Neuropharmacology 110 (2016) 519e529

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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Stimulation of brain glucose uptake by cannabinoid CB2 receptors and

its therapeutic potential in Alzheimer's disease
€ falvi a, b, Cristina Lemos a, 2, Ana M. Martín-Moreno c, d, 1, Ba
Attila Ko rbara S. Pinheiro a, 2,
^
Luis García-García e, Miguel A. Pozo e, f, Angela rio-Fernandes a, Rui O. Beleza a,
Vale
a, g
Paula Agostinho , Ricardo J. Rodrigues a, b  c, h, Rodrigo A. Cunha a, g,
, Susana J. Pasquare
c, d, *
María L. de Ceballos
a
CNC e Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-504 Coimbra, Portugal
b
Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal
c
Neurodegeneration Group, Department of Cellular, Molecular and Developmental Neurobiology, Instituto Cajal, CSIC, Doctor Arce, 37, 28002 Madrid, Spain
d
CIBERNED; Centre for Biomedical Research on Neurodegenerative Diseases, Spain
e
CAI de Cartografía Cerebral, Instituto Pluridisciplinar, UCM, Madrid, Spain
f
PET Technology Institute, Madrid, Spain
g
FMUC, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
h
Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), CONICET-Bahía Blanca and Universidad Nacional del Sur, Edificio E1, Camino La
Carrindanga km 7, 8000 Bahía Blanca, Argentina

a r t i c l e i n f o a b s t r a c t

Article history: Cannabinoid CB2 receptors (CB2Rs) are emerging as important therapeutic targets in brain disorders that
Received 20 June 2015 typically involve neurometabolic alterations. We here addressed the possible role of CB2Rs in the
Received in revised form regulation of glucose uptake in the mouse brain. To that aim, we have undertaken 1) measurement of 3H-
18 February 2016 deoxyglucose uptake in cultured cortical astrocytes and neurons and in acute hippocampal slices; 2) real-
Accepted 7 March 2016
time visualization of fluorescently labeled deoxyglucose uptake in superfused hippocampal slices; and 3)
Available online 11 March 2016
in vivo PET imaging of cerebral 18F-fluorodeoxyglucose uptake. We now show that both selective
(JWH133 and GP1a) as well as non-selective (WIN55212-2) CB2R agonists, but not the CB1R-selective
Chemical compounds studied in this article: agonist, ACEA, stimulate glucose uptake, in a manner that is sensitive to the CB2R-selective antagonist,
AM630 (PubChem CID
AM630. Glucose uptake is stimulated in astrocytes and neurons in culture, in acute hippocampal slices, in
4302963)
Anandamide (PubChem CID
different brain areas of young adult male C57Bl/6j and CD-1 mice, as well as in middle-aged C57Bl/6j
5281969) mice. Among the endocannabinoid metabolizing enzymes, the selective inhibition of COX-2, rather than
Arachidonyl-2-chloroethylamide (PubChem that of FAAH, MAGL or a,bDH6/12, also stimulates the uptake of glucose in hippocampal slices of middle-
CID aged mice, an effect that was again prevented by AM630. However, we found the levels of the endo-
5311006) cannabinoid, anandamide reduced in the hippocampus of TgAPP-2576 mice (a model of b-amyloidosis),
2-Arachidonoylglycerol (PubChem CID and likely as a consequence, COX-2 inhibition failed to stimulate glucose uptake in these mice. Together,
5282280) these results reveal a novel general glucoregulatory role for CB2Rs in the brain, raising therapeutic in-
2-Deoxy-D-glucose (PubChem CID
terest in CB2R agonists as nootropic agents.
108223)
© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
DuP697 (PubChem CID
3177) license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
GP1a (PubChem CID
10252734)
JWH133 (PubChem CID

Abbreviations: 2-AG, 2-arachidonoylglycerol; 3Rs, replacement, refinement and reduction of animals in research; ABDH6/12, a/b-hydrolase domain 6/12; ACEA, arach-
idonyl-20 -chloroethylamide; AEA, anandamide (N-arachidonoylethanolamine); CB1Rs and CB2Rs, cannabinoid CB1 and CB2 receptors; COX-2, cycloxygenase-2; DMEM,
Dulbecco's modified eagle medium; 3HDG, 3H-2-deoxyglucose; DMSO, dimethyl-sulfoxide; FAAH, fatty acid amide hydrolase; 18FDG, 18F-fluoro-deoxyglucose; HEPES, N-(2-
hydroxyethyl)piperazine-N0 -(2-ethanesulfonic acid); MAGL, monoacylglycerol lipase; 2-NBDG, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; TgAPP,
transgenic amyloid precursor protein; WT, wild-type.
* Corresponding author. Neurodegeneration Group, Dept. of Cellular, Molecular and Developmental Neurobiology Cajal Institute, CSIC, Doctor Arce, 37, 28002 Madrid,
Spain.
E-mail address: mceballos@cajal.csic.es (M.L. de Ceballos).
1
Present address: MD Anderson Cancer Center, Arturo Soria 270, 28033 Madrid, Spain.
2
Present address: Experimental Psychiatry Unit, Center for Psychiatry and Psychotherapy, Innsbruck Medical University, Austria.

