Cellular/Molecular

Brain-Derived Neurotrophic Factor Stimulates Energy
Metabolism in Developing Cortical Neurons

Julia Burkhalter, Hubert Fiumelli, Igor Allaman, Jean-Yves Chatton, and Jean-Luc Martin
Institute of Physiology, Faculty of Medicine, University of Lausanne, CH-1005 Lausanne, Switzerland

Brain-derived neurotrophic factor (BDNF) promotes the biochemical and morphological differentiation of selective populations of
neurons during development. In this study we examined the energy requirements associated with the effects of BDNF on neuronal
differentiation. Because glucose is the preferred energy substrate in the brain, the effect of BDNF on glucose utilization was investigated
in developing cortical neurons via biochemical and imaging studies. Results revealed that BDNF increases glucose utilization and the
expression of the neuronal glucose transporter GLUT3. Stimulation of glucose utilization by BDNF was shown to result from the activa-
tion of Na �/K �-ATPase via an increase in Na � influx that is mediated, at least in part, by the stimulation of Na�-dependent amino acid
transport. The increased Na �-dependent amino acid uptake by BDNF is followed by an enhancement of overall protein synthesis
associated with the differentiation of cortical neurons. Together, these data demonstrate the ability of BDNF to stimulate glucose
utilization in response to an enhanced energy demand resulting from increases in amino acid uptake and protein synthesis associated
with the promotion of neuronal differentiation by BDNF.

Key words: BDNF; glucose utilization; 2-deoxyglucose; Na� influx; amino acid uptake; protein synthesis; GLUT3

Introduction
Brain-derived neurotrophic factor (BDNF) belongs to a family of
closely related neurotrophic factors termed neurotrophins
(Lewin and Barde, 1996). There is ample evidence that BDNF
regulates the survival and differentiation of selective populations
of neurons in the peripheral and central nervous systems (Davies,
1994; Jones et al., 1994; Lewin and Barde, 1996). Thus BDNF
knock-out mice exhibit substantially reduced numbers of cranial
and spinal sensory neurons as well as marked decreases in the
expression of neuropeptide Y (NPY) and calcium-binding pro-
teins in specific brain regions, including cerebral cortex and hip-
pocampus (Jones et al., 1994). It is also well established that
BDNF increases peptidergic differentiation of GABA-containing
neurons as it stimulates the expression of specific neuropeptides
such as somatostatin, substance P, NPY, and cholecystokinin
both in vitro and in vivo (Nawa et al., 1993; Croll et al., 1994). In
the CNS BDNF promotes structural changes in axonal and den-
dritic arborization (McAllister et al., 1999). Thus BDNF increases
the length and complexity of dendrites of cortical pyramidal neu-
rons (McAllister et al., 1995).

Besides these actions on neuronal survival and differentiation,
BDNF also modulates both short-term and long-term synaptic
transmission (Schuman, 1999). For instance, hippocampal long-

term potentiation (LTP) is impaired markedly in BDNF knock-
out mice (Korte et al., 1995; Patterson et al., 1996), whereas reex-
pression of BDNF in the CA1 region (Korte et al., 1996) or
treatment with recombinant BDNF restores LTP (Patterson et al.,
1996). Recently, BDNF has been shown to evoke rapid neuronal
depolarization by activating the tetrodotoxin-insensitive sodium
channel Nav1.9 (Kafitz et al., 1999; Blum et al., 2002).

In the mammalian brain �50% of the total energy consump-
tion is associated with neural signaling, including maintenance of
resting potentials and neurotransmitter recycling (Attwell and
Laughlin, 2001). Indeed, the majority of energy consumption is
used to restore the ion gradients and resting membrane potentials
by Na�/K�-ATPase, a plasma membrane enzyme that catalyzes
the active transport of Na� and K� across the cell membrane
(Astrup et al., 1981). In addition to the energy expenditure linked
to neural signaling, a lower fraction of energy consumption is
used to sustain basic cellular processes that are not coupled
tightly to neural signaling, such as turnover of macromolecules
(proteins, oligonucleotides, lipids), axoplasmic transport, and
mitochondrial proton leak (Attwell and Laughlin, 2001). In the
brain, glucose is the predominant substrate for energy metabo-
lism (Sokoloff, 1960). Delivery of glucose from the blood to the
brain requires its transport across the endothelial cells of the
blood– brain barrier and the plasma membrane of neurons and
glia. The entry of glucose into these cells is mediated by two
facilitative glucose transporter (GLUT) isoforms i.e., GLUT1 and
GLUT3. GLUT1 is the major glucose transporter in the blood–
brain barrier, choroid plexus, ependyma, and glia, whereas
GLUT3 is the neuronal glucose transporter (Maher et al., 1994;
Vannucci et al., 1997). Cerebral glucose utilization increases dur-
ing development of the human, mouse, and rat brain (Chugani
and Phelps, 1986; Nehlig et al., 1988; Khan et al., 1999). Thus

Received Feb. 4, 2003; revised May 21, 2003; accepted May 21, 2003.
This work was supported by Swiss National Science Foundation Grant 3200-061364.00 (to J.-L.M.). We thank Dr.

M. Kiraly for stimulating discussions, Professor B. Thorens for the generous gift of the anti-GLUT8 antibody, and Dr.
D. Jabaudon for preliminary experiments.

Correspondence should be addressed to Jean-Luc Martin, Institute of Physiology, Faculty of Medicine, University
of Lausanne, 7 Rue du Bugnon, CH-1005 Lausanne, Switzerland. E-mail: Jean-Luc.Martin@iphysiol.unil.ch.

H. Fiumelli’s present address: Department of Molecular and Cell Biology, University of California at Berkeley,
Berkeley, CA 94720.
Copyright © 2003 Society for Neuroscience 0270-6474/03/238212-09$15.00/0

8212 • The Journal of Neuroscience, September 10, 2003 • 23(23):8212– 8220



cerebral glucose utilization increases by 50 –100% in most re-
gions of the rat brain from postnatal day (P) 17 to P21 (Nehlig et
al., 1988). Enhancement of glucose utilization during postnatal
brain development relates to stimulation of neuronal differenti-
ation and increases in neuronal activity (Nehlig et al., 1988; Van-
nucci et al., 1998). The interesting observation in the rat visual
cortex revealing that the expression of BDNF is increased mark-
edly during the same developmental period (Bozzi et al., 1995;
Schoups et al., 1995; Tropea et al., 2001) led us to examine
whether BDNF regulates glucose utilization in developing corti-
cal neurons. In this study we show that BDNF increases glucose
utilization and GLUT3 expression in cortical neurons. We also
provide evidence that this effect results from the activation of
Na�/K�-ATPase by a mechanism that involves the stimulation
of Na�-dependent amino acid uptake and the increase in intra-
cellular Na� concentration.

