Olanzapine-Induced Hyperphagia and Weight Gain
Associate with Orexigenic Hypothalamic Neuropeptide
Signaling without Concomitant AMPK Phosphorylation
Johan Fernø1,2*, Luis Varela3,4., Silje Skrede1,2., Marı ´a Jesu ´s Va ´zquez3,4, Rube ´n Nogueiras3,4, Carlos
Die ´guez3,4, Antonio Vidal-Puig5, Vidar M. Steen1,2, Miguel Lo ´pez3,4*
1Dr. Einar Martens’ Research Group for Biological Psychiatry, Department of Clinical Medicine, University of Bergen, Bergen, Norway, 2Center for Medical Genetics and
Molecular Medicine, Haukeland University Hospital, Bergen, Norway, 3Department of Physiology, School of Medicine, University of Santiago de Compostela-Instituto de
Investigacio ´n Sanitaria (IDIS), Santiago de Compostela, Spain, 4CIBER Fisiopatologı ´a de la Obesidad y Nutricio ´n (CIBERobn), Santiago de Compostela, Spain, 5Institute of
Metabolic Science, Metabolic Research Laboratories, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom
The success of antipsychotic drug treatment in patients with schizophrenia is limited by the propensity of these drugs to
induce hyperphagia, weight gain and other metabolic disturbances, particularly evident for olanzapine and clozapine.
However, the molecular mechanisms involved in antipsychotic-induced hyperphagia remain unclear. Here, we investigate
the effect of olanzapine administration on the regulation of hypothalamic mechanisms controlling food intake, namely
neuropeptide expression and AMP-activated protein kinase (AMPK) phosphorylation in rats. Our results show that
subchronic exposure to olanzapine upregulates neuropeptide Y (NPY) and agouti related protein (AgRP) and downregulates
proopiomelanocortin (POMC) in the arcuate nucleus of the hypothalamus (ARC). This effect was evident both in rats fed ad
libitum and in pair-fed rats. Of note, despite weight gain and increased expression of orexigenic neuropeptides, subchronic
administration of olanzapine decreased AMPK phosphorylation levels. This reduction in AMPK was not observed after acute
administration of either olanzapine or clozapine. Overall, our data suggest that olanzapine-induced hyperphagia is
mediated through appropriate changes in hypothalamic neuropeptides, and that this effect does not require concomitant
AMPK activation. Our data shed new light on the hypothalamic mechanism underlying antipsychotic-induced hyperphagia
and weight gain, and provide the basis for alternative targets to control energy balance.
Citation: Fernø J, Varela L, Skrede S, Va ´zquez MJ, Nogueiras R, et al. (2011) Olanzapine-Induced Hyperphagia and Weight Gain Associate with Orexigenic
Hypothalamic Neuropeptide Signaling without Concomitant AMPK Phosphorylation. PLoS ONE 6(6): e20571. doi:10.1371/journal.pone.0020571
Editor: Silvana Gaetani, Sapienza University of Rome, Italy
Received February 25, 2011; Accepted May 4, 2011; Published June 13, 2011
Copyright: ? 2011 Fernø ¸ et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors acknowledge the research infrastructure provided by the Norwegian Microarray Consortium (NMC; www.microarray.no), a national FUGE
technology platform (Functional Genomics in Norway; www.fuge.no). The present study has been supported by grants from the Research Council of Norway (incl.
the FUGE program and ‘‘PSYKISK HELSE’’ program), Norwegian Council for Mental Health, Extrastiftelsen Helse og Rehabilitering (JF), Helse Vest RHF, Dr. Einar
Martens Fund, European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreements nu 245009 and 223713 (CD, ML and RN) and nu
018734 (AVP), Xunta de Galicia (ML: 10PXIB208164PR; RN: 2010/14)), Fondo Investigationes Sanitarias (ML: PS09/01880), Ministerio de Educacion y Ciencia (CD:
BFU2008-02001; ML: RyC-2007-00211; RN: RyC-2008-02219 and SAF2009-07049;), Medical Research Council (AVP) and Welcome Trust (AVP). CIBER de
Fisiopatologı ´a de la Obesidad y Nutricio ´n is an initiative of ISCIII. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (JF); email@example.com (ML)
. These authors contributed equally to this work.
The successful use of antipsychotic drugs such as clozapine and
olanzapine in the treatment of schizophrenia is hampered by their
unwanted obesogenic effect and associated metabolic side effects
[1,2]. It is clear that in a medium to long-term perspective,
metabolic dysregulation predisposes to cardiovascular disease
(CVD) and premature death , but even in a shorter perspective,
weight gain may reduce treatment compliance, increasing the risk
of relapse of psychosis . The underlying mechanisms of
antipsychotic-induced weight gain are incompletely understood;
however, their elucidation may identify alternative targetable
pathways controlling energy balance.