http://dx.doi.org/10.1016/j.neuropharm.2016.03.015
0028-3908/© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
520 €falvi et al. / Neuropharmacology 110 (2016) 519e529
A. Ko
6918505)
JZL184 (PubChem CID
25021165)
LY2183240 (PubChem CID
11507802)
Ouabain (PubChem CID
439501)
WIN55212-2 (PubChem CID
5311501)
WWL70 (PubChem CID:17759121)
Keywords:
Anandamide (AEA)
b-amyloid
Cerebral glucose uptake
Cannabinoid CB2 receptor
Cyclooxygenase-2 (COX-2)
Positron emission tomography (PET)
1. Introduction
Brain disorders, including Alzheimer's disease (AD), often
involve specific early alterations in the metabolism of glucose in the
brain (Mosconi, 2005; Teune et al., 2010). The idea of alleviating
symptoms of dementia by boosting cerebral energy metabolism
has been toyed with for decades (Branconnier, 1983), yet safe
pharmacological agents with well characterized mechanism of
action are still lacking. In this sense, we have investigated here the
local cerebral glucoregulatory potential of the endocannabinoid
system in rodents.
The endocannabinoid system mainly consists of two established
(CB1 and CB2) receptors that are activated by endocannabinoid
signalling molecules, such as N-arachidonoylethanolamine (anan-
damide, AEA) and 2-arachidonoylglycerol (2-AG) (Di Marzo and De
Petrocellis, 2012; Pertwee, 2012). At the cellular level, endocanna-
binoid signalling via CB1 receptors (CB1Rs) regulates peripheral
energy metabolism and glucose uptake (Matias et al., 2008). In the
brain, acute activation of the psychoactive CB1R inhibits mito-
chondrial respiration and glucose metabolism in neurons and as-
trocytes, without robust effect on the uptake of glucose (Be nard
et al., 2012; Duarte et al., 2012; Lemos et al., 2012). Similarly, the
peripheral glucoregulatory role of the non-psychoactive CB2 re-
ceptors (CB2Rs) has also been recognized (Agudo et al., 2010;
Romero-Zerbo et al., 2012), although this remains to be docu-
mented in the brain.
CB2Rs were first described in peripheral organs and cells
(Galiegue et al., 1995), and subsequently in activated microglial
cells (Carlisle et al., 2002) - the resident immune cell of the brain.
However, during the last decade, the presence and function of
CB2Rs have been described in other cell types in the brain. An early
evidence was provided by Van Sickle et al. (2005) who identified
and functionally characterized CB2Rs in brainstem neurons. Other
studies have reported CB2R involvement in neurogenesis, axon
guidance, and neuronal excitability (Den Boon et al., 2012; Duff
et al., 2013; Sierra et al., 2014; Zhang et al., 2014). Furthermore,
astrocytes (in particular, activated astrocytes) are also endowed
with CB2R (Sheng et al., 2005; Benito et al., 2008; Bari et al., 2011;
Rodríguez-Cueto et al., 2014). However, a recent report added that
the vast majority of specific CB2R immunoreactivity in the healthy
mouse neocortex is neuronal (Savonenko et al., 2015). Nonetheless,
conventional CB2R immunstaining techniques may have possible
limitations (Ashton, 2011). Therefore, it is important that the highly
sensitive and specific RNAscope technique also proved the presence
of CB2R mRNA in excitatory and inhibitory neurons (but hardly in
resting microglia) throughout the hippocampus of adult mice (Li
and Kim, 2015). Furthermore, these hippocampal (and medial en-
torhinal) CB2Rs appear to control the synaptic release of GABA
(Morgan et al., 2009; Ando  et al., 2012), and the deletion of CB2Rs is
associated with impaired synaptic plasticity at excitatory synapses,
too, in the mouse hippocampus (Li and Kim, 2016a). Not surpris-
ingly, the CB2R KO mice exhibit memory impairments as well (Li
and Kim, 2016b).
Besides these indefinite physiological roles, the therapeutic
potential of central CB2Rs have also been recognized. There is an
increasing clinical interest in endorsing novel selective CB2R ago-
nists to treat different illnesses (Atwood and Mackie, 2010;
Pertwee, 2012; Han et al., 2013), including AD. Indeed, prolonged
oral treatment with a CB2R selective agonist conferred anti-
inflammatory effects and reduced microglial activation, which
have been demonstrated to mitigate the cognitive deficits of TgAPP
mice (Martín-Moreno et al., 2012). Similarly, subchronic treatment
with another CB2R agonist promoted b-amyloid (Ab) clearance, and
restored synaptic plasticity, cognition, and memory in Ab injected
rats (Wu et al., 2013). These results were extended in other reports
using TgAPP/PS1 mice (Aso and Ferrer, 2014; Cheng et al., 2014).
Additionally, the severity of Ab pathology correlates positively with
the density of the CB2R rather than the CB1R (Esposito et al., 2007;
Solas et al., 2013; Savonenko et al., 2015). Therefore, to map the
putative glucoregulatory potential of CB2Rs in healthy mice and in a
model of b-amyloidosis, we have taken advantage of recently
optimized in vitro and in vivo protocols (Lemos et al., 2012; Martín-
Moreno et al., 2012). Here we report that acute CB2R activation
increases glucose uptake in the brain of young and middle-aged
mice both in vitro and in vivo, which highlights a novel cerebral
role for the CB2R with therapeutic potential.
2. Materials and methods
2.1. Chemicals
3
H-2-deoxy-D-glucose (3HDG; specific activity: 60 Ci/mmol),
arachidonylethanolamide [ethanolamide 1e3H] (50 Ci/mmol) and
unlabelled AEA were obtained from American Radiolabeled
Chemicals, Inc. (Saint Louis, MO, USA). 2-arachidonoyl-[1,2,3-3H]
glycerol (3H-2-AG; 40 Ci/mmol) was bought from PerkinElmer,
Madrid, Spain). 2-NBDG was purchased from Invitrogen (Carslbad,
California, USA). Non-organic reagents, HEPES and DMSO were
purchased from Sigma-Aldrich Portugal (Sintra, Portugal) or Merck
Biosciences (Darmstadt, Germany). ACEA, AM630, DuP697, GP1a,
JWH133, JZL184, LY2183240, WIN55212-2 and WWL70 were ob-
tained from Tocris Bioscience (Bristol, U.K.). All non-water soluble
 €falvi et al. / Neuropharmacology 110 (2016) 519e529
A. Ko
substances and 2-NBDG were reconstituted in DMSO at 1000 the
final concentration, and stored in aliquots at 20  C.
2.2. Animals
All studies were carried out in accordance with the EU (2010/63/
EU) and FELASA guidelines, and they were approved by the Spanish
and Portuguese Ministries of Agriculture, as well as the local
institutional Animal Care Committees (license No. 280279-31-A
and 025781). All studies involving animals are reported in accor-
dance with the ARRIVE guidelines for reporting experiments
involving animals (Kilkenny et al., 2010; McGrath et al., 2010). A
total of 73 animals were used in the experiments described here.
For the experiments which are summarized in Figs. 3AB, 4 and 5,
we used 12-month-old (middle-aged) C57Bl/6j mice (i.e. wild type
[WT] littermates as controls), and 12-month-old C57Bl/6j trans-
genic 2576 mice, containing the human APP695 transgene with
mutations at KM670/671NL (hereafter, TgAPP mice). TgAPP mice
have b-amyloid-burden but no evident cell loss (Hsiao et al., 1996).
At 12 months, TgAPP mice did not differ in weight from the WT
C57Bl/6j mice (WT: 39.6 ± 2.4 g; TgAPP: 38.6 ± 2.3 g), yet they
displayed compromised new object recognition compared to WT
mice (data not shown). Data shown in Fig. 3C were obtained from
10-week-old male WT CD-1 mice and their CB1R global KO litter-
mates (Ledent et al., 1999). All these transgenic and WT mice were
genotyped from tail tip biopsies.
Data shown in Fig. 1 were obtained from young adult (8-10
week-old) male C57bl/6 mice (Charles-River, Barcelona, Spain),
while to obtain embryos for cell cultures, we mated young adult
C57Bl/6 mice (Charles-River).
The animals were group-housed under controlled temperature
(23 ± 2  C) and subjected to a fixed 12 h light/dark cycle, with free
access to food and water. We applied the principles of the ARRIVE
guideline in designing and performing the in vivo and in vitro
pharmacological experiments (see below), as well as for data
management and interpretation. All efforts were made to reduce
the number of animals used and to minimize their stress and
discomfort. The animals used to perform the in vitro studies were
deeply anesthetized with halothane before sacrifice (no reaction to
handling or tail pinching, while still breathing).
2.3. In vitro real-time fluorescent measurement of deoxyglucose
uptake in hippocampal slices
Experiments were performed as before (Lemos et al., 2015). Five
young C57bl/6 mice were sacrificed, and their brain was quickly
removed and placed in ice-cold Krebs-HEPES solution (in mM: NaCl
113, KCl 3, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, NaHCO3 25, glucose 5.5,
HEPES 1.5, pH 7.2). Coronal vibratome slices (300 mm) were left to
recover for 1 h at room temperature in Krebs-HEPES solution
gassed with 5% CO2 and 95% O2, and then gently mounted on
coverslips with the hippocampus in the center, and placed in a RC-
20 superfusion chamber on a PH3 platform (Warner Instruments,
Harvard, UK). The slices were superfused with gassed Krebs-HEPES
solution at a rate of 0.5 mL min-1 in a closed circuit, and they were
then photographed with a CoolSNAP digital camera (Roper Scien-
tific, Trenton, NJ, USA) every 30 s over the following 30 min (using a
5 PlanNeofluar-objective [NA 0.25] on an inverted Axiovert 200 M
fluorescence microscope [Carl Zeiss, Germany], coupled to a
Lambda DG-4 integrated 175 Watt light source and wavelength
switching excitation system [Sutter Instrument Company, Novato,
CA, USA] to allow real-time video imaging). The data was band-pass
filtered for excitation (BP470/40) and emission (BP525/50).
After recording 4 images for auto-fluorescence (to establish the
baseline), 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-
521
deoxyglucose (2-NBDG; 30 mM) was applied through the reser-
voir and 15 min later, JWH133 (1 mM), GP1a (100 nM) or the DMSO
vehicle alone (0.1% v/v) were administered. All measurements were
obtained in duplicate from each animal (see Supplementary Fig. 1
for additional details).
2.4. Cell culture preparation
Because the embryonic hippocampus is too small to provide
enough cells for our assays, neuronal and astrocyte primary cul-
tures were prepared from the neocortex as before (Lemos et al.,
2015). The neocortex of E18 male and female C57Bl/6 rat embryos
was digested for 15 min with 0.125% trypsin (type II-S, from porcine
pancreas, Sigma-Aldrich Portugal) and 50 mg/mL DNAse (Sigma-
Aldrich) in Hank's balanced salt solution without calcium and
magnesium (in mM: NaCl 137, KCl 5.36, KH2PO4 0.44, NaHCO3 4.16,
Na2HPO4 0.34, D-glucose 5, pH 7.2). After dissociation, astrocytes
were grown in plastic Petri dishes (p100) for 14 days in DMEM with
10% fetal bovine serum, 50 U/mL penicillin and 50 mg/mL strepto-
mycin (Sigma-Aldrich), at 37  C in an atmosphere of 95%/5% air/
CO2, replacing half of the medium at DIV 7. The cells were then
trypsinized and plated onto poly-D-lysine (100 mg/mL, Sigma-
Aldrich) and laminin (10 mg/mL, Sigma-Aldrich) coated 24-well
culture plates at a cell density of 150,000/cm2. Uptake assays
were performed 24 h later. For neuronal cultures, cells were plated
directly onto poly-D-lysine and laminin coated 24-well culture
plates, at the same cell density, in DMEM plus 10% fetal bovine
serum, 50 U/mL penicillin and 50 mg/mL streptomycin. Two hours
after seeding, the medium was replaced by Neurobasal medium
with 2% B27 supplement (GIBCO, Life technologies), 50 U/mL
penicillin, 50 mg/ml streptomycin and 2 mM glutamine (Sigma-
Aldrich), and cells were grown at 37  C in an atmosphere of 95%/5%
air/CO2 until they were used 14 days later. The medium was
partially (40%) replaced every 4 days. On average, 15% of cells were
found positive for glial fibrillar acidic protein immunostaining in
randomly chosen wells (Lemos et al., 2015).
2.5. In vitro 3H-deoxyglucose uptake in neocortical neuron and
astrocyte cultures
The assays were carried out according to Lemos et al. (2015).
For these assays, we used 7 different astrocyte cultures and 5
neuron cultures from 7 and 5 independent preparations (pregnant
dams). After 15 days in culture, the culture medium was replaced
with 250 mL Krebs-HEPES assay solution (pH 7.4; 37  C) (see
above), containing either the vehicle alone (DMSO 0.1%, in half of
the plate), or the CB2R-selective antagonist, AM630 (1 mM, in the
other 12 wells). The cultures were incubated for 60 min, at 37  C,
and the CB2R agonists JWH133 (100 nM) (4 wells/plate) or GP1a
(100 nM) (4 wells/plate), or their vehicle (DMSO 4 wells/plate),
was gently added to the wells in a volume of 250 mL, together with
AM630 (i.e. 3  4 wells/plate) or its vehicle (i.e. 3  4 wells/plate),
and the radioactive glucose analogue 3H-2-deoxyglucose (3HDG;
16.7 nM, final concentration). After a further 30 min incubation at
37  C, the plates were transferred onto ice and the 3H content was
measured in an aliquot from each well (to assess the small dif-
ferences in the individual 3H concentration in each well that
pipetting and mixing solutions may introduce). The cells were
then washed gently 3 times with 1 mL ice-cold Krebs-HEPES,
300 mL NaOH (0.5 M) was added to each well, and the plates were
left on a shaker for an hour to recover the proteins and 3HDG
taken up by the cells. Tritium was measured in a Tricarb b-counter
(PerkinElmer, USA), and these values were adjusted for protein
quantity and the 3H concentrations in each individual wells, and
multiplied by 3.3  105 (the ratio between 3HDG and D-glucose in
522 €falvi et al. / Neuropharmacology 110 (2016) 519e529
A. Ko