Materials and Methods
Preparation of primary cultures of mouse cortical neurons. All experiments
were performed in accordance with the European Communities Council
Directive regarding the care and use of animals for experimental
procedures.

Primary cultures of cerebral cortical neurons were prepared from 17-
d-old Swiss mice embryos as previously described (Fiumelli et al., 1999)
with minor modifications. Cells were plated at a density of 2 � 10 5/cm 2

on 60 � 15 mm (Northern blots) or on 35 � 10 mm (2-DG uptake,
Western blots, protein synthesis, [Na �]i measurements and [ 14C]-AIB
uptake) culture plates in Neurobasal medium supplemented with B27
(Invitrogen, Basel, Switzerland) and 500 �M glutamine. For immunocy-
tochemical studies the cultures were plated at a density of 105/cm 2 on
glass coverslips in 24-well cell culture clusters in the above-mentioned
medium. Cells were maintained at 37°C in a humidified atmosphere of
95% air/5% CO2 and were subjected to experiments after 6 d in vitro.

[ 3H]2-deoxyglucose uptake. [ 3H]2-deoxyglucose ([ 3H]2-DG) uptake
was determined as previously described (Yu et al., 1993) with minor
modifications. The culture medium was replaced by 2 ml of DMEM
(5030, Sigma, Buchs, Switzerland) supplemented with (in mM) 5 glucose,
7.5 NaHCO3, 5 HEPES buffer, pH 7.0, and 0.045 phenol red (DMEM5).
After 1 hr of stabilization at 37°C in a water-saturated atmosphere con-
taining 5% CO2/95% air, cortical neurons were exposed to BDNF
(Alomone Labs, Jerusalem, Israel) for 2 hr (except for time course anal-
ysis). When tested, 200 nM K252a (Biomol, Wangen, Switzerland), 50 �M

PD98059 (Tocris, Wangen, Switzerland), 10 �M LY294002 (Calbiochem,
Lucerne, Switzerland), 10 �M U73122 (Calbiochem), 10 �M U73343
(Sigma), 10 nM–1 �M ouabain (Sigma), 1 �M TTX (Latoxan, Lucerne,
Switzerland), 1 �M MK-801 (Tocris), and 100 �M CNQX (Calbiochem)
were added 20 min before BDNF. After replacement of the medium by 2
ml of DMEM5 containing [ 3H]2-DG (American Radiolabeled Chemi-
cals, Wangen, Switzerland) at a tracer concentration of 1 �Ci/ml (33 nM),
cortical neurons were incubated for 20 min at 37°C. [ 3H]2-DG uptake
was stopped by aspiration of the medium; the cells were rinsed three
times with ice-cold PBS, pH 7.4, and 2 ml of 10 mM NaOH/0.1% Triton
X-100 was added to lyse the cells. Aliquots of 500 �l were assayed for
radioactivity by liquid scintillation counting, whereas 30 �l aliquots were
used for measurement of protein content by a BCA protein assay kit
(Pierce, Lausanne, Switzerland). Results, which represent glucose
transporter-mediated uptake and subsequent phosphorylation by hex-
okinase, were calculated by subtracting from total counts the portion that
was not inhibited by 50 �M of the glucose transporter inhibitor cytocha-
lasin B (Sigma). The cytochalasin B-sensitive uptake accounted for
�70% of total [ 3H]2-DG uptake. For Na � replacement experiments an
HEPES-buffered salt solution with the following composition (in mM)
was used: 10 HEPES, pH 7.0, 7.5 NaHCO3, 2 KCl, 2 CaCl2, 3 MgSO4, 2
KH2PO4, 5 glucose, 0.045 phenol red, and 140 NaCl. For experiments
performed in low extracellular Na �, the NaCl was replaced by an
equimolar concentration of choline.

As to the kinetic analysis, [ 3H]2-DG uptake was determined in

DMEM (5030, Sigma) supplemented with 7.5 mM NaHCO3, 5 mM

HEPES buffer, pH 7.0, and 0.045 mM phenol red that contained the
appropriate concentration of glucose. For each glucose concentration
[ 3H]2-DG concentration was adjusted to maintain the 2-DG/glucose
concentration ratio constant. Kinetic parameters of glucose transport
were determined by a double-reciprocal plot of [ 3H]2-DG uptake rate
versus extracellular glucose concentration. This Lineweaver–Burk plot
yields a straight line with a slope of Km/Vmax, an x-intercept of –1/Km,
and a y-intercept of 1/Vmax.

Northern blot analysis. Cortical neurons were exposed to BDNF for 2
hr except for the time course analysis in which time varied. At the end of
the stimulation period the cells were washed twice with ice-cold PBS, and
total RNA was extracted from cultured cortical neurons by using Trizol
reagent according to the manufacturer’s instructions (Invitrogen). Then
10 �g of total RNA was electrophoresed on a 1.2% agarose/2 M formal-
dehyde gel and transferred onto nylon membrane (NEN, Geneva, Swit-
zerland) as previously described (Fiumelli et al., 1999). Hybridization
was performed with a 32P-antisense GLUT3 riboprobe. Filters then were
washed under high-stringency conditions (twice with 2� SSC/0.1% SDS
at 65°C for 15 min and twice with 0.1� SSC/0.1% SDS at 65°C for 30
min) and apposed to autoradiography films at –70°C with an intensifying
screen. Differences in RNA gel loading and blotting were assessed
by rehybridizing the filters with a 32P-antisense glyceraldehyde-3-
phosphate-dehydrogenase (GAPDH) riboprobe (Fiumelli et al., 1999).
After autoradiography the films were scanned densitometrically, and the
results were quantified by using the Macintosh-based NIH Image pro-
gram, version 1.61. Densitometric values for GLUT3 mRNA were nor-
malized to corresponding GAPDH mRNA values.