Current evidence indicates that antipsychotic-induced weight
gain and lipid disturbances may be explained by the antipsychotics’
and in animal models [5,6,7,8,9]. The molecular events involved in
antipsychotic-induced hyperphagia remain unclear, but the pro-
pensity of the different antipsychotics to increase food intake and
weight gain is correlated with particular patterns of affinity for
serotonergic, histaminergic and muscarinic receptors in the central
nervous system (CNS) . In particular, antagonism at serotonin
5HT2C and histamine H1 receptors in the hypothalamus seems to
of energy intake and expenditure, the hypothalamus integrates a
wide array of afferent signals, including hormones such as leptin,
ghrelin and insulin, by modifying the expression of specific
neuromodulators including orexigenic and anorexigenic neuropep-
tides. These include the orexigenic neuropeptide Y (NPY) and
agouti-related peptide (AgRP), and the anorexigenic neuropeptide
precursors proopiomelanocortin (POMC) and cocaine and am-
phetamine-regulated transcript (CART) [12,13,14,15,16,17]. The
PLoS ONE | www.plosone.org1June 2011 | Volume 6 | Issue 6 | e20571
hypothalamus is organized in anatomically discrete neuronal
clusters known as nuclei, with the arcuate nucleus (ARC) considered
the ‘‘master hypothalamic centre’’ for feeding control [16,17]. The
effect of antipsychotic drugs on the expression of appetite-regulating
hypothalamic neuropeptides has been investigated in rodent
models, but with equivocal results. Hypothalamic expression of
NPY was increased by clozapine  but decreased by olanzapine
 although neither of these studies reported effects on food intake
or weight gain. On the other hand, in other studies monitoring
antipsychotic-induced hyperphagia and weight gain, no transcrip-
tional changes of hypothalamic neuropeptides were found [20,21].
Recent investigations have also linked antipsychotic drug
treatment to alterations in hypothalamic lipid metabolism. In an
acute study on mice, it was proposed that H1 receptor-mediated
activation of hypothalamic AMP-activated protein kinase (AMPK)
represents an important mechanism of action for antipsychotic-
induced hyperphagia . AMPK, a sensor of energy homeostasis
at the cellular level, integrates metabolic signals and regulates
energy balance via modulation of hypothalamic fatty acid
metabolism within the hypothalamus [15,23,24,25,26]. At the
molecular level, AMPK phosphorylation (activation) in the
hypothalamus leads to phosphorylation (inhibition) of acetyl-CoA
carboxylase (ACC), thus reducing the flux of substrates through the
fatty acid biosynthesis pathway and, most importantly, lowering
levels of malonyl-CoA with resultant orexigenic effects [13,27].
Despite the fact that rodent models of antipsychotic-induced
metabolic disturbances do not consistently recapitulate the human
clinical phenotype, they are still extensively used preclinically (for
review; see ). In rats, olanzapine frequently mimics the weight-
promoting effect observed in patients, whereas comparable effects
of clozapine are typically not reproduced in rodents [29,30].
Furthermore, the olanzapine-induced hyperphagia and weight
[29,31,32,33,34,35,36,37] are less robustly demonstrated in male
littermates [29,38,39]. To study potential molecular mechanisms
involved in antipsychotic-induced hyperphagia, we therefore chose
to use female Sprague-Dawley rats subchronically treated with
olanzapine. In addition, acute effects of both olanzapine and
clozapine were investigated in female rats. We demonstrate that
subchronic exposure to olanzapine upregulates the orexigenic
neuropeptides NPY and AgRP and downregulates the anorexi-
genic neuropeptide precursor POMC in the ARC. This effect was
evident in both ad libitum and pair-fed female rats. Notably, despite
weight gain and increases in orexigenic neuropeptides, AMPK
phosphorylation levels were decreased by olanzapine in ad libitum-
fed female rats, suggesting that olanzapine-induced orexigenic
effects and neuropeptide expression changes in the subchronic
setting may be regulated without concomitant AMPK activation.
female ratsand mice
Effect of acute olanzapine administration on
hypothalamic AMPK phosphorylation
Intracerebroventricular (ICV) injection of olanzapine induced no
clear sedative effects at doses up to 20 mg (evaluated through visual
inspection), whereas a clear, but transient sedative effect was evident
at 50 mg. We observed no effect on food intake, measured 1 h or
24 h after injection, at any of the doses tested (data not shown). It
has been demonstrated that in an acute setting, antipsychotic agents
induce hypothalamic activation of AMPK in rodents when
administered at relatively high doses [22,40]. We therefore
measured the levels of phosphorylated (activated) AMPK (pAMPK)
after ICV injection with 50 mg (the highest dose of olanzapine used
in our experiment). No significant alteration in phosphorylation
status was observed 30 minutes after the olanzapine injection
relative to vehicle-treated controls, although we did see a trend
towards increased levels of pAMPK (133617%, P=0.17)
(Figure 1a). No significant effect was observed for phosphorylated
acetyl-CoA carboxylase (pACC; 118626%, P=0.52), a down-
stream target of pAMPK (Figure 1a). In the same experimental
setting, administration of the AMPK activator AICAR induced a
significant increase of both pAMPK (170617%, P,0.01) and
pACC (295654%, P,0.01) (Figure 1b). Similar data were obtained
both for olanzapine and for AICAR 90 minutes after ICV injection
(data not shown). Notably, antipsychotic-induced elevation of
hypothalamic pAMPK levels has consistently been demonstrated
in the acute setting after peripheral injection [22,40]. We therefore
performed an acute IP experiment, where we also included
clozapine at a dose previously shown to induce marked metabolic
changes in peripheral tissues . In order to induce direct drug
effects on AMPK phosphorylation, we chose to use relatively high
doses of both olanzapine (10 mg/kg) and clozapine (25 mg/kg) in
the IP experiment. It should be noted that sedative effects were
evident (by visual inspection) for both drugs, precluding measure-
ments of food intake. Neither clozapine nor olanzapine induced
significant changes in the levels of pAMPK (Figure 2a) or pACC
(Figure 2b), 15 and 30 minutes after injection. Still, a non-significant
trend towards increased pACC levels was observed for both
olanzapine (135610%, P=0.09) and clozapine (142617%,
P=0.11) 15 minutes after injection (Figure 2a).
