a role for hypothalamic amp-activated protein kinase in the mediation of hyperphagia and weight gain...
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ORIGINAL RESEARCH
A Role for Hypothalamic AMP-Activated Protein Kinasein the Mediation of Hyperphagia and Weight Gain Inducedby Chronic Treatment with Olanzapine in Female Rats
Ei Sejima • Atsushi Yamauchi • Tsuyoshi Nishioku •
Mitsuhisa Koga • Kengo Nakagama •
Shinya Dohgu • Kojiro Futagami • Yasufumi Kataoka
Received: 3 December 2010 / Accepted: 12 February 2011 / Published online: 17 June 2011
� Springer Science+Business Media, LLC 2011
Abstract Olanzapine is known to be advantageous with
respect to outcome and drug compliance in patients with
schizophrenia. However, olanzapine has adverse effects,
including a higher incidence of weight gain and metabolic
disturbances, when compared with those of other antipsy-
chotic agents. The mechanisms underlying these adverse
events remain obscure. Female rats were orally adminis-
tered olanzapine (2 mg/kg) or vehicle once a day for
2 weeks to ascertain if hypothalamic AMP-activated pro-
tein kinase (AMPK) mediates olanzapine-induced weight
gain and hyperphagia. Body weight and food intake in each
rat were evaluated every day and every two days, respec-
tively. After the termination of drug treatment, we mea-
sured the protein levels of AMPK and phosphorylated
AMPK in the hypothalamus using western blot analyses.
Olanzapine significantly increased body weight and food
intake. The phosphorylation levels of AMPK were signif-
icantly elevated by olanzapine. These results suggest that
activation of hypothalamic AMPK may mediate hyper-
phagia and weight gain induced by chronic treatment with
olanzapine.
Keywords Olanzapine � Weight gain � Hyperphagia �AMP-activated protein kinase � Hypothalamus
Introduction
Atypical antipsychotic drugs are known to have significant
advantages in the treatment of schizophrenia. However, most
of these drugs induce a disturbance in energy homeostasis
which can lead to hyperphagia, weight gain, obesity, diabetes
mellitus, and lipid abnormalities (Lieberman et al. 2005).
Among these drugs, olanzapine produces the most serious
weight gain as revealed by several clinical trials and meta-
analyses (Allison et al. 1999; Lieberman et al. 2005; Leucht
et al. 2009). Olanzapine-induced weight gain has become
one of the major causes of non-compliance for drug intake,
thereby exposing the patient to an increased risk of relapse to
psychotic symptoms. The mechanisms underlying olanza-
pine-induced weight gain are not completely understood.
Several reports suggest that drug-induced changes in appe-
tite and food intake are involved in these adverse effects
(Cooper et al. 2005; Coccurello et al. 2006; Davoodi et al.
2009). Among many in vivo and in vitro studies on this
mechanism, some authors have suggested that antagonism of
the receptors for monoamine neurotransmitters, including
dopamine, histamine, and serotonin, contributes to drug-
induced weight gain (Kirk et al. 2009; Yoon et al. 2010; Deng
et al. 2010). However, other mechanisms also have been
proposed in the mediation of olanzapine-induced weight
gain (Starrenburg and Bogers 2009).
Adenosine monophosphate-activated protein kinase
(AMPK) is an ‘‘energy sensor’’ that responds to hormone and
nutrition status and regulates a systemic energy homeostasis
in vivo. In the brain, hypothalamic AMPK has emerged as a
‘‘master regulator’’ of energy metabolism. Recent studies
E. Sejima � A. Yamauchi � T. Nishioku � M. Koga �K. Nakagama � S. Dohgu � Y. Kataoka (&)
Department of Pharmaceutical Care and Health Sciences,
Fukuoka University, 8-19-1 Nanakuma, Jonan-ku,
Fukuoka 814-0180, Japan
e-mail: ykataoka@fukuoka-u.ac.jp
E. Sejima � K. Futagami
Department of Pharmacy, Fukuoka University Hospital,
7-45-1, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
Y. Kataoka
BBB Laboratory, PharmaCo-Cell Co. Ltd,
Nagasaki 852-8523, Japan
123
Cell Mol Neurobiol (2011) 31:985–989
DOI 10.1007/s10571-011-9663-8
have suggested that the stimulatory or inhibitory effects of
various agents on appetite are mediated by hypothalamic
AMPK. Orexigenic compounds (adiponectin, ghrelin, etc.)
and anorexigenic compounds (leptin, insulin, a-lipoic acid,
metformin, etc. ) activate and inhibit hypothalamic AMPK,
respectively (Andersson et al. 2004; Minokoshi et al. 2004;
Kim et al. 2004; Chau-Van et al. 2007; Kubota et al. 2007).