32P-antisense GLUT3 riboprobe was generated by using T7 RNA poly-
merase and 32P-�-UTP from a linearized pGEM-T vector containing an
802 bp GLUT3 cDNA fragment. GLUT3 cDNA fragment was obtained
by reverse transcription and PCR amplification of total RNA from pri-
mary cultures of mouse cortical neurons with a set of oligonucleotide
primers (5�-TTGGTTTGGACTTTATT-3� and 5�-CATCTTCGCTGC-
CTTCCT-3�) located at 594 – 610 and 1378 –1395 bp in the coding region
of the mouse GLUT3 cDNA sequence (Nagamatsu et al., 1992). The
identity of the amplified GLUT3 cDNA fragment was confirmed by DNA
sequencing.

Western blot analysis. Mouse cortical neurons were exposed to 5 ng/ml
BDNF for various periods of time. At the end of the incubation period the
cells were washed twice with ice-cold PBS; lysed by adding 150 �l of 62.5
mM Tris-HCl, pH 6.8, 50 mM DTT, 0.3% SDS supplemented with a
mixture of protease inhibitors (Sigma); heated for 5 min at 95°C; and
sonicated to shear genomic DNA. The sample protein concentration was
measured by the Bio-Rad (Glattbrugg, Switzerland) protein assay. Then
10 �g of each sample diluted in 62.5 mM Tris-HCl, pH 6.8, 50 mM DTT,
2% SDS, 10% glycerol, 0.1% bromophenol blue was electrophoresed
through a 12% SDS-polyacrylamide minigel and blotted onto polyvinyli-
dene difluoride membrane (NEN), using a semi-dry blotting system
(Bio-Rad) as previously described (Cardinaux et al., 1997).

For immunodetection of GLUT3 the blots were blocked in 50 mM

Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20 complemented with 5%
skim milk, and 1% BSA for 1 hr at room temperature. The blots subse-
quently were incubated overnight at 4°C in 50 mM Tris-HCl, pH 7.5, 150
mM NaCl, and 0.1% Tween-20 with 1% skim milk in the presence of a
polyclonal rabbit anti-GLUT3 antibody (Chemicon, Lucerne, Switzer-
land) diluted at 1:5000. Immunolabeled GLUT3 was detected via the ECL
detection system (Pharmacia, Dübendorf, Switzerland). Differences in
protein gel loading and blotting were determined by reprobing the blots
with a monoclonal mouse anti-�-tubulin antibody (Sigma) at a dilution
of 1:100. Results were quantified by the Macintosh-based NIH Image
program, version 1.61. Densitometric values for GLUT3 protein were
normalized to corresponding �-tubulin values.

[Na �]i measurements. Na �
i imaging experiments were performed on

the stage of an inverted epifluorescence microscope (Carl Zeiss, Jena,
Germany) and observed through a 40 � 1.3 numerical aperture oil-
immersion Neofluar objective lens (Carl Zeiss) as previously described
(Chatton et al., 2000). Fluorescence excitation wavelengths were selected
via a holographic monochromator (Polychrome II, Till Photonics,

Burkhalter et al. • BDNF Stimulates Energy Metabolism in Cortical Neurons J. Neurosci., September 10, 2003 • 23(23):8212– 8220 • 8213



Planegg, Germany), and fluorescence was detected with a 12-bit cooled
CCD camera (Micromax, Princeton Instruments, Trenton, NJ). Acqui-
sition of images and time series were computer-controlled with the soft-
ware Metafluor (Universal Imaging, West Chester, PA) running on a
Pentium computer. The acquisition rate of ratio images was varied be-
tween 0.5 and 0.1 Hz.

[Na �]i was measured in single neurons after the cells were loaded with
the Na �-sensitive fluorescent dye sodium-binding benzofuran isophta-
late (SBFI-AM, Teflabs, Austin, TX). Cell loading was performed at 37°C,
using 15 �M SBFI-AM in a HEPES-buffered balanced solution with the
following composition (in mM): 135 NaCl, 5.4 KCl, 20 HEPES, 1.3 CaCl2,
0.8 MgSO4, 0.78 NaH2PO4, 20 glucose supplemented with 0.1% Pluronic
F-127 (Molecular Probes, Eugene, OR). Once loaded with SBFI, the neu-
rons were placed in a thermostated perfusion chamber (Harris et al.,
1994) and superfused at 35°C in DMEM5 without phenol red. Fluores-
cence was excited sequentially at 340 and 380 nm and detected at 510 nm
by using a bandpass filter (80 nm bandwidth). Fluorescence excitation
ratios (F340 nm/F380 nm) were computed for each image pixel, and ratio
images of cells proportional to [Na �]i were produced. In situ calibration
was performed after each experiment as previously described (Chatton et
al., 2000). Briefly, cells were permeabilized for monovalent cations with 6
�g/ml gramicidin (Sigma) and 10 �M monensin (Sigma) with simulta-
neous inhibition of the Na �/K �-ATPase by the use of 1 mM ouabain.
Cells then were perfused sequentially with solutions buffered at pH 7.2
with 20 mM HEPES and containing 0, 5, 10, 20, and 50 mM Na �, respec-
tively, plus 30 mM Cl � and 136 mM gluconate with a constant total
concentration of Na � and K � of 165 mM. A five point calibration curve
was computed for each selected cell in the field of view and used
to convert fluorescence ratio values (F340 nm/F380 nm) into Na �

concentrations.
Experiments were repeated on four different coverslips from two in-

dependent cultures. Data are presented as changes in [Na �]i evoked by
10 ng/ml BDNF. At the end of each experiment the cortical neurons were
exposed to 25 �M veratridine to elicit Na � entry through voltage-gated
Na � channels, thereby enabling us to verify the responsiveness of the
neurons. The few neurons that did not respond to veratridine were dis-
carded from the analysis.