Fig. 1. Time-course and sub-regional variation of the effect of CB2R agonists on the uptake of the fluorescent glucose analogue, 2-NBDG in hippocampal slices of young C57Bl/6j
male mice. For technical details and additional information, see Supplementary Methods, Supplementary Fig. 1 and Supplementary Video. (A,A0 ) Representative images illustrating
the distribution of the fluorescence signal, using the false color scale of blue (low signal), green (moderate signal) and yellow (strong signal). As indicated in the time-course graph
(B), image (A) was taken just before the treatment (minute 15 of baseline), and image (A0 ) was photographed close to the end of the exposure to GP1a (100 nM: see also Sup-
plementary Video). (B) JWH133 (1 mM) and GP1a (100 nM) both increased the uptake in astrocyte-rich zones of the hippocampus. The displayed curves represent the mean þ S.E.M.
of individual net effects (in duplicate) in each animal, which was obtained after the subtraction of the respective DMSO controls (in duplicates per animal) from the raw treatment
data. Therefore, the DMSO control is represented with the dashed line crossing the zero value. (C) The individual points represent the average of 2 different slices from the same
mouse in duplicate (n ¼ 5): *P < 0.05 and **P < 0.01.

specificity of the transport process (Lemos et al., 2015). Addition-

ally, the plasma membrane Naþ/Kþ-ATPase inhibitor, ouabain (10
mM), also inhibited neuronal glucose uptake in this assay by
42.8 ± 2.1% (n ¼ 3, P < 0.01; figure not shown).