Subchronic administration of olanzapine increases food
intake and body weight
Next, we investigated the effect of subchronic olanzapine
exposure (6 mg/kg/day) on food intake (Figure 3a) and weight
gain (Figure 3b) in female rats. Repeated-measures two-way
ANOVA was performed for daily food intake with treatment (2
groups) and time (6 days, including day 0, when starting the
treatment) as factors. The analysis for six different time points
revealed a significant main effect of the treatment [F(1,21)=14.27;
p,0.01] and a significant treatment x time interaction effect
[F(5,17)=5.24; p,0.01]. Each time point was subsequently
analysed using Student’s t-test (since only two treatment groups
were present), revealing that daily food intake was significantly
increased in the olanzapine ad libitum group from day 2 onwards
(p,0.05). Similarly, cumulative body weight gain was analyzed
using a two-way ANOVA repeated measures with treatment (3
groups) and time (6 days) as factors. Both significant main effect
[F(2,45)=8.15; p,0.01] and significant treatment x time interac-
tion [F(10,82)=2.52; p,0.05] effect were observed. Olanzapine-
induced body weight gain was significantly increased from control
from day 2 onwards (p,0.05) as determined from one-way
ANOVA analysis, followed by Tukey’s Post-hoc test. Any sedation
caused by the moderately high dose of olanzapine used (6 mg/kg/
day) may potentially have weight-promoting effects. Locomotor
activity was measured only by visual inspection, which is a weakness
in this study. Still, in pair-fed rats offered the same amount food as
control rats, olanzapine exposure did not induce significant weight
Thesedata suggestedthat the weight-promoting effectofolanzapine
is dependent on its orexigenic effects.
Subchronic administration of olanzapine does not affect
serum leptin, insulin, or adiponectin levels
Antipsychotic-induced weight gain has been suggested to be
related to alterations in leptin, adiponectin and insulin serum
levels [28,42]. In our study, subchronic olanzapine exposure
Olanzapine and Hypothalamic AMPK and Neuropeptides
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did not significantly alter serum levels of any of these
endocrine factors, despite marked hyperphagia and weight
gain (Table 1).
Subchronic olanzapine administration decreases
hypothalamic AMPK phosphorylation
In the subchronic setting, hypothalamic pAMPK levels were
measured after 5 days of olanzapine exposure. Interestingly,
we found that pAMPK levels in the hypothalamus of
olanzapine-treated, ad libitum-fed rats were significantly re-
duced (4263%, P,0.0001) relative to vehicle-treated controls
(Figure 4a). Accordingly, olanzapine reduced the levels of
phosphorylated acetyl-CoA carboxylase (pACC;
P,0.05) in ad libitum-fed rats. No significant changes were
observed in pair-fed rats, neither for pAMPK nor for pACC
Figure 1. Effect of ICV olanzapine and AICAR administration on phosphorylation of hypothalamic AMPK and ACC. Western blot
analysis of hypothalamic pAMPK and pACC from rats sacrificed 30 minutes after ICV injection of a) olanzapine or b) AICAR, relative to control rats
(DMSO). Calculations are based on results from 6 rats for each treatment group, run in duplicate. Representative images for the calculated difference
were selected. Each lane (pACC, pAMPK and b-actin) always represents results on the same gel from the same rat. * P#0.05 vs. vehicle. ** P#0.01 vs.
vehicle. *** P#0.001 vs. vehicle.
Figure 2. Effect of IP olanzapine and clozapine administration on phosphorylation of hypothalamic AMPK and ACC. Western blot
analysis of hypothalamic levels of a) pACC or b) pAMPK in rats following IP injection of vehicle (ctrl) olanzapine (olanz; 10 mg/kg), clozapine (cloz;
25 mg/kg). Protein levels were normalized against b-actin as the endogenous control. Statistical calculations were based on results from n=6 rats in
each control group. * P#0.05 vs. vehicle. ** P#0.01 vs. vehicle. *** P#0.001 vs. vehicle.
Olanzapine and Hypothalamic AMPK and Neuropeptides
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Subchronic olanzapine treatment increases mRNA
expression of AgRP and NPY and decreases POMC in the
The observation that pAMPK levels were reduced in the
subchronic experiment was inconsistent with a role of AMPK
activation in olanzapine-induced hyperphagia. We therefore assayed
the expression of key ARC neuropeptides involved in the control of
food intake by using insitu hybridization analysis,considered the most
suitable and robust approach for quantitative mRNA studies in the
hypothalamus. In line with the elevated food intake observed in ad
libitum-fed rats, olanzapine treatment increased mRNA levels of the
orexigenic neuropeptides NPY (147618%, P,0.05; Figure 5a) and
AgRP (12769%, P,0.05; Figure 5b) and reduced mRNA levels of
the anorexigenic POMC (71610%, P,0.05) in the ARC (Figure 5c)
Similar results were observed in pair-fed rats that had not gained
weight, with a marked increase in NPY (160612%, P#0.01) and
AgRP (143612%, P#0.05) and reduced levels of POMC (7668%,
P#0.05). Overall, these data suggest that the changes in neuropep-
tides do not represent secondary effects of olanzapine-induced
hyperphagia. The expression level of the anorexigenic neuropeptide
precursor CART did not change significantly in any of the
olanzapine-treated groups (Figure 5d).