[ 14C]-aminoisobutyric acid uptake. [ 14C]-aminoisobutyric acid
([ 14C]-AIB) uptake was performed as follows. Cortical neurons were
exposed to 5 ng/ml BDNF for various time periods in HEPES buffer
containing 140 mM NaCl or choline, respectively. At the end of the stim-
ulation period the neurons were incubated for 20 min at 37°C in the same
buffer, but containing [ 14C]-AIB (specific activity, 51.5 mCi/mmol;
NEN) at a concentration of 0.5 �Ci/ml. [ 14C]-AIB uptake was stopped
by aspirating the medium; the cells were rinsed three times with ice-cold
PBS, pH 7.4, and 2 ml of 10 mM NaOH/0.1% Triton X-100 was added to
lyse the cells. Aliquots of 500 �l were assayed for radioactivity by liquid
scintillation counting, whereas 30 �l aliquots were used for measurement
of protein content by a BCA protein assay kit (Pierce). For Na � replace-
ment experiments a HEPES-buffered salt solution with the following
composition (in mM) was used: 10 HEPES, pH 7.0, 7.5 NaHCO3, 2 KCl,
2 CaCl2, 3 MgSO4, 2 KH2PO4, 5 glucose, and 140 NaCl. For experiments
that were performed in low extracellular Na �, the NaCl was replaced by
an equimolar concentration of choline.

Protein synthesis. Cortical neurons were washed once with DMEM
lacking methionine and cysteine (Invitrogen) complemented with 5 mM

HEPES, pH 7.0, and preincubated for 1 hr in 1 ml of the same medium at
37°C in a humidified atmosphere of 95% air/5% CO2. For 35S radioactive
labeling studies the medium was replaced by 800 �l of a mixture consist-
ing of 99% of the above-mentioned medium and 1% DMEM (5030,
Sigma) containing 0.125 mCi/ml L-[ 35S]methionine and L-[ 35S]cysteine
(met-labeling mix, Hartmann, Braunschweig, Germany; specific activity,
1000 Ci/mmol); BDNF was added immediately thereafter. After incuba-
tion with BDNF for various time periods the cells were washed twice with
ice-cold PBS; lysed with 500 �l of 62.5 mM Tris, pH 6.8, 50 mM DTT, and
0.3% SDS; heated for 5 min at 95°C; and sonicated.

Cell suspension (50 �l) was added to 500 �l of 0.1 mg/ml BSA con-
taining 0.02% NaN3 and placed on ice. To precipitate labeled proteins,
we added 500 �l of ice-cold 20% TCA to the samples, vortex-mixed them

vigorously, and incubated them for 30 min on ice. Suspensions then were
filtered onto glass microfiber disks; the disks were washed twice with
ice-cold 10% TCA and 100% EtOH. Air-dried filters were assayed for
radioactivity by liquid scintillation counting.

Immunocytochemistry. Cortical neurons cultured on glass coverslips
were exposed to 5 ng/ml BDNF for various time periods without chang-
ing the medium. Cells then were fixed in 4% paraformaldehyde for 20
min, washed twice in PBS, and preincubated in PBS containing 0.2%
Triton X-100 (PBS-TX) and 4% normal donkey serum (Milan Analytica,
La Roche, Switzerland) for 1 hr. Immunodetection of NPY was per-
formed as follows: a polyclonal rabbit anti-NPY antibody (Sigma) di-
luted in PBS-TX at 1:5000 was applied overnight. The next day the cul-
tures were rinsed twice with PBS-TX and once with PBS and then treated
with Cy3-conjugated donkey anti-rabbit IgG (Milan Analytica) at a di-
lution of 1:1000 (PBS) for 30 min. All incubations and rinses were per-
formed at room temperature. The coverslips were mounted on slides in
Vectashield (Vector Laboratories, Servion, Switzerland). Cultures were
observed with an Axioplan 2 microscope (Carl Zeiss) equipped with
epifluorescence and appropriate filters.

The percentage of NPY-immunoreactive neurons was determined by
counting the number of labeled cells in a surface identical for control and
treated cultures and corresponding to �30% of the total surface of the
coverslip. The number of cells counted in BDNF-treated cultures was
expressed as a percentage of control cultures. Three coverslips were
quantified at each time period in two independent experiments.

Results
BDNF increases 2-DG uptake in cortical neurons
Regulation of glucose utilization by BDNF was examined in pri-
mary cultures of cortical neurons by using the 2-DG method
(Sokoloff et al., 1977). 2-DG is a glucose analog that has been
shown to be transported specifically by facilitative glucose trans-
porters of the GLUT family, but not by Na�-dependent glucose
transporters (Kimmich and Randles, 1976; Hediger and Rhoads,
1994). Treatment of cortical neurons with BDNF resulted in a
marked increase in 2-DG uptake (Fig. 1). Time course analysis
revealed that 2-DG uptake increased significantly 20 min after
exposure to BDNF (122.2 � 3.9% of control), reached maximal
levels after 2 hr (182.1 � 19.1% of control), and remained ele-
vated up to at least 7 hr of treatment with BDNF (152.1 � 6.1% of
control; Fig. 1A). As shown in Figure 1B, BDNF causes a statis-
tically significant increase in 2-DG uptake at 0.1 ng/ml and elicits
a maximal uptake at 1 ng/ml BDNF. Because cultures of neurons
also contain a few percentages of astrocytes, a glial cell type in-
volved in cerebral glucose metabolism, we investigated the effect
of BDNF on glucose utilization in primary cultures of mouse
cortical astrocytes. In marked contrast to its effect in cortical
neurons, BDNF did not affect glucose utilization in cultured cor-
tical astrocytes (data not shown). BDNF promotes neuronal sur-
vival and differentiation by binding and activating the receptor
tyrosine kinase TrkB (Barbacid, 1994; Bothwell, 1995). To exam-
ine whether stimulation of 2-DG uptake by BDNF resulted from
the activation of the tyrosine kinase TrkB receptor, we tested the
effect of the tyrosine kinase inhibitor K252a. Data presented in
Figure 2A show that BDNF-induced 2-DG uptake is abolished
completely by K252a, consistent with a requirement for TrkB
receptor activation. Binding of BDNF to TrkB produces biologi-
cal responses via activation of three well described intracellular
signaling pathways, including Ras/mitogen-activated protein ki-
nase (MAPK), phospholipase C-� (PLC-�), and phosphatidyl-
inositol 3-kinase (PI3K) (Segal and Greenberg, 1996). To deter-
mine the signaling pathway by which BDNF stimulates 2-DG
uptake, we treated cortical neurons with BDNF in the presence of
inhibitors of the Ras/MAPK, PLC-�, or PI3K pathways. Results
revealed that the PLC inhibitor U73122 completely suppressed

8214 • J. Neurosci., September 10, 2003 • 23(23):8212– 8220 Burkhalter et al. • BDNF Stimulates Energy Metabolism in Cortical Neurons



the effect of BDNF on 2-DG uptake, whereas the inactive analog
U73343 was ineffective (Fig. 2C). In contrast to U73122, the
MAPK kinase inhibitor PD98059 and the PI3K inhibitor
LY294002 did not prevent the BDNF-induced 2-DG uptake (Fig.
2B). Together, these data indicate that stimulation of glucose
utilization by BDNF requires the activation of PLC-�.