Recently, Kim et al. have reported that acute treatment with
olanzapine activated AMPK in the hypothalamic slices in
mice (Kim et al. 2007). In addition, Martins et al. reported
that phosphorylation of hypothalamic AMPK was increased
in rats acutely injected with olanzapine (Martins et al. 2010).
However, with special reference to effects of chronic treat-
ment with olanzapine, the relationship between the altered
activities of the hypothalamic AMPK and increased food
intake and body weight has not yet been clarified.
In this study, we investigated the phosphorylation levels
of hypothalamic AMPK after chronic treatment with
olanzapine in female rats to ascertain if the active status of
hypothalamic AMPK was involved in olanzapine-induced
weight gain and hyperphagia.
Materials and Methods
Ethical Approval of the Study Protocol
All the procedures involving experimental animals adhered
to the law (number 105) and notification (number 6) of the
Japanese Government. The study protocol was approved
by the Laboratory Animal Care and Use Committee of
Fukuoka University (Fukuoka, Japan).
Animals
Female Sprague–Dawley rats (age, 6 weeks) were pur-
chased from Kyudo (Saga, Japan). They were housed
individually in cages under a 12-h light/dark cycle (lights
on at 7 am) at a temperature of 23 ± 2�C with free access
to food and water.
Drugs
Olanzapine orally disintegrating tablets (Zyprexa�, Zydis�;
Eli Lilly, Tokyo, Japan) was dissolved in 0.2% acetic acid
in 0.9% saline and adjusted to pH 6 with 1 M NaOH.
Vehicle solutions consisted of the same solution utilized to
dissolve olanzapine.
Chronic treatment with olanzapine
Rats were divided into two groups (each group with equal
mean body weight). Olanzapine or vehicle was orally
administered once a day (between 8 and 10 am) for
2 weeks. The dosage of olanzapine was 2 mg/kg. This dose
was determined according to reports showing that olanza-
pine (2 mg/kg) caused significant hyperphagia and gain in
body weight similar to our present paradigms (Davoodi
et al. 2009; Cooper et al. 2005).
Body weight was measured every day, just before
olanzapine administration. Food intake was calculated as
the difference between before feeding and 2 days after
feeding in the amount of food placed on the grid cover. The
amount of food intake included food spillage because food
spillage was negligible.
After 2 weeks, rats were killed by decapitation and
brains immediately removed. The hypothalamus was dis-
sected on an ice-cold glass plate and tissues rapidly frozen
in liquid nitrogen. The frozen tissues were stored at -80�C
until western blot analyses.
Western Blot Analyses
The phosphorylation levels of hypothalamic AMPK in rats
were investigated by western blot analyses. Frozen tissues
were homogenized in lysis buffer (10 mM Tris–HCl
[pH = 6.8], 100 mM NaCl, 1 mM ethylenediamine tetra-
acetic acid (EDTA), 10% glycerol, 1% TritonX, 0.1%
sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate,
20 mM sodium pyrophosphate dechohydrate (Napp),
2 mM Na3VO4, 1 mM NaF, 1 mM phenylmethylsulfonyl
fluoride (PMSF), 0.4% protease inhibitor cocktail, and
0.2% phosphatase inhibitor cocktail of I and II). Protein
concentrations of the tissue lysates were determined by the
bicinchoninic acid (BCA) method. Tissue lysates were
separated by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) and transferred to polyvi-
nylidene difluoride membranes. Membranes were immu-
noblotted with rabbit anti-AMPK (Cell Signaling
Technology, Beverly, MA, USA) and rabbit anti-phospho-
AMPK (Thr172; Cell Signaling Technology) antibodies.
Immunoblots were then exposed to peroxidase-conjugated
secondary antibodies (GE Healthcare, Chalfont, UK) and
visualized using an Amersham ECL PlusTM Western
Blotting Detection System (GE Healthcare). Densitometric
analyses were undertaken using a FluorChem SP imaging
system with AlphaEaseFC software (Alpha Innotech, San
Leandro, CA).
Statistical Analyses
Data are means ± S.E.M. Statistical analyses were carried
out using the Student’s t-test. The differences between the
means were considered significant at P \ 0.05.
986 Cell Mol Neurobiol (2011) 31:985–989
123
Results
Effects of olanzapine on body weight and food intake
Figure 1a shows time-course of body weight in rats
treated with olanzapine (2 mg/kg, p.o.) or vehicle once a
day for 2 weeks. The body weight in rats administered
with olanzapine was larger (though not significantly) than
that in rats administered with vehicle. However, the total
gain in body weight in rats treated with olanzapine for
2 weeks was significantly increased when compared with
vehicle (67.2 ± 3.3 g and 54.8 ± 3.4 g, respectively;
Fig. 1b).