2.6. In vitro 3H-deoxyglucose uptake in hippocampal slices

To better characterize the glucoregulator role of CB2R signalling

Fig. 2. CB2R activation stimulates hippocampal glucose uptake in astrocytes and
neurons in vitro. The CB2R-selective agonists JWH133 (30 nM) and GP1a (100 nM)
stimulated glucose uptake in (A) primary cortical astrocyte and (B) neuronal cultures
from C57Bl/6 mice, as assessed using the 3HDG uptake assay (30 min). The CB2R se-
lective antagonist, AM630 (1 mM) prevented the action of the CB2R agonists, while it
had no effect on the uptake per se. D-glucose uptake is expressed as mean þ SEM in
nmol/mg protein (n ¼ 7 astrocyte and n ¼ 5 neuron cultures, performed in quadru-
plicate, at a density of 15  104 cells/cm in 24-well plates). *P < 0.05 and **P < 0.01 vs.
DMSO control; n.s., not significant.
pharmacologically, we moved to an in vitro glucose uptake protocol
previously optimized in acute hippocampal slices which allows the
effect of various treatments to be compared simultaneously in
different strains in a pairwise arrangement (Lemos et al., 2012,
2015). Animals were anesthetized with halothane, sacrificed
(around 14:00 o'clock each experimental day to reduce potential
circadian hormonal effects), and their brain was immediately
placed in ice-cold Krebs-HEPES assay. The hippocampus was cut
into 450 mm-thick transverse slices with the help of a McIlwain
tissue chopper, and the slices were gently separated in ice-cold
carboxygenated (95% O2 and 5% CO2) assay solution, and main-
tained at 37  C in a multichamber slice incubator with 50 ml of
carboxygenated assay solution until the end of the experiment.
Four slices were used from each animal and in each experimental
condition. The CB2R-selective antagonist, AM630 or its vehicle
(DMSO 0.1%) were bath-applied from min 55 of the preincubation
period. WIN55212-2, JWH133, ACEA or DuP697, LY2183240, as well
as JZL184 and WWL70 combined or their vehicle (DMSO 0.1%) were
added from min 60 of the preincubation period, and the radioactive
glucose analogue, 3H-2-deoxy-D-glucose (3HDG; final cc. 2 nM) was
applied to the bath 5 min later. After 30 min, the slices were washed
twice in ice-cold assay solution for 5 min and collected in 1 mL
NaOH (0.5 M) to dissolve the slices. An aliquot of 800 mL was then
assayed for 3H in a Tricarb b-counter, while the remaining solution
was used to quantify the protein in a bicinchoninic acid assay
(Merck Biosciences, Germany). Tritium uptake was multiplied by a
factor of 2.75  106 to estimate the total glucose uptake (corre-
sponding to the concentration difference between D-glucose and
3
HDG in the uptake medium; Lemos et al., 2012).

2.7. Blood glucose measurements

the medium).
In this experimental model, cytochalasin B (10 mM) largely in- Blood glucose from non-fasted WT and TgAPP mice was
hibits glucose uptake in both cell cultures, thus confirming the assessed before and one hour following the acute administration of
JWH133 0.2 mg/kg i.p. Glucose was measured in one drop of blood
 €falvi et al. / Neuropharmacology 110 (2016) 519e529
A. Ko 523

Fig. 3. CB2R activation stimulates glucose uptake in hippocampal slices of both middle-aged and young mice ex vivo. A) The CB2R-selective agonist, JWH133 (1 mM), the mixed CB1R/
CB2R agonist, WIN55212-2 (1 mM), as well as the COX-2 inhibitor, DuP697 (500 nM) all increased glucose uptake in 12-month-old wild-type (WT) C57Bl/6j mice. All these effects
were prevented by a 5 min preincubation with the CB2R-selective antagonist, AM630 (1 mM). Inhibition of other endocannabinoid metabolizing enzymes, namely, FAAH, MAGL or
a,bDH6, with LY2183240, JZL184 and WWL70 (all at 1 mM) respectively, did not affect glucose uptake. B) By contrast, only JWH133 and WIN55212-2 but not DuP697 stimulated
glucose uptake in the TgAPP 2567 mice. Data represent the mean þ SEM of individual measurements from 6 to 12 animals, with the dashed line indicating the DMSO control values:
*P < 0.05 vs. DMSO-treated slices; n.s., not significant. C) JWH133 (1 mM), stimulated the uptake of glucose in acute hippocampal slices of 10 week-old CB1R KO mice and their WT
littermates. Each of the connected pairs of squares and circles represent one animal. *P < 0.05 vs. DMSO-treated slices (n ¼ 6).

standard, the samples were diluted to 70% water and centrifuged.

The pellets were kept for total protein determination. Solid-phase
extraction (Strata-X 33 mm columns, Phenomenex) was per-
formed before the LC/MS analysis on a Thermo Surveyor liquid
chromatographic system coupled to an electrospray ionization
triple quadrupole TSQ Quantum mass spectrometer operated in
positive ionization mode. Chromatographic analysis was performed
on an ACE 5 mm C8 (150  2.1 mm, Hichrom) column and the
analytes were eluted using an isocratic mobile phase of water/
methanol/formic acid (15/85/0.5 v/v/v) at a flow rate of 200 mL/min.
The column was maintained at 30  C and the sample tray at 4  C
during the analysis. The extraction efficiency was >95% and the
limit of quantification was 0.01 pmol for AEA and <25 pmol for 2-
AG. The sample concentrations were determined from calibration
curves and the endocannabinoid levels were expressed per mg
protein (Bradford assay).