BDNF stimulates GLUT3 expression
To characterize further the increased glucose utilization by
BDNF, we determined the rate of 2-DG uptake as a function of
increasing concentrations of extracellular glucose in control and
BDNF-treated cortical neurons. As shown in Figure 3A, the rates
of 2-DG uptake in control and BDNF-treated cultures exhibit
typical Michaelis–Menten kinetics. Double-reciprocal plots of
2-DG uptake rate versus extracellular glucose concentration
(Lineweaver–Burk plots) were used to determine kinetic param-
eters of 2-DG uptake in control and BDNF-treated cultures. The
maximal velocity (Vmax) of 2-DG uptake increased significantly
from 37.7 � 4.3 fmol/mg of protein/min to 55.3 � 3.9 fmol/mg of
protein/min ( p � 0.05, unpaired Student’s t test) when cortical
neurons were exposed to BDNF (Fig. 3B). In contrast, the
Michaelis–Menten constant (Km) did not change significantly
after treatment with BDNF because Km was 0.36 � 0.1 mM in
control neurons and 0.39 � 0.16 mM in BDNF-treated neurons
(Fig. 3B). The increased Vmax value of 2-DG uptake in response to
BDNF suggests that BDNF enhances the number of functional

glucose transporters in the plasma membrane of cortical neu-
rons. Because GLUT3 is the predominant glucose transporter
expressed in neurons (Maher et al., 1991; Nagamatsu et al., 1992),
we examined the regulation of its expression by BDNF. Treat-
ment of cultured cortical neurons with BDNF caused an upregu-
lation of GLUT3 mRNA expression in a time- and concentration-
dependent manner (Fig. 4A–D). Thus GLUT3 mRNA levels
transiently increased, reaching maximal levels after 2 hr and re-
turning to control values 7 hr after stimulation by BDNF (Fig.
4A,C). Induction of GLUT3 mRNA expression was dependent
on the concentration of BDNF, with a maximal rise achieved at a
concentration of 3 ng/ml (Fig. 4B,D). The transient increase in
GLUT3 mRNA level by BDNF was accompanied by a sustained
elevation of GLUT3 protein expression, as revealed by Western
blot analysis (Fig. 4E,F). Subcellular fractionation of cortical
neurons followed by Western blot analysis showed that the large
majority (�95%) of GLUT3 protein was associated with plasma
membranes and did not reveal a translocation of GLUT3 from
intracellular stores to the plasma membrane of cortical neurons
in response to BDNF (data not shown). Because there are several
lines of evidence that GLUT1 and GLUT8 may be expressed in
neurons (Maher and Simpson, 1994; Ibberson et al., 2002), we
examined the regulation of GLUT1 and GLUT8 expression by

Figure 1. BDNF increases [ 3H]2-DG uptake in cortical neurons. A, Time course analysis.
Cultures of cortical neurons were stimulated by 5 ng/ml BDNF for various periods of time.
[ 3H]2-DG uptake was measured as described in Materials and Methods. Data are the mean �
SEM percentages of control (Ctrl) values of triplicate determinations from three independent
experiments. *p � 0.05, **p � 0.01 by unpaired Student’s t test between Ctrl and BDNF-
treated cultures. B, Concentration dependency. Cultures of cortical neurons were treated for 2 hr
with increasing concentrations of BDNF. [ 3H]2-DG uptake was measured as described in Mate-
rials and Methods. Data are the mean � SEM percentages of Ctrl values of triplicate determi-
nations from three independent experiments. **p � 0.01, ***p � 0.001 by unpaired Stu-
dent’s t test between Ctrl and BDNF-treated cultures.

Figure 2. Effects of inhibitors of TrkB-mediated signaling pathways on BDNF-induced
[ 3H]2-DG uptake. Cultures of cortical neurons were exposed to 5 ng/ml BDNF for 2 hr in the
presence of different inhibitors. Inhibitors also were tested on basal [ 3H]2-DG uptake. A, Effect
of the tyrosine kinase inhibitor K252a (200 nM) on BDNF-induced [ 3H]2-DG uptake. Data are the
mean � SEM percentages of Ctrl values of triplicate determinations from three independent
experiments. ***p � 0.001 by unpaired Student’s t test between BDNF- and BDNF � K252a-
treated cultures. B, Effects of the MAPK kinase inhibitor PD98059 (50 �M) and the PI3K inhibitor
LY294002 (10 �M) on BDNF-induced [ 3H]2-DG uptake. Data are the mean � SEM percentages
of Ctrl values of triplicate determinations from at least three independent experiments. C, Ef-
fects of the PLC inhibitor U73122 (10 �M) and of its inactive analog U73343 (10 �M) on BDNF-
stimulated [ 3H]2-DG uptake. Data represent the mean � SEM percentages of Ctrl values of
triplicate determinations from at least three independent experiments. ***p � 0.001 by un-
paired Student’s t test between BDNF- and BDNF � U73122-treated cultures.

Burkhalter et al. • BDNF Stimulates Energy Metabolism in Cortical Neurons J. Neurosci., September 10, 2003 • 23(23):8212– 8220 • 8215



BDNF. Western blot analysis revealed that in cultured cortical
neurons GLUT1 was barely detectable and GLUT8 was not de-
tected and that the expression of these two facilitative glucose
transporters was not affected by BDNF (data not shown).