In this study, we investigated acute and subchronic effects of
olanzapine exposure on hypothalamic AMPK as well as
subchronic effects on satiety-regulating neuropeptides in female
rats. In accordance with olanzapine-induced hyperphagia and
increased body weight in the subchronic setting, we observed
increased mRNA expression of the orexigenic neuropeptides NPY
and AgRP, and decreased expression of the anorexigenic
neuropeptide precursor POMC in the ARC. Interestingly, these
changes were also observed in pair-fed rats, with restricted food
intake and no weight gain, demonstrating that the olanzapine-
induced transcriptional changes were primarily caused by
antipsychotic treatment and did not occur secondary to alterations
in feeding pattern and weight changes. Contrary to our initial
hypothesis, we found that olanzapine reduced phosphorylated
levels of AMPK, suggesting that hypothalamic AMPK activation is
not the primary mechanism mediating olanzapine-induced
neuropeptide expression and thus hyperphagia and weight gain
in the subchronic setting. With respect to our acute experiments,
no significant effect on AMPK phosphorylation status was
It has been proposed that the molecular mechanisms underlying
the appetite-stimulating effects of antipsychotic drugs may involve
H1 receptor-mediated activation of hypothalamic AMPK .
This was supported by a recent study demonstrating AMPK
activation in the hypothalamus of male rats following intravenous
injection of olanzapine . In our acute experiments, we
observed a subtle trend towards increased levels of pAMPK after
an acute ICV olanzapine injection and elevated pACC after an
acute IP olanzapine injection. However, no accompanying
measurements of food intake and body weight were reported in
the aforementioned acute study . In our acute study, the
relatively high drug doses induced sedative effects, which
potentially blunted hyperphagic effects.
Based on the recently established orexigenic effects of
hypothalamic AMPK activation [15,23,25] and the previously
suggested role of increased AMPK phosphorylation in antipsy-
chotic-induced weight gain , it was somewhat unexpected that
hypothalamic pAMPK levels and its molecular substrate pACC
were reduced in our experimental setting. It is counterintuitive
that AMPK does not mediate the hyperphagic and weight-
promoting effects of olanzapine, and we speculate that AMPK
phosphorylation may have been stimulated by olanzapine in the
Figure 3. Food intake and body weight following subchronic administration of olanzapine. a) Daily average food intake in groups of rats
(n=8) exposed to olanzapine or vehicle by gavage (b.i.d) for 5 consecutive days. Rats were fasted over night and sacrificed in the morning on day 6.
b) Cumulative weight gain in groups of rats (n=8) treated with vehicle or olanzapine for 5 consecutive days. Total relative weight gain (mean6SEM),
relative to treatment day 0 was as follows: control 3.061.8 g, olanzapine ad libitum 10.861.6 g, olanzapine pair-fed 1.261.4 g. * P#0.05 vs. vehicle.
** P#0.01 vs. vehicle. *** P#0.001 vs. vehicle.
Table 1. Leptin, adiponectin and insulin plasma levels in
control and olanzapine (ad libitum and pair-fed) treated rats.
ctrl olanz pair-fedolanz ad lib
Leptin (ng/ml) 1.7360.19 1.4460.14 1.9460.26
Insulin (ng/ml) 0.4460.040.3660.020.4160.02
Data are expressed as mean6SEM.
Olanzapine and Hypothalamic AMPK and Neuropeptides
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very short term (at initial time points) both in the acute and
subchronic experiments, but that the elevation was not sustained
at the time of dissection, around 20 hours after the last drug dose
in subchronically treated rats. The reduction of pAMPK levels
after subchronic olanzapine treatment was most pronounced in ad
libitum-fed rats, which may suggest the involvement of negative
feedback mechanisms triggered by increased body weight rather
than a direct drug effect. Additionally, sedative effects may
contribute to the weight gain observed in the hyperphagic
olanzapine-treated ad libitum rats. However, the lack of significant
weight gain in olanzapine-treated pair-fed rats strongly suggests
that weight-inducing effect of sedation alone is unlikely.
The transcriptional changes of the appetite-regulating neuropep-
tidesobserved inoursubchronicexperimentare in accordancewith a
recent study in which acute ICV administration of olanzapine
increased hypothalamic expression of both NPY and AgRP .
However, the expression of the anorexigenic POMC was not affected
by olanzapine in this acute study, in contrast with our observation
that POMC expression is reduced. In another subchronic study with
an experimental design resembling ours, no effect was observed on
the expression of hypothalamic neuropeptides after 7 days of
olanzapine treatment in female rats, despite marked hyperphagia
and weight gain . These discrepancies are probably related to
differences in experimental setup, including different drug doses
(2 mg/kg/day versus 6 mg/kg/day in our study), the number of
hours between the last drug dose and sacrifice, the duration of fasting
before sacrifice, and particularly the use of real-time PCR analysis
instead of the more sensitive in situ hybridization when assessing
neuropeptide expression levels in specific neuronal populations. In
this sense, in situ hybridization is a more suitable technique for
studying neuropeptide expression, particularly relevant for neuro-
peptides expressed in more than one hypothalamic nucleus. This is
the case of NPY, which is expressed both in the arcuate (ARC) and
the dorsomedial nuclei (DMH), with ARC expression predominantly
relevant in terms of feeding control [43,44].
Additionally, our findings suggest that regulation of antipsychotic-
induced appetite-controlling neuropeptides may occur without
concomitant AMPK activation. This is supported by the aforemen-
tioned acute study by Martins et al. , demonstrating that
hypothalamic AMPK activation by olanzapine occurs independently
of food intake and without detectable neuropeptide expression
changes following intravenous injection. Indeed, former studies have
demonstrated that regulation of food intake and hypothalamic
neuropeptides does not necessarily depend upon AMPK phosphor-
ylation status. For instance, we previously showed that the anorectic
effect of the drug tamoxifen is exerted by modulation of ARC
neuropeptides through an AMPK-independent mechanism .
notion of a positive correlation between hyperphagia and AMPK
activity, as demonstrated by reduced AMPK activation in hyper-
phagic, hyperthyroid rats  and by resistin-induced AMPK
activation despite the anorexigenic effects of this hormone .