2.9. Endocannabinoid hydrolysis assay

Experiments were carried out as before (Mulder et al., 2011),

Fig. 4. AEA levels were reduced in the hippocampus of TgAPP mice. Of the two
principal endocannabinoids, the levels of AEA (B) but not of 2-AG (A) were significantly
lower (by 38%) in the TgAPP mice (dark bars) than in the WT (light bars), as assessed by
combined liquid chromatography-mass spectrometry. Data represent the mean þ SEM
of individual measurements from 7 animals. 2-AG hydrolysis (C) was also unaltered in
hippocampal membranes of TgAPP mice in vitro (n ¼ 6), however, AEA metabolism (D)
by FAAH into ethanolamine was significantly smaller in the transgenic hippocampi
in vitro (n ¼ 6). *P < 0.05, ***P < 0.001; n.s., not significant.
from the tail using Glucocard Memory meter strips (Menarini,
Spain), in a range of 40e500 mg/mL.
2.8. Endocannabinoid level measurement in mouse hippocampus
Endocannabinoids were measured externally (Division of
Applied Medicine, School of Medicine and Dentistry, University of
Aberdeen, UK). Lipid extraction was performed by homogenizing
the frozen hippocampi on ice in 600 mL of acetonitrile/methanol
(50/50) using an Ultra-Turrax. Upon addition of 6 pmol of N-(2-
hydroxyethyl-1,1,2,2-d4)-5Z,8Z-,11Z,14Z-eicosatetraenamide
[deuterated (d4)-AEA; Tocris Bioscience, UK] as an internal
and adapted to mice (Pascual et al., 2014). Cortices were mechan-
ically homogenized in TriseHCl (20 mM, pH 7.2) containing 0.32 M
sucrose and protease inhibitors, and centrifuged to eliminate nuclei
and cellular debris (1000g, 10 min, 4  C). Protein concentrations
were determined by Bradford's colorimetric method. The 2-AG-
degrading activity was assessed by incubating sample fractions
(50 mg protein) in a solution containing TriseHCl (10 mM, pH 7.2),
1 mM EDTA, fatty acid-free bovine serum albumin (1.25 mg/mL),
and 3H-2-AG in a final volume of 200 ml for 15 min at 37  C. Identical
conditions were used to assess AEA degrading activity using 3H-
AEA ([1-H]ethanolamine, 25 mM, 40 Ci/mmol specific activity; ARC
Inc.). Reactions were stopped by the addition of 400 mL chlor-
oform:methanol (2:1, v/v) and vigorous vortexing, after centrifu-
gation (2000g, 10 min, 4  C), ethanolamine or glycerol, the AEA and
2-AG hydrolysis products respectively, were obtained in the upper
phase, and they were transferred into scintillation vials and
radioactivity measured by liquid scintillation spectroscopy.
The experimental conditions were selected from pilot experi-
ments establishing the relationship of substrate and protein con-
centrations to identify optimal AEA-degrading enzymatic activity
(data not shown), allowing 50% of AEA converted into
ethanolamine.
Blanks were prepared identically except that the membranes
were omitted or inactivated by boiling for 10 min or by the addition
of chloroform:methanol (1:1, v/v) before use. No differences were
observed among the different blanks. These values were subtracted
524 €falvi et al. / Neuropharmacology 110 (2016) 519e529
A. Ko

Fig. 5. Evidence for the stimulation of brain glucose uptake by a single low-dose injection of JWH133 in middle-aged mice in vivo. Basal (A1,B1) and JWH133-stimulated A2,B2) 18F-
fluoro-deoxy-glucose (18FDG) uptake in the frontal and temporal cortices, and the hippocampus. A1) Representative color-coded PET of basal 18FDG uptake in 12 month old wild-
type (WT) and B1) TgAPP 2567 mice. Panels A10,B10 ) illustrate the same images merged with the respective CT images, seen from three directions. The effect of acute JWH133
injection (0.2 mg/kg, i.p.) is shown in panels A2,A20 ,B1,B20 ). Color code for FDG activity can be seen vertically on the left of the figure. C) Mean basal uptake values in the three
regions of interest (n ¼ 9 mice). D) Acute JWH133 injection does not alter the blood glucose levels in the two strains and hence, JWH133 does not appear to influence glucose uptake
in the brain through systemic mechanisms. E-G0 ) Before-after graphs summarizing the basal uptake, and 4 days later, the JWH133-stimulated (JWH) uptake in the frontal E, E0 ) and
temporal F, F0 ) cortices, as well as in the hippocampus G, G0 ), of 4 wild-type (WT, E-G) and 4 TgAPP 2567 (E0 -G0 ) mice. Uptake values were corrected for the dose injected and for
body weight, *P < 0.05.