Role of Na �/K �-ATPase in the stimulation of glucose
utilization by BDNF
Because glucose uptake and phosphorylation are linked directly
to glucose consumption, it was of interest to determine the origin
of the energy-consuming processes activated by BDNF. The cost-
liest energy-consuming process of the brain, which represents
�50% of the total cerebral energy consumption, is linked to the
active transport of Na� and K� through Na�/K�-ATPase, a
pump that maintains the intracellular concentrations of Na� and
K� ions in animal cells (Ames, 2000). To study the involvement
of Na�/K�-ATPase in the stimulation of glucose utilization by
BDNF, we pretreated cortical neurons for 20 min with increasing
concentrations of ouabain, a specific inhibitor of Na�/K�-
ATPase. As shown in Figure 5A, the BDNF-induced 2-DG uptake
was blocked almost completely by 1 �M ouabain (75% inhibi-
tion), indicating that activation of the Na�/K�-ATPase accounts
for this large increase in glucose utilization. In addition, partial
replacement of extracellular Na� with the impermeant cation
choline completely abolished the stimulation of 2-DG uptake by
BDNF, indicating that this effect is dependent on extracellular
Na� (Fig. 5B).

BDNF increases Na � influx in cortical neurons
On the basis of these data, we addressed the question of whether
BDNF enhanced Na� influx in cortical neurons. Studies that
used fluorescence Na� imaging revealed that treatment of corti-
cal neurons with BDNF caused a rise in intracellular Na� con-
centration ([Na�]i) with a latency of 13.4 � 1.2 min (n 	 22) in
60% of the neurons that were studied (Fig. 6). Na� enters cells by
many routes, including voltage-dependent Na� channels [e.g.,
tetrodotoxin (TTX)-sensitive Na� channels], ligand-gated Na�

channels (e.g., glutamate-gated ionotropic channels), and differ-
ent Na�-dependent transporters (e.g., Na�-dependent amino
acid transporters). To investigate the route through which BDNF
stimulates Na� influx and thereby increases glucose utilization,
we pretreated cortical neurons with the Na� channel blocker
TTX, the NMDA receptor antagonist MK-801, or the AMPA/

kainate receptor antagonist CNQX; glucose utilization was deter-
mined by the 2-DG method. Data showed that application of
TTX, MK-801, and CNQX did not reduce 2-DG uptake stimu-
lated by 5 ng/ml BDNF (Ctrl 	 100 � 1.67%, BDNF 	 137.44 �
3.41%, BDNF�TTX 	 131.98 � 5.53%, BDNF�MK-801 	
131.99 � 5.61%, BDNF�CNQX 	 128.96 � 6.06%; n 	 6).
These results rule out the involvement of TTX-sensitive Na�

channels and ionotropic glutamate receptors in the stimulation
of glucose utilization by BDNF.

BDNF increases Na �-dependent amino acid transport
To investigate further the route of entry of Na� ions, we exam-
ined whether BDNF stimulated amino acid transport in cortical
neurons. Amino acid transport across plasma membranes is me-

Figure 3. Kinetic analysis of the increase in [ 3H]2-DG uptake by BDNF. Cultures of cortical
neurons were treated for 2 hr with 5 ng/ml BDNF in DMEM containing increasing concentrations
of glucose. A, Rate of [ 3H]2-DG uptake as a function of increasing concentrations of glucose in
control (E) or BDNF-treated (F) neurons. Data are the mean � SEM values of triplicate deter-
minations from three independent experiments. B, Double-reciprocal plots of the results pre-
sented in A. Lineweaver–Burk plots yield straight lines, where x- and y-intercepts are –1/Km

and 1/Vmax , respectively.

Figure 4. BDNF stimulates GLUT3 mRNA and protein expression in cortical neurons. A, Time
course analysis of GLUT3 mRNA changes induced by BDNF. Cultures of cortical neurons were
exposed for various periods of time to 5 ng/ml BDNF. Extractions of total RNA and Northern blot
analysis were performed as described in Materials and Methods. B, Concentration dependency
of GLUT3 mRNA changes in response to BDNF. Cultures of cortical neurons were treated for 2 hr
with increasing concentrations of BDNF. C, D, Quantitative analysis of the results presented in A
and B. Data are the mean � SEM percentages of Ctrl values from three independent experi-
ments. E, Time course analysis of GLUT3 protein changes in response to BDNF. Cultures of
cortical neurons were exposed for various periods of time to 5 ng/ml BDNF. Western blot anal-
ysis was performed as described in Materials and Methods. F, Quantitative analysis of the results
presented in E.

8216 • J. Neurosci., September 10, 2003 • 23(23):8212– 8220 Burkhalter et al. • BDNF Stimulates Energy Metabolism in Cortical Neurons



diated via Na�-dependent and Na�-independent transporters
(Bode, 2001). Na�-dependent transporters use the transmem-
brane Na� electrochemical gradient, maintained mainly by
Na�/K�-ATPase, to drive the uptake of amino acids against their
concentration gradient (Bode, 2001). On the basis of these obser-
vations, we hypothesized that stimulation of Na�-dependent
amino acid uptake by BDNF could contribute to the increased
[Na�]i and thereby to the stimulation of Na�/K�-ATPase and
glucose utilization observed in this study. To address this issue,
we examined whether BDNF enhanced the uptake of [ 14C]-AIB,
a non-metabolizable amino acid analog, which has affinity for
both Na�-dependent and Na�-independent amino acid trans-
porters (Brookes, 1988). Data showed that BDNF increased
[ 14C]-AIB uptake in cortical neurons (Fig. 7). Time course anal-
ysis revealed that [ 14C]-AIB uptake was increased significantly
after 20 min and reached maximal levels (175 � 5.3% of control)
after 5 hr of exposure to BDNF (Fig. 7). Partial substitution of
extracellular Na� with choline almost completely inhibited basal
[ 14C]-AIB uptake (�90% inhibition) and abolished the in-
creased [ 14C]-AIB uptake in response to BDNF (Fig. 7). The time
course of [ 14C]-AIB uptake by BDNF indicates that stimulation
of Na�-dependent amino acid uptake contributes to the in-
creased [Na�]i (Figs. 6, 7).