is not mediated by increased AMPK activity and is also independent
ofneuropeptide tone,contrarytoobservations madeinthe acute
setting [23,25]. In fact, it was recently proposed both by us and by
others that in long-term altered nutritional conditions, AMPK-
induced changes in hypothalamic fatty acid metabolismmay not play
been suggested that hypothalamic fatty acid metabolism could be a
regulatory mechanism maintaining energy homeostasis in starvation
In summary, we show in this study that subchronic olanzapine
exposure in female rats induces alterations in the expression of
satiety-controlling neuropeptides in the ARC of hyperphagic rats,
indicating that antipsychotic-induced weight gain may be
mediated via changes in ‘‘classical’’ appetite-regulating neuropep-
tides. Of note, altered neuropeptide expression levels were also
evident in food-restricted rats that did not gain weight,
demonstrating that the olanzapine-induced changes are not
secondary to changes in body weight and/or feeding patterns. In
addition, we demonstrate that phosphorylation levels of AMPK
are reduced by subchronic olanzapine exposure, suggesting that
the role of AMPK in long-term antipsychotic-induced weight gain
Figure 4. Effect of subchronic olanzapine administration on phosphorylation of hypothalamic AMPK and ACC. Western blot analysis
of hypothalamic pAMPK and pACC from rats following an over night fast after 5 consecutive days of administration by gavage (b.i.d) with a)
olanzapine (ad libitum fed) or b) olanzapine (pair-fed), relative to control rats. Calculations are based on results from 6 rats for each treatment group,
run in duplicate. Representative images for the calculated difference were selected. Each lane (pACC, pAMPK and b-actin) always represents results on
the same gel from the same rat. * P#0.05 vs. vehicle. ** P#0.01 vs. vehicle. *** P#0.001 vs. vehicle.
Olanzapine and Hypothalamic AMPK and Neuropeptides
PLoS ONE | www.plosone.org5 June 2011 | Volume 6 | Issue 6 | e20571
may be less robust than anticipated in previous acute studies.
Overall, these data provide new insight into the hypothalamic
mechanism underlying antipsychotic-induced hyperphagia and
weight gain and provide a rationale for the search for alternative
therapeutic targets to control energy balance.
Materials and Methods
All experiments were carried out in accordance with the
guidelines of the Norwegian and Spanish Committees for
Experiments on Animals. In accordance, experiments performed
in Norway were approved by the Norwegian Committee for
Experiments on Animals (Forsøksdyrutvalget, FDU), following
standardized application through the animal facility at Haukeland
University Hospital with ID 20092167. In the same way, all
procedures performed in Spain were also approved by the
University of Santiago de Compostela Institutional Bioethics
Committee, the Xunta de Galicia (Local Government) and the
Ministry of Science and Innovation with ID PS09/01880. Female,
outbred Sprague-Dawley rats (Mollegaard, Denmark and the
University of Santiago de Compostela Animal House) weighing
Figure 5. Effect of subchronic olanzapine administration on appetite-regulating neuropeptides. Expression levels of the appetite-
regulating neuropeptides in the arcuate nucleus following 5 days of treatment (b.i.d) with vehicle (ctrl), olanzapine with food restriction (olanz pair-
fed) or olanzapine with free access to food (olanz ad libitum). Calculations are based on results from groups of rats (n=8) from each treatment group
fasted over night and killed in the morning on day 6. Representative images demonstrating the calculated differences were selected. Delineated areas
are shown at higher magnification at the bottom. * P#0.05 vs. vehicle. ** P#0.01 vs. vehicle. *** P#0.001 vs. vehicle.
Olanzapine and Hypothalamic AMPK and Neuropeptides
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between 230 g and 250 g on the first day of treatment were
housed individually under standard conditions with an artificial
12:12 hrs light/dark cycle under constant 48% humidity. Animals
were allowed free access to tap water and fed with standard
laboratory chow during the experimental periods, as described
Olanzapine was dissolved in 0.1 M hydrochloric acid (HCl) and
pH was adjusted to 5.5 using 0.1 M sodium hydroxide (NaOH).
Stock solutions of 1.5 mg/ml were prepared and ,0.5 ml of this
solution was administered to the rats via gavage, twice daily (the
actual volume was corrected for variation in body weight so that
for each rat, each of the two daily doses was 3 mg/kg). For IP
experiments, both olanzapine and clozapine solutions were
prepared the same way, with the appropriate concentrations.
For ICV injections, olanzapine and AICAR were dissolved in
DMSO, which was used as vehicle.
Female rats had free access (ad libitum) to food and tap water
throughout the experiment. Rats were acutely administered with
olanzapine, either by intracerebroventricular (ICV) injection or by
intraperitoneal (IP) injection. In the ICV experiment, cannulae
were surgically implanted in
[23,24,25,50]. After 3 days of recovery, rats were injected ICV
with vehicle (DMSO, 10 ml), olanzapine (50 mg) or the AMPK
(AICAR; 50 mg) and sacrificed after 30 or 90 minutes. In the
intraperitoneal (IP) experiment, female rats were sacrificed 15 or
30 minutes after administration of vehicle (saline), olanzapine
(10 mg/kg) or clozapine (25 mg/kg). Whole brain was dissected
out, frozen immediately on dry ice and stored at 280uC until
Female rats were exposed to either vehicle (saline) or olanzapine
(3 mg/kg), administered twice daily (total daily dose: 6 mg/kg) by
gavage (9 a.m. and 3 p.m.) for 6 days, and sacrificed on day 7 after
an overnight fast. The dose used is relatively high as compared to
other studies, but has been shown to robustly induce hyperphagia
and weight gain in mice  as well as in rats in our laboratory
(unpublished results). In order to explore whether olanzapine
could induce metabolic alterations independent of weight gain, we
also included a pair-fed olanzapine-treated group in which the
animals received an amount of food corresponding to that
consumed by the control group during the previous 24 hours.