from each enzymatic activity.
Enzyme activity was expressed as picomole [1,2,3-3H]glycerol
formed  mg protein  15 min and picomole [3H]ethanolamine
formed  mg protein  15 min respectively.
2.10. 18F-fluorodeoxy-D-glucose positron emission tomography
(18FDG-PET)
These experiments were carried out as described previously
(Martín-Moreno et al., 2012) with only slight modifications. In the
PET studies the images of basal (no treatment) uptake were ac-
quired, and 4 days later the animals were injected with JWH133
0.2 mg/kg ip, and after 30 min, the radiotracer was injected and
new PET images were acquired (see below).
PET images were acquired in fasted mice injected (i.p.) with
18
FDG (approx. 11.1 MBq or 300 mCi/200 ml saline, PET Technology
Institute, Madrid). After a 30 min period for radiotracer uptake, the
animals were anesthetized by isoflurane (2%) inhalation, to assure
immobilization during the scanning process, and they were placed
on the bed of the tomograph. Images of 18FDG uptake were ac-
quired over 30 min, immediately followed by a CT (computed to-
mography) acquisition (Albira ARS small animal PET/CT dual
scanner, Oncovision, Spain). PET images were reconstructed using
the ordered subset expectation maximization algorithm (OSEM, 3
iterations), applying corrections for dead time, radioactive decay,
random coincidences and scattering. For the CT images a high
resolution filtered back projection (FBP) algorithm was used.
Metabolic activity was quantified by matching the CT image of
the skull of each animal to a common magnetic resonance (MR)
mouse brain template, in which regions of interest were previously
delineated. After saving the spatial transformation, it was applied
to the corresponding fused PET image, to allow the correct co-
registration of the PET image to the MR brain template. Results
were normalized to the actual radioactivity dose injected and the
body weight of each animal (Kuntner et al., 2009). All these pro-
cesses were carried out with PMOD software version 3.0 (PMOD
Technologies, Switzerland).
2.11. Data presentation and statistical analysis
All data are expressed as the means ± or þ SEM of the number of
independent observations (n) indicated. Normalized data were
tested for normality with the Kolmogorov-Smirnov normality tests
and statistical significance was calculated by one sample t-test
 €falvi et al. / Neuropharmacology 110 (2016) 519e529
A. Ko
against the hypothetical value of zero (Figs. 1C and 2). Alternatively,
a paired t-test (Fig. 3C) or repeated measures of ANOVA followed by
Bonferroni's post-hoc test were used, and a value of P < 0.05 was
accepted as significant.
3. Results
3.1. CB2R activation rapidly enhances glucose uptake in
hippocampal slices
First we assessed whether CB2R activation affects glucose up-
take in superfused hippocampal slices, and the rate of fluorescence-
labeled deoxyglucose (2-NBDG) accumulation was tested at 30 s
resolution in such slices. The basal accumulation of 2-NBDG was
most evident in astrocyte-rich areas of transverse hippocampal
slices, i.e. in the in the stratum lacunosum, followed by the strata
radiatum and moleculare, and the smallest signal was found in the
strata pyramidale and granulare (Fig. 1AeA0 ), consistent with pre-
vious findings (Jakoby et al., 2014). Importantly, both of the two
selective CB2R agonists, GP1a (100 nM, P < 0.01) and JWH133 (1 mM,
P < 0.05), rapidly increased the velocity of 2-NBDG accumulation in
these hippocampal slices (Fig. 1 B,C, and Supplementary Video).
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.neuropharm.2016.03.015.
3.2. CB2R activation stimulates glucose transport in cultured
neurons and astrocytes
Since the use of 2-NBDG underrepresents neuronal glucose
transport (Jakoby et al., 2014), we measured the effect of acute
in vitro CB2R activation on glucose uptake by primary mouse
cortical astrocytes and neurons using a 3HDG transport assay. In the
presence of JWH133 (30 nM) over 30 min, glucose uptake
augmented by 35.4 ± 7.5% in astrocytes (n ¼ 7, P < 0.01 vs. DMSO
control) and by 13.5 ± 4.0% in neurons (n ¼ 5, P < 0.05: 2 A,B).
Notably, JWH133 exhibits an EC50 value close to 1 mM at the CB1R
(Huffman et al., 1999). To avoid the possible non-specific co-acti-
vation of CB1R in these cell cultures, we opted for testing JWH133
here at 30 nM, i.e. 9-times above its EC50 at the CB2R but more than
an order of magnitude below its EC50 at the CB1R (Huffman et al.,
1999).
Similarly, GP1a at a concentration ~2700-times above its EC50 at
the CB2R but ~4-fold below its EC50 at the CB1R, i.e. at 100 nM
(Murineddu et al., 2006) also increased glucose uptake in astrocytes
by 22.4 ± 10.7% (n ¼ 7, P < 0.05) and by 13.7 ± 2.4% in neurons
(n ¼ 5, P < 0.01: Fig. 2 A,B). Therefore, with the chosen ligand
concentrations we achieved good selectivity toward the CB2R while
still observing maximal effect amplitudes. The CB2R antagonist
AM630 (1 mM) did not alter glucose uptake per se in cultures of
astrocytes (n ¼ 7) or neurons (n ¼ 5). However, in the presence of
AM630, neither GP1a nor JWH133 significantly altered glucose
uptake in either of these two cell types (P > 0.05 vs. AM630 control;
astrocytes n ¼ 5, neurons n ¼ 4: Fig. 2 A,B). In summary, CB2R
activation was more effective at increasing glucose uptake in as-
trocytes compared with neurons. As stated previously, our neuron
cultures contained 15% of astrocytes, which likely contributed to
the effect of the CB2R agonists. However, this low astrocytic
contamination cannot solely account for the effect amplitudes
observed neuronal cultures.
3.3. b-amyloidosis hinders endogenous CB2R activation
A similar pharmacological analysis was then performed in hip-
pocampal slices of 12-month-old TgAPP mice and their age-
matched controls. The normalized control uptake values of the
525
TgAPP hippocampus amounted to 105.4 ± 8.9% of the WT control
uptake (i.e. þ5.4 ± 8.9% over DMSO control) when assessed in a
pairwise arrangement (n ¼ 19 pairs, P > 0.05: Fig. 3 A,B). Glucose
uptake was significantly enhanced by the CB2R-selective agonist
JWH133 (1 mM) in both the WT (þ26.3 ± 10.0%, n ¼ 12, P < 0.05:
Fig. 3A) and the TgAPP slices (þ16.7 ± 6.7%, n ¼ 12, P < 0.05: Fig. 3B).
Due to the limited availability of 12-month old TgAPP mice, we
made a risk-free decision when testing JWH133 at 1 mM, i.e. at the
maximal concentration where good CB2R-selectivity is still ach-
ieved (Huffman et al., 1999).
Importantly, glucose uptake was also stimulated by the non-
selective (“mixed”) CB1R/CB2R agonist, WIN55212-2 (1 mM), in
both WT (þ22.4 ± 9.2%, n ¼ 12, P < 0.05: Fig. 3A) and TgAPP slices
(þ17.5 ± 7.1%, n ¼ 12, P < 0.05: Fig. 3B). While the selective CB2R
antagonist, AM630 (1 mM) had no effect on glucose uptake per se in
either strain (n ¼ 12), it did prevent JWH133 and WIN55212-2 from
stimulating glucose uptake in both WT and TgAPP slices (n ¼ 6:
Fig. 3 A,B).
Cannabinoid agonists exert their actions predominantly via
CB1R activation in the mammalian brain (Katona and Freund, 2012).