BDNF increases overall protein synthesis
Because stimulation of amino acid transport is required for in-
creased protein synthesis, it was important to examine the effect
of BDNF on overall protein synthesis. Results showed that BDNF
caused a time-dependent rise in the incorporation of [ 35S]methi-
onine and [ 35S]cysteine into newly synthesized proteins (Fig.
8A). A significant increase in [ 35S]methionine and [ 35S]cysteine
incorporation was measured 5 hr after stimulation by BDNF
(124.1 � 4.3% of control), and maximal incorporation was ob-
served after 7 hr (132.7 � 6.1% of control; Fig. 8A). Because
cortical neurons used throughout this study are postmitotic, the
enhanced overall protein synthesis by BDNF must reflect the

activation of mechanisms associated with neuronal differentia-
tion. In agreement with this, BDNF was shown to promote dif-
ferentiation of cortical neurons (Nawa et al., 1993; Takei et al.,
1997; Fiumelli et al., 2000). These effects were accompanied by

Figure 5. Stimulation of [ 3H]2-DG uptake by BDNF is inhibited by applying ouabain and by
lowering the extracellular Na � concentration. A, Cultures of cortical neurons were stimulated
for 2 hr by 5 ng/ml BDNF in the presence of increasing concentrations of the Na �/K �-ATPase
inhibitor ouabain. Ouabain also was tested on basal [ 3H]2-DG uptake. Data represent the
mean � SEM percentages of Ctrl values of triplicate determinations from two independent
experiments. ***p � 0.001 by unpaired Student’s t test between cultures treated with BDNF
and BDNF � 1 �M ouabain. B, Cultures of cortical neurons were exposed for 2 hr to 5 ng/ml
BDNF in a medium containing 140 mM NaCl or 140 mM choline, respectively. Data represent the
mean � SEM percentages of Ctrl values of triplicate determinations from two independent
experiments. ***p � 0.001 by unpaired Student’s t test between Ctrl and BDNF-treated
cultures.

Figure 6. BDNF increases the intracellular Na � concentration ([Na �]i ) in cortical neurons.
A, Images of SBFI-loaded neurons showing a raw fluorescence image excited at 380 nm (top
left) and false color excitation ratio images (340/380 nm) recorded under control conditions
(top right) after 15 min (bottom left) and 30 min (bottom right), respectively, of treatment with
10 ng/ml BDNF. Scale bar, 20 �m. B, Changes in [Na �]i were measured in SBFI-AM-loaded
neurons stimulated by 10 ng/ml BDNF. At the indicated time points [Na �]i values of 13–22
neurons from four independent experiments were averaged and presented as the mean � SEM
values. *p � 0.05, **p � 0.001 by paired Student’s t test between t 	 0 min and t 	 2– 40
min after treatment with BDNF.

Figure 7. BDNF increases Na �-dependent [ 14C]-AIB uptake in cortical neurons. Cultures of corti-
cal neurons were exposed for different time periods to 5 ng/ml BDNF in a medium containing either
140 mM NaCl to determine total [ 14C]-AIB uptake or 140 mM choline to determine Na �-independent
[ 14C]-AIB uptake. [ 14C]-AIB uptake was determined as described in Materials and Methods. Data are
the mean � SEM values of six determinations from two independent experiments. *p � 0.05,
***p � 0.001 by unpaired Student’s t test between Ctrl and BDNF-treated cultures.

Burkhalter et al. • BDNF Stimulates Energy Metabolism in Cortical Neurons J. Neurosci., September 10, 2003 • 23(23):8212– 8220 • 8217



the increased synthesis of certain proteins such as calcium-
binding proteins (Fiumelli et al., 2000), synaptic vesicle proteins
(Takei et al., 1997), and neuropeptides (Nawa et al., 1993).
Among the neuropeptides NPY was reported to be induced after
several days of treatment with BDNF (Nawa et al., 1993). This led
us to examine the expression of NPY after acute treatment with
BDNF. Immunocytochemical analysis revealed an early increase
in the number of NPY-positive neurons resulting in a fivefold
elevation of the number of NPYergic neurons after 7 hr of stim-
ulation by BDNF (Fig. 8B).

Discussion
The present study provides evidence that BDNF increases Na�-
dependent amino acid transport, causing a rise in [Na�]i that in
turn leads to the activation of Na�/K�-ATPase, energy con-
sumption, and the stimulation of glucose uptake and GLUT3
expression. The increased Na�-dependent amino acid transport
ensures an adequate substrate supply for the enhanced protein
synthesis induced by BDNF.

These data demonstrate for the first time that BDNF increases
energy metabolism in developing CNS neurons. NGF, another
member of the neurotrophin family, was shown previously to
increase glucose utilization in PC12 cells (Morelli et al., 1986).
However, the effect of NGF on glucose utilization was observed
�2 d after chronic exposure to this neurotrophin, contrasting
significantly with the time course of the BDNF effect reported in
this study, in which a significant stimulation of glucose utiliza-
tion was detected 20 min after exposure to BDNF (Fig. 1A).

Northern and Western blot analyses showed that stimulation
of glucose utilization by BDNF was accompanied by an increase
in GLUT3 mRNA and protein levels (Fig. 4). However, time
course analysis of GLUT3 induction by BDNF (Fig. 4E,F) sug-
gests that the upregulation of GLUT3 protein expression does not
contribute to the stimulation of glucose utilization during the
first hour of treatment with BDNF. It also is interesting to note
that, although GLUT3 expression markedly increases after 2 hr of
stimulation by BDNF (Fig. 4), glucose utilization remains rela-
tively constant (Fig. 1A). A possible explanation for this observa-

tion is that 2-DG uptake is determined by the rates of both glu-
cose transport and glucose phosphorylation (Whitesell et al.,
1995). In addition, glucose phosphorylation has been shown to
be the rate-limiting step for glucose utilization in the brain
(Whitesell et al., 1995), suggesting that stimulation of GLUT3
expression by BDNF may not be coupled to a proportional in-
crease in glucose phosphorylation.

In the brain �50% of the total energy consumption is used to
restore ion gradients and resting membrane potentials by Na�/
K�-ATPase (Ames, 2000). Our findings of an almost complete
inhibition of BDNF-induced 2-DG uptake by ouabain are sup-
portive of the view that the increased glucose demand is linked
directly to the activation of Na�/K�-ATPase.