To avoid binge eating, the pair-fed animals received 1/3 of the
relevant amount of chow at 9.30 a.m., and the remaining 2/3 at 3
p.m. each day. Food intake and weight were measured daily for
each animal. The last drug dose prior to sacrifice was administered
18–20 hours prior to decapitation. All animals were fasted from 9
p.m. on the day prior to euthanasia, with dissection starting at 9
a.m. the following day. Prior to decapitation, animals were
anesthesized using isoflourane. Like in the acute experiment,
whole brain was dissected out from all animals, frozen immedi-
ately on dry ice and stored at 280uC until processed. The brains
were either used for in situ hybridization analysis (half of the
animals) or western blot analysis (other half).
Serum insulin, leptin and adiponectin measurements
Truncal vein blood was collected in EDTA tubes, left on ice for
30 minutes and centrifuged at 3,000 rpm for 10 minutes. Serum
was transferred to pre-cooled Eppendorf tubes immediately after
centrifugation and stored at -20uC. Serum insulin, leptin and
adiponectin levels were assessed by means of a double-antibody
radioimmunoassay (Linco Research, USA), as previously de-
scribed [23,25,46]. All samples were assayed in duplicate within
one assay, and the results were expressed in terms of the insulin,
leptin or adiponectin standards.
In situ hybridization
Coronal hypothalamic sections (16 mm) were cut on a cryostat
and immediately stored at 280uC until hybridization. We used
specific oligos for detection of AgRP, NPY, CART and POMC
mRNAs. These probes were 39-end labeled with 35S-adATP using
terminal deoxynucleotidyl transferase (Amersham Biosciences,
UK). We performed in situ hybridizations as previously published
[24,25,52]. Similar anatomical regions were analyzed using the rat
brain atlas of Paxinos & Watson . The slides from all
experimental groups were exposed on the same autoradiographic
film. All sections were scanned and the specific hybridization
signal was quantified by densitometry using the ImageJ software
(National Institute of Health, USA). We determined the optical
density of the hybridization signal and subsequently corrected by
the optical density of its adjacent background value. A rectangle,
with the same dimensions in each case, was drawn enclosing the
hybridization signal over each nucleus and over adjacent brain
areas of each section (background) as previously described
[24,25,52]. For the in situ analysis we included 8 animals per
experimental group. We used between 16 and 20 sections for each
animal (4–5 slides with four sections per slide). The mean of these
16–20 values was used as the densitometry value for each animal.
Dissected hypothalami were homogenized in lysis buffer and
centrifuged at 12000 g for 10 minutes at 4uC. 40 mg of total
protein from each sample were separated on SDS-PAGE gels and
blotted onto PVDF membranes. PVDF membranes were blocked
with 5% BSA in 0.1% TBST prior to incubation with primary
antibody at 4uC overnight, followed by incubation with secondary
antibody at room temperature for one hour, as previously
described [23,52]. The primary antibodies used were: pACCa-
Ser79(Upstate, USA), pAMPKa-Thr172(Cell signalling Technol-
ogy, USA) and b-actin (Abcam, UK). Signal intensity measure-
ments were performed using the ImageJ software (National
Institutes of Health, USA).
Food intake in the subchronic experiment was analyzed by two-
way ANOVA repeated measures with treatment (2 groups; control
and olanzapine ad libitum fed) as between-subject variable and
time (6 days) as within-subject variable. Body weight changes was
analyzed using the same method, with treatment (3 groups;
control, olanzapine ad libitum fed and olanzapine pair-fed) and
time (6 days) as factors. When a significant interaction effect from
the two-way ANOVA was obtained, Student’s t-test or one-way
ANOVA, followed by Tukey’s post-hoc test, was used to analyze
statistical significance for each time point. All data are expressed as
mean6SEM. All tests were conducted with PASW Statistics
Version 18 (PASW statistics; SPSS, USA) software. A significance
level of P=0.05 was used.
We highly appreciate the excellent technical assistance from Marianne S.
Nævdal in the animal facility.
Olanzapine and Hypothalamic AMPK and Neuropeptides
PLoS ONE | www.plosone.org7June 2011 | Volume 6 | Issue 6 | e20571
Conceived and designed the experiments: JF SS RN CD AV-P VMS ML.
Performed the experiments: JF LV SS MJV. Analyzed the data: JF LV SS
MJV. Wrote the paper: JF ML. Discussed the manuscript: JF SS RN CD
AV-P VMS ML.
1. Allison DB, Mentore JL, Heo M, Chandler LP, Cappelleri JC, et al. (1999)
Antipsychotic-induced weight gain: a comprehensive research synthesis.
Am J Psychiatry 156: 1686–1696.
2. American Diabetes Association APA, American Association of Clinical
Endocrinologists, North American Association for the Study of Obesity. (2004)
Consensus development conference on antipsychotic drugs and obesity and
diabetes. J Clin Psychiatry 65: 267–272.
3. Colton CW, Manderscheid RW (2006) Congruencies in increased mortality
rates, years of potential life lost, and causes of death among public mental health
clients in eight states. Prev Chronic Dis 3: A42.
4. Weiden PJ, Mackell JA, McDonnell DD (2004) Obesity as a risk factor for
antipsychotic noncompliance. Schizophr Res 66: 51–57.
5. Blouin M, Tremblay A, Jalbert ME, Venables H, Bouchard RH, et al. (2008)
Adiposity and eating behaviors in patients under second generation antipsy-
chotics. Obesity (Silver Spring) 16: 1780–1787.
