacetate (IA), 200 nM AA

Glia. Author manuscript; available in PMC 2013 January 01.

Glia. Author manuscript; available in PMC 2013 January 01.

subjected to changes in glucose concentrations (horizontal bars, from 2 to 10 mM glucose)

to 10 mM glucose alone or in combination with the following blockers: 200 µM 10panx1,

Glia. Author manuscript; available in PMC 2013 January 01.

Figure 4. Increased Etd uptake induced by glucose is mediated by a glucokinase/KATP channel-

Glia. Author manuscript; available in PMC 2013 January 01.

Glia. Author manuscript; available in PMC 2013 January 01.

Glia. Author manuscript; available in PMC 2013 January 01.

Glia. Author manuscript; available in PMC 2013 January 01.