Our data also show that activation of Na�/K�-ATPase by
BDNF results from an increase in [Na�]i (Fig. 6). The similar
onsets of increases in Na� influx (Fig. 6) and [ 14C]-AIB uptake
(Fig. 7) strongly support the involvement of Na�-dependent
amino acid transport in the elevation of [Na�]i. The relative
contribution of this mechanism to the total increase in [Na�]i

could not be determined because no high-affinity inhibitor for
Na�-dependent amino acid transporters is presently available
(Palacin et al., 1998). Therefore, the implication of other Na�-
dependent transport systems in the increased glucose utilization
by BDNF cannot be ruled out. Because glucose also might be
transported by Na�-dependent glucose transporters such as
SGLT1 (Poppe et al., 1997), we examined the regulation of Na�-
dependent glucose transport activity by BDNF. However, our
results ruled out the implication of this Na�-dependent trans-
port system in the increased [Na�]i and glucose utilization in
response to BDNF (data not shown). Together, these results re-
veal a novel mechanism by which BDNF stimulates Na� influx in
CNS neurons. The latency (13.4 � 1.2 min; n 	 22) of the in-
crease in [Na�]i in response to BDNF contrasts with the recently
described Na� current activated by BDNF within milliseconds
(Kafitz et al., 1999). The latency observed in this study might be
explained by the translocation of amino acid transporters to the
plasma membrane of cortical neurons. In support of this hypoth-
esis is the recent observation that insulin, a hormone that also
activates receptor tyrosine kinase, promotes the recruitment of
SAT2, a Na�-dependent amino acid transporter, from an endo-
somal compartment to the plasma membrane of skeletal muscle
cells (Hyde et al., 2002) with a latency consistent with the en-
hanced [Na�]i observed in our study.

The increase in Na�-dependent amino acid uptake by BDNF
reported in this study precedes the enhancement of protein syn-
thesis (Figs. 7, 8A). Thus a significant increase in [ 14C]-AIB up-
take is observed after 20 min of exposure to BDNF, whereas a
significant enhancement of [ 35S]methionine and [ 35S]cysteine
incorporation into newly synthesized proteins requires 5 hr of
stimulation by BDNF (Figs. 7, 8A). Our data suggest that BDNF
acts by a concerted mechanism to induce an early entry of amino
acids and a delayed enhancement of protein synthesis. The early
entry of amino acids in response to BDNF ensures an adequate
substrate supply for the increased protein synthesis, as previously
shown for insulin (DeFronzo and Ferrannini, 1992). Among the
proteins induced by BDNF with a time course similar to the
stimulation of overall protein synthesis, we found an early in-
crease in NPY expression (Fig. 8B). These data contrast with
previous studies reporting an increased NPY expression in cul-
tured cortical neurons after several days of treatment with BDNF
(Nawa et al., 1993; Barnea et al., 1995; Takei et al., 1996). The
increased number of NPYergic neurons by BDNF observed in
our study (Fig. 8B) results from the enhancement of NPY mRNA

Figure 8. BDNF increases overall protein synthesis and the number of NPY-positive neurons.
A, Cultures of cortical neurons were stimulated for different time periods by 5 ng/ml BDNF.
Overall protein synthesis was determined by measuring the incorporation of [ 35S]methionine
and [ 35S]cysteine into newly synthesized proteins in Ctrl (E) or BDNF-treated (F) neurons.
Data represent the mean � SEM values of six determinations from two independent experi-
ments. *p � 0.05, ***p� 0.001 by unpaired Student’s t test between Ctrl and BDNF-treated
cultures. B, Cultures of cortical neurons were exposed to 5 ng/ml BDNF for different time peri-
ods, and the detection of NPY-positive neurons was performed by immunocytochemistry. Data
are the mean � SEM percentages of Ctrl values of six determinations from two independent
experiments. **p � 0.01, ***p � 0.001 by unpaired Student’s t test between Ctrl and BDNF-
treated cultures.

8218 • J. Neurosci., September 10, 2003 • 23(23):8212– 8220 Burkhalter et al. • BDNF Stimulates Energy Metabolism in Cortical Neurons



levels maintained for at least 2 d (data not shown), indicating that
BDNF stimulates the phenotypic differentiation of NPYergic
neurons. With regard to the induction by BDNF of proteins as-
sociated with neuronal differentiation, we have reported previ-
ously that, on acute treatment of cortical neurons, BDNF stimu-
lates the expression and the release of tissue-type plasminogen
activator (tPA) (Fiumelli et al., 1999) with a time course similar to
that of the enhancement of overall protein synthesis found in the
present study. The fact that tPA is a serine protease capable of
facilitating neurite outgrowth and neuronal migration during
nervous system development (Seeds et al., 1997) suggests that
activation of tPA by BDNF may contribute to structural changes
associated with neuronal development (Fiumelli et al., 1999).

Stimulation of glucose utilization and GLUT3 expression by
BDNF, as observed in this study, may be relevant to brain devel-
opment. Indeed, a series of functional and structural modifica-
tions that affects the energetic demands of the brain and therefore
the rate of glucose utilization occurs during brain development
(Chugani and Phelps, 1986; Nehlig et al., 1988; Khan et al., 1999).
Thus glucose utilization in the rat brain is low at birth and in-
creases during postnatal maturation (Vannucci et al., 1994). In
particular, glucose utilization sharply enhances between P14 and
P21, a developmental stage that coincides with synaptogenesis
and neuronal maturation (Vannucci et al., 1994). Interestingly,
this developmental increase in cerebral glucose utilization is par-
alleled by a similar increase in the expression of GLUT3 in the rat
brain (Vannucci, 1994; Vannucci et al., 1994). These results in-
dicate that cerebral glucose utilization and GLUT3 expression
increase in concert with synaptogenesis and neuronal matura-
tion. Moreover, developmental studies have demonstrated that
in the rat visual cortex the expression of BDNF increases sharply
during the second and third postnatal week (Bozzi et al., 1995;
Schoups et al., 1995; Rossi et al., 1999). Together, these data
reported by different studies reveal a temporal link between the
appearance of BDNF and the increase in glucose utilization and
GLUT3 expression in the cerebral cortex in vivo. Our in vitro
observations provide evidence that BDNF plays a determinant
role as a stimulator of glucose utilization and GLUT3 expression
in response to the increased energy demand resulting from the
enhanced amino acid uptake and protein synthesis associated
with the promotion of neuronal development by this neurotro-
phic factor.

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