To check if CB1Rs are also involved in the stimulation of glucose
uptake, first we tested the CB1R-selective agonist ACEA at a con-
centration half way between its Kd values at the CB1R and the CB2R,
i.e. at 1 mM (Hillard et al., 1999). ACEA did not affect glucose uptake
in the middle-aged C57Bl/6 mice (þ5.3 ± 7.9%, n ¼ 7, P > 0.05:
Fig. 3A). Subsequently, we asked if CB1Rs were involved in the effect
of JWH133, taken that this ligand has some affinity toward the CB1R
at 1 mM (Huffman et al., 1999). Thus, we tested JWH133 (1 mM) in
acute hippocampal slices of young adult CB1R KO mice and their
WT littermates on the CD-1 strain. JWH133 (1 mM) significantly
stimulated glucose uptake þ13.4 ± 3.7% (n ¼ 6, P < 0.05) in the WT
mice and þ26.7 ± 9.0% (n ¼ 6, P < 0.05) in the CB1R KO, thus
virtually, JWH133 was more twice as more effective in the absence
of the CB1R. These data together with the results with ACEA also
confirm the absence of acute modulation of hippocampal glucose
uptake by CB1Rs.
Next, we assessed whether endogenous activation of CB2R by
the major endocannabinoids, AEA and 2-AG also bolstered glucose
uptake. To that end, we inhibited either fatty acid amide hydrolase
(FAAH) or cyclooxygenase-2 (COX-2), two enzymes known to be
involved in AEA degradation (Egertova  et al., 2003; Fowler et al.,
2013). The treatment of slices with DuP697 (500 nM), a selective
COX-2 inhibitor (IC50 at COX-2, 10 nM; IC50 at COX-1, 800 nM;
Gierse et al., 1995), augmented glucose uptake by þ16.6 ± 7.7% in
the WT mice (n ¼ 6, P < 0.05: Fig. 3A) - an effect that was prevented
by CB2R blockade (n ¼ 6: Fig. 3A). However, unlike in the WT,
DuP697 failed to modify glucose uptake in the TgAPP mice (n ¼ 6,
Fig. 3B). In contrast, LY2183240 (100 nM), a potent dual inhibitor of
FAAH and AEA reuptake (Nicolussi et al., 2014), did not affect
glucose uptake in WT mice, and therefore, it was not tested in
TgAPP mice slices. The major enzymes that degrade 2-AG in the
mammalian brain are monoacylglycerol lipase (MAGL) and a/b-
hydrolase domain 6/12 (ABDH6/12) (Savinainen et al., 2012;
Murataeva et al., 2014). The inhibition of 2-AG hydrolysis by the
concurrent application of JZL184 (1 mM), a MAGL inhibitor (Long
et al., 2009), and WWL70 (1 mM), an a,bDH6/12 inhibitor (Li et al.,
2007), also failed to modify glucose uptake in WT mice (Fig. 3 A).
3.4. Lower AEA levels in the hippocampus of TgAPP mice
The inability of COX-2 inhibition to stimulate glucose uptake in a
CB2R-dependent fashion in the TgAPP hippocampus suggests a loss
of endocannabinoid function, which prompted us to measure
endocannabinoid levels in these two strains. The hippocampal
levels of AEA and 2-AG in the WT mice (Fig. 4) were similar to those
526 €falvi et al. / Neuropharmacology 110 (2016) 519e529
A. Ko
reported in the rat brain (Stella et al., 1997), and while no difference
in the 2-AG levels were detected between WT and TgAPP mice
(n ¼ 7, P > 0.05), AEA levels in the latter were 37.8 ± 11.4% lower
than in WT (n ¼ 7, P < 0.05) (Fig. 4A,B). Accordingly, there was a
2.4 ± 0.3 fold increase in the 2-AG/AEA ratio in TgAPP mice (n ¼ 7,
P < 0.001). The degradation of 2-AG was also not statistically
different between the WT and TgAPP (n ¼ 6, P > 0.05) (Fig. 4D).
Interestingly, the degradation of AEA into ethanolamine by FAAH
was significantly reduced in TgAPP by 33.5 ± 8.9% (n ¼ 6, P < 0.001)
(Fig. 4D).
18
3.5. Effect of CB2R activation on FDG uptake e a PET analysis
in vivo
Finally, we tested the feasibility of targeting CB2R to stimulate
brain glucose uptake in middle-aged mice in vivo. We first evalu-
ated the basal uptake of 18FDG in the brain regions typically bearing
the hallmarks of the diminished 18FDG signal in the Alzheimer's
disease-affected human brain, i.e. the frontal and temporal cortices,
and the hippocampus (Mosconi, 2005). Like the ex vivo data, we
observed no difference in the 18FDG signal in the aforementioned
areas of WT and TgAPP mice (n ¼ 9: Fig. 5 A,A0 ,B,B0 ,C). Four days
later, the same animals were injected with a small dose of JWH133
(0.2 mg/kg, i.p.) and they were re-evaluated for 18FDG uptake.
While this CB2R agonist did not alter blood glucose levels (Fig. 5 D),
it did stimulate glucose uptake by an average of 31.5% in each of the
three regions in the WT mice (n ¼ 4, P < 0.05: Fig. 5EeG), as well as
by an average of 21.3% in the TgAPP mice (n ¼ 4, P < 0.05:
Fig. 5E0 eG0 ).
4. Discussion
4.1. A novel role for cannabinoid CB2 receptors: stimulating glucose
uptake in the brain
We show here that CB2R activation in vitro, and more impor-
tantly, in vivo rapidly enhances cerebral glucose uptake by ~30%
over the basal levels. These data are in concert with a recent finding
that the intraperitoneal injection of the mixed CB1R/CB2R agonist,
HU210 increased cerebral 18FDG PET signal by 21% in the rat
(Nguyen et al., 2012). Since CB1R activation decreases mitochon-
drial glucose metabolism (Duarte et al., 2012) CB2Rs are likely
involved in the stimulation of brain metabolism in the above study.
These data support the view of the recently described antagonistic
interaction between CB1Rs and CB2R in neurons (Calle n et al., 2012).
Given that brain glucose availability controls cognition and
memory in humans (Messier, 2004) and that central metabolic
boosting alleviates the cognitive symptoms of dementia, these data
suggest that CB2R agonists might represent a novel class of noot-
ropic drugs (Branconnier, 1983). This raises the interesting hy-
pothesis that by promoting AEA release, neural activity can activate
central CB2Rs and thereby refuel circuits under heavy load. Indeed,
it was recently found that JWH133 enhanced aversive memory
consolidation in mice, while the genetic ablation or pharmacolog-
ical inhibition of CB2R impaired it (García-Gutie rrez et al., 2013).
2-NBDG uptake in slices suggested a contribution of astrocytes
in the effects of CB2R activation. Consistent with this, our data in
cultures show that CB2R influences both astrocytic and neuronal
glucoregulation. CB2R are found at low density on most cell types in
the brain, including activated microglia and astrocytes controlling
neuroinflammation (Ramírez et al., 2005; Martín-Moreno et al.,
2012), and inhibiting astrocytoma growth (Sa nchez et al., 2001).
Moreover, CB2R mRNA or protein has also been found in several
types of neurons, including cortical, hippocampal and midbrain
neurons (Atwood and Mackie, 2010; Calle n et al., 2012; Den Boon
et al., 2012; Zhang et al., 2014). Interestingly, CB2R expression and
density in neurons apparently surpasses that in microglia or as-
trocytes in the mouse neocortex (Savonenko et al., 2015) and hip-
pocampus (Li and Kim, 2015).
The therapeutic activation of CB2R is safe and it does not trigger
psychoactivity (Atwood and Mackie, 2010; Pertwee, 2012).
Furthermore, CB2R density is known to increase in Alzheimer's
disease and Down's syndrome, as well as in b-amyloidosis in vitro
(Esposito et al., 2007; Ruiz-Valdepen ~ as et al., 2010; Solas et al.,
2013). This probably reflects an adaptive self-defense process