6. Cooper GD, Pickavance LC, Wilding JP, Halford JC, Goudie AJ (2005) A
parametric analysis of olanzapine-induced weight gain in female rats.
Psychopharmacology (Berl) 181: 80–89.
7. Pouzet B, Mow T, Kreilgaard M, Velschow S (2003) Chronic treatment with
antipsychotics in rats as a model for antipsychotic-induced weight gain in
human. Pharmacol Biochem Behav 75: 133–140.
8. Hartfield AW, Moore NA, Clifton PG (2003) Effects of clozapine, olanzapine
and haloperidol on the microstructure of ingestive behaviour in the rat.
Psychopharmacology (Berl) 167: 115–122.
9. Hartfield AW, Moore NA, Clifton PG (2006) Effects of atypical antipsychotic
drugs on intralipid intake and cocaine-induced hyperactivity in rats. Neuropsy-
chopharmacology 31: 1938–1945.
10. Nasrallah HA (2008) Atypical antipsychotic-induced metabolic side effects:
insights from receptor-binding profiles. Mol Psychiatry 13: 27–35.
11. Reynolds GP, Kirk SL (2010) Metabolic side effects of antipsychotic drug
treatment--pharmacological mechanisms. Pharmacol Ther 125: 169–179.
12. Carling D (2004) The AMP-activated protein kinase cascade--a unifying system
for energy control. Trends Biochem Sci 29: 18–24.
13. Hu Z, Cha SH, Chohnan S, Lane MD (2003) Hypothalamic malonyl-CoA as a
mediator of feeding behavior. Proc Natl Acad Sci U S A 100: 12624–12629.
14. Kahn BB, Alquier T, Carling D, Hardie DG (2005) AMP-activated protein
kinase: ancient energy gauge provides clues to modern understanding of
metabolism. Cell Metab 1: 15–25.
15. Lage R, Die ´guez C, Vidal-Puig A, Lo ´pez M (2008) AMPK: a metabolic gauge
regulating whole-body energy homeostasis. Trends Mol Med 14: 539–549.
16. Lo ´pez M, Lelliott CJ, Vidal-Puig A (2007) Hypothalamic fatty acid metabolism:
a housekeeping pathway that regulates food intake. Bioessays 29: 248–261.
17. Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW (2006)
Central nervous system control of food intake and body weight. Nature 443:
18. Kirk SL, Cahir M, Reynolds GP (2006) Clozapine, but not haloperidol,
increases neuropeptide Y neuronal expression in the rat hypothalamus.
J Psychopharmacol 20: 577–579.
19. Huang XF, Deng C, Zavitsanou K (2006) Neuropeptide Y mRNA expression
levels following chronic olanzapine, clozapine and haloperidol administration in
rats. Neuropeptides 40: 213–219.
20. Davoodi N, Kalinichev M, Korneev SA, Clifton PG (2009) Hyperphagia and
increased meal size are responsible for weight gain in rats treated sub-chronically
with olanzapine. Psychopharmacology (Berl) 203: 693–702.
21. Guesdon B, Denis RG, Richard D (2010) Additive effects of olanzapine and
melanin-concentrating hormone agonism on energy balance. Behav Brain Res
Antipsychotic drug-induced weight gain mediated by histamine H1 receptor-linked
activation of hypothalamic AMP-kinase. Proc Natl Acad Sci U S A 104: 3456–3459.
23. Lo ´pez M, Lage R, Saha AK, Pe ´rez-Tilve D, Va ´zquez MJ, et al. (2008)
Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin.
Cell Metab 7: 389–399.
24. Lo ´pez M, Lelliott CJ, Tovar S, Kimber W, Gallego R, et al. (2006) Tamoxifen-
induced anorexia is associated with fatty acid synthase inhibition in the
ventromedial nucleus of the hypothalamus and accumulation of malonyl-CoA.
Diabetes 55: 1327–1336.
25. Lo ´pez M, Varela L, Va ´zquez MJ, Rodriguez-Cuenca S, Gonzalez CR, et al.
(2010) Hypothalamic AMPK and fatty acid metabolism mediate thyroid
regulation of energy balance. Nat Med 16: 1001–1008.
26. Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, et al. (2004) AMP-kinase
regulates food intake by responding to hormonal and nutrient signals in the
hypothalamus. Nature 428: 569–574.
27. Lane MD, Wolfgang M, Cha SH, Dai Y (2008) Regulation of food intake and
energy expenditure by hypothalamic malonyl-CoA. Int J Obes (Lond) 32 Suppl
28. Boyda HN, Tse L, Procyshyn RM, Honer WG, Barr AM (2010) Preclinical
models of antipsychotic drug-induced metabolic side effects. Trends Pharmacol
Sci 31: 484–497.
29. Albaugh VL, Henry CR, Bello NT, Hajnal A, Lynch SL, et al. (2006) Hormonal
and metabolic effects of olanzapine and clozapine related to body weight in
rodents. Obesity (Silver Spring) 14: 36–51.
30. Cooper GD, Harrold JA, Halford JC, Goudie AJ (2008) Chronic clozapine
treatment in female rats does not induce weight gain or metabolic abnormalities
but enhances adiposity: implications for animal models of antipsychotic-induced
weight gain. Prog Neuropsychopharmacol Biol Psychiatry 32: 428–436.
31. Goudie AJ, Smith JA, Halford JC (2002) Characterization of olanzapine-induced
weight gain in rats. J Psychopharmacol 16: 291–296.
32. Arjona AA, Zhang SX, Adamson B, Wurtman RJ (2004) An animal model of
antipsychotic-induced weight gain. Behav Brain Res 152: 121–127.