since CB2R activation confers neuroprotection in several experi-
mental models of Alzheimer's disease (Ramírez et al., 2005;
Esposito et al., 2007; Ruiz-Valdepen ~ as et al., 2010; Martín-
Moreno et al., 2012).
4.2. AEA and b-amyloidosis
The first step in common pathomechanisms toward AD pa-
thology is excessive b-amyloid accumulation (Sperling et al., 2013;
Zahs and Ashe, 2013). Remarkably, the antagonistic relationship
between AEA and b-amyloid appears to be bi-directional: b-amy-
loid toxicity is prevented by AEA in human cell lines in vitro (Milton,
2002) and conversely, b-amyloid treatment decreases AEA levels in
C6 rat astroglioma cells (Esposito et al., 2007). Accordingly, AEA
levels decrease in the AD brain and they are inversely correlated
with b-amyloid levels (Jung et al., 2012), consistent with the lower
hippocampal AEA levels in TgAPP mice. b-amyloidosis may lower
AEA levels by either bolstering catabolism and/or by impairing its
synthesis. COX-2 is an important enzyme in AEA metabolism
(Glaser and Kaczocha, 2010; Pamplona et al., 2010), furthermore, b-
amyloidosis induces COX-2 expression in astrocytes (Giovannini
et al., 2002) and in AD-affected neurons. Hence, COX-2 inhibition
may help to decrease the incidence or slow the progress of AD (Berk
et al., 2013). In fact, in a previous work we treated TgAPP 2576 mice
orally for four months with the CB2R-selective agonist JWH133 and
we observed an attenuation of the AD phenotype, including re-
covery from memory impairment and neuroinflammation, as well
as a normalization of the increase in COX-2 protein levels (Martín-
Moreno et al., 2012).
Strikingly, COX-2 inhibition by the selective DuP697 antagonist
facilitated glucose uptake via CB2R activation in WT but not in the
TgAPP mice, probably due to the rescue of AEA from COX-2-driven
tonic metabolism in WT mice (Glaser and Kaczocha, 2010;
Pamplona et al., 2010). In the TgAPP mice, we found a 38%
decrease of AEA, suggesting that these levels were already too low
to stimulate the CB2Rs, even following COX-2 blockade. In fact, this
may illustrate a deficit in synthesis, since AEA production requires
the concurrent stimulation of both NMDA and acetylcholine re-
ceptors (Stella and Piomelli, 2001), both of which become hypo-
functional in AD (Pavía et al., 1998; Giovannini et al., 2002). In the
present work FAAH activity was reduced in TgAPP mice, as judged
by the decrease in AEA hydrolysis, again in agreement with its
reduction in AD brain, which we recently reported (Mulder et al.,
2011; Pascual et al., 2014). This reduction is mimicked by patho-
logically relevant Ab1-40 peptide concentrations (Pascual et al.,
2014) and may serve as to preserve AEA levels under lower AEA
production, as a compensatory mechanism. Even though, FAAH
inhibition did not stimulate glucose uptake, suggesting that AEA
metabolism by FAAH is not a rate limiting factor for CB2R activation
in the WT hippocampus.
4.3. Cannabinoid receptors and glucose uptake
Although the role of cannabinoid receptors in peripheral glu-
coregulation is extensively studied (Matias et al., 2008), the
 €falvi et al. / Neuropharmacology 110 (2016) 519e529
A. Ko
mechanism through which CB2R activation facilitates glucose
transport in the brain awaits further interrogation. Several mech-
anisms could be invoked on that respect. For instance, cannabi-
noids stimulate AMP-activated protein kinase (Dagon et al., 2007), a
major intracellular energy sensor, which leads to enhanced glucose
uptake by stimulating glucose transporter (GLUT) membrane
expression and transport efficiency (Rutter et al., 2003; Weisova  KO mice and their WT littermates.
et al., 2009). More importantly, the effect was absent in CB2R
knockout mice (Dagon et al., 2007). A possible direct effect of CB2R
agonists on glucose transporters remains unexplored. New syn-
thesis of transporters would not parallel the acute pharmacological
effects reported here. Therefore, we suggest that CB2R agonists may
favor transporter localization at the plasma membrane. On the
other hand, it is less likely that CB2Rs increased glucose uptake
driven by the stimulation of its metabolism, because we found
previously that WIN55212-2 slows rather than facilitates the
mitochondrial oxidative metabolism of glucose via CB1R activation
both in neurons and astrocytes (Duarte et al., 2012).
4.4. Conclusions
In summary, the present results provide the first direct phar-
macological evidence in vitro and in vivo of a role of CB2R in central
glucoregulation. Additionally, we found that glucoregulation by
endogenous CB2R signalling is negatively affected by b-amyloidosis,
thought to be the first pathological step in AD. Therefore, it would
be interesting to perform further studies to define how CB2R
mediated glucoregulation contributes to the recently discovered
therapeutic potential of CB2R agonists in animal models of AD
(Martín-Moreno et al., 2012; Wu et al., 2013; Aso and Ferrer, 2014;
Cheng et al., 2014).
Conflicts of interest
The authors have no conflict of interests to report, and received
no financial support or compensation from any individual or
corporate entity over the past 3 years for research or professional
services, and there are no personal holdings that could be perceived
as constituting a potential conflict of interest.
Author contributions
AK and MLC conceived and designed the work, performed data
analysis, writing and edition of the manuscript; AK, CL, BSP and AV-
F performed glucose uptake in slices and in the cell cultures; ROB,
PA and RJR made the cell cultures; AMM-M assessed glucose in
plasma, genotyped by PCR and performed the respective data
analysis; LG-G and MAP conducted the PET studies and the
respective data analysis; MLC and SJP performed the endocanna-
binoid hydrolysis assay; and RAC interpreted results, and contrib-
uted to the writing and the edition of the manuscript, as well as to
the purchase of some supplies. All authors have reviewed and
commented on the manuscript.
Acknowledgements
This work was supported by the Council of Madrid (S-BIO/0170/
2006 and P2010/BMD-2349 to MLC and MAP). AMM-M received a
fellowship from the Spanish Ministry of Education and Science.
Authors from the University of Coimbra acknowledge funding from
the following sources: NARSAD, Santa Casa da Miserco rdia, DARPA
(09-68-ESR-FP-010), FEDER (QREN) (CENTRO-07-ST24-FEDER-
002006), Programa Mais Centro under project CENTRO-07-ST24-
FEDER-002006 and Programa Operacional Factores de Com-
petitividade - COMPETE, and national funds (PTDC/SAU-OSM/
527
105663/2008 and Pest-C/SAU/LA0001/2013e2014) via
FCTeFundaç~ ^ncia e a Tecnologia.
ao para a Cie
We thank Dr M. Delgado for excellent assistance in the PET
studies, and Drs L. M. García-Segura and M. A. Arevalo for com-
ments on the manuscript. We are grateful to Drs Catherine Ledent
and Catherine Vroonen (IRIBHM, Brussels) for providing the CB1R
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.neuropharm.2016.03.015.
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