33. Coccurello R, Brina D, Caprioli A, Conti R, Ghirardi O, et al. (2009) 30 days of
continuous olanzapine infusion determines energy imbalance, glucose intoler-
ance, insulin resistance, and dyslipidemia in mice. J Clin Psychopharmacol 29:
34. Coccurello R, Caprioli A, Conti R, Ghirardi O, Borsini F, et al. (2008)
Olanzapine (LY170053, 2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-
b][1,5] benzodiazepine), but not the novel atypical antipsychotic ST2472 (9-
piperazin-1-ylpyrrolo[2,1-b][1,3]benzothiazepine), chronic administration in-
duces weight gain, hyperphagia, and metabolic dysregulation in mice.
J Pharmacol Exp Ther 326: 905–911.
35. Coccurello R, Caprioli A, Ghirardi O, Conti R, Ciani B, et al. (2006) Chronic
administration of olanzapine induces metabolic and food intake alterations: a
mouse model of the atypical antipsychotic-associated adverse effects. Psycho-
pharmacology (Berl) 186: 561–571.
36. Coccurello R, D’Amato FR, Moles A (2008) Chronic administration of
olanzapine affects Behavioral Satiety Sequence and feeding behavior in female
mice. Eat Weight Disord 13: e55–60.
37. Fell MJ, Marshall KM, Williams J, Neill JC (2004) Effects of the atypical
antipsychotic olanzapine on reproductive function and weight gain in female
rats. J Psychopharmacol 18: 149–155.
38. Minet-Ringuet J, Even PC, Goubern M, Tome D, de Beaurepaire R (2006)
Long term treatment with olanzapine mixed with the food in male rats induces
body fat deposition with no increase in body weight and no thermogenic
alteration. Appetite 46: 254–262.
39. Minet-Ringuet J, Even PC, Lacroix M, Tome D, de Beaurepaire R (2006) A
model for antipsychotic-induced obesity in the male rat. Psychopharmacology
(Berl) 187: 447–454.
40. Martins PJ, Haas M, Obici S (2010) Central nervous system delivery of the
antipsychotic olanzapine induces hepatic insulin resistance. Diabetes 59:
41. Ferno J, Vik-Mo AO, Jassim G, Havik B, Berge K, et al. (2009) Acute clozapine
exposure in vivo induces lipid accumulation and marked sequential changes in
the expression of SREBP, PPAR, and LXR target genes in rat liver.
Psychopharmacology (Berl) 203: 73–84.
42. Jin H, Meyer JM, Mudaliar S, Jeste DV (2008) Impact of atypical antipsychotic
therapy on leptin, ghrelin, and adiponectin. Schizophr Res 100: 70–85.
43. Lo ´pez M, Seoane LM, Tovar S, Garcı ´a MC, Nogueiras R, et al. (2005) A
possible role of neuropeptide Y, agouti-related protein and leptin receptor
isoforms in hypothalamic programming by perinatal feeding in the rat.
Diabetologia 48: 140–148.
44. Lo ´pez M, Tovar S, Va ´zquez MJ, Williams LM, Die ´guez C (2007) Peripheral
tissue-brain interactions in the regulation of food intake. Proc Nutr Soc 66:
45. Vazquez MJ, Gonzalez CR, Varela L, Lage R, Tovar S, et al. (2008) Central
resistin regulates hypothalamic and peripheral lipid metabolism in a nutritional-
dependent fashion. Endocrinology 149: 4534–4543.
46. Sangiao-Alvarellos S, Varela L, Vazquez MJ, Da Boit K, Saha AK, et al. (2010)
Influence of ghrelin and growth hormone deficiency on AMP-activated protein
kinase and hypothalamic lipid metabolism. Journal of neuroendocrinology 22:
47. Andrews ZB, Liu ZW, Walllingford N, Erion DM, Borok E, et al. (2008) UCP2
mediates ghrelin’s action on NPY/AgRP neurons by lowering free radicals.
Nature 454: 846–851.
48. Varela L, Vazquez MJ, Cordido F, Nogueiras R, Vidal-Puig A, et al. (2011)
Ghrelin and lipid metabolism: key partners in energy balance. Journal of
molecular endocrinology 46: R43–63.
49. Lo ´pez M (2008) The AMPK-malonyl-CoA-CPT1 axis in the control of
hypothalamic neuronal function. Cell Metab 8: 176–176.
50. Lage R, Vazquez MJ, Varela L, Saha AK, Vidal-Puig A, et al. (2010) Ghrelin
effects on neuropeptides in the rat hypothalamus depend on fatty acid
metabolism actions on BSX but not on gender. The FASEB journal : official
Olanzapine and Hypothalamic AMPK and Neuropeptides
PLoS ONE | www.plosone.org8 June 2011 | Volume 6 | Issue 6 | e20571
publication of the Federation of American Societies for Experimental Biology Download full-text
51. Cope MB, Nagy TR, Fernandez JR, Geary N, Casey DE, et al. (2005)
Antipsychotic drug-induced weight gain: development of an animal model.
Int J Obes (Lond) 29: 607–614.
52. Chakravarthy MV, Zhu Y, Lo ´pez M, Yin L, Wozniak DF, et al. (2007) Brain
fatty acid synthase activates PPARalpha to maintain energy homeostasis. The
Journal of clinical investigation 117: 2539–2552.
53. Paxinos G (1986) The rat brain in stereotaxic coordinates. Sydney: Academic
Olanzapine and Hypothalamic AMPK and Neuropeptides
PLoS ONE | www.plosone.org9 June 2011 | Volume 6 | Issue 6 | e20571