Food intake measured every 2 days for 2 weeks in rats
treated with olanzapine (2 mg/kg) or vehicle is shown in
Fig. 1c. Olanzapine, but not vehicle, time-dependently
increased food intake. This increased food intake over time
became significant at 12 days after the start of adminis-
tration. The total food intake during a 2-week period in
olanzapine-treated rats was significantly higher than that in
the vehicle group (262.6 ± 11.2 g and 230.5 ± 8.0 g,
respectively; Fig. 1d).
Effect of olanzapine on hypothalamic AMPK
phosphorylation
Chronic treatment with olanzapine (2 mg/kg) for 2 weeks
caused a significant increase in the phosphorylation levels
of hypothalamic AMPK in rats when compared with
vehicle (Fig. 2). The ratio of phosphorylated AMPK to
total AMPK in olanzapine-treated rats was increased by
33% compared to that of vehicle.
Discussion
This study demonstrated that chronic administration of
olanzapine (2 mg/kg, p.o.) significantly increased the gain
in body weight and total food intake in female rats. The
phosphorylation levels of hypothalamic AMPK in these
rats were significantly higher than those in vehicle-treated
rats.
Serious gains in body weight and hyperphagia are
frequently observed in patients treated with olanzapine
(Allison et al. 1999; Lieberman et al. 2005; Leucht et al.
Fig. 1 Effects of chronic
treatment with olanzapine on
the body weight and food intake
in female rats. Body weight
(a) and food intake (c) were
measured every day and every
2 days, respectively, in female
rats treated with olanzapine
(2 mg/kg, p.o.) or vehicle (p.o.)
once a day for 2 weeks. From
12 days after the first
administration, the amounts of
food intake in olanzapine-
treated rats were significantly
increased when compared with
vehicle-treated rats. Total
weight gain (b) and food intake
(d) during a 2-week period were
significantly increased when
compared with each vehicle
group. Values are
means ± S.E.M. (n = 5).
*P \ 0.05, significantly
different from vehicle-treated
rats
Cell Mol Neurobiol (2011) 31:985–989 987
123
2009). Several animal studies have been undertaken to
elucidate the mechanisms underlying these adverse effects.
In this study, the increases in body weight in rats became
detectable 7 days after the first administration of olanza-
pine. This phenomenon was related to the early occurrence
of olanzapine-induced hyperphagia within 4 days after the
first administration. These findings with female rats are
consistent with observations by other authors (Cooper et al.
2005; Kalinichev et al. 2005; Albaugh et al. 2006; Choi
et al. 2007; Davoodi et al. 2009). Figure 1c shows that
olanzapine-induced significant hyperphagia started 12 days
after the first administration. Based on those findings
together with this report, the drug levels that accumulate
over several days after the first administration are likely to
be required for olanzapine-induced hyperphagia. This
hyperphagia may, at least in part, contribute to olanzapine-
induced weight gain. Arjona et al. reported that olanzapine-
induced hyperphagia showed a delayed onset (Arjona et al.
2004), supporting the results of this study. The mechanisms
underlying this delayed appearance are unknown.
Kim et al. and Martins et al. have already shown olan-
zapine-increased phosphorylation of AMPK in the hypo-
thalamic slices in vitro and in the rat hypothalamus in vivo,
respectively (Kim et al. 2007; Martins et al. 2010). A short
period of olanzapine was exposed to tissues in vitro (Kim
et al. 2007). A single treatment with olanzapine was carried
out, and food intake and body weight were not evaluated in
vivo. Therefore, we investigated if the phosphorylation
levels of hypothalamic AMPK were related to olanzapine-
induced weight gain and hyperphagia in rats. These levels
were significantly elevated in rats treated with olanzapine
for 2 weeks. AMPK is activated when phosphorylated at
Thy172 of a subunit. The increased phosphorylation of
hypothalamic AMPK is followed by an increase in food
intake and weight gain in rodents (Andersson et al. 2004;
Minokoshi et al. 2004; Kim et al. 2004; Kubota et al.
2007). This study together with those reports suggests that
olanzapine triggers the induction of increased weight gain
and hyperphagia by activating hypothalamic AMPK. The
oral antidiabetic agent metformin inhibited AMPK activity
and the expression of the orexigenic peptide NPY in cul-
tured hypothalamic neurons (Chau-Van et al. 2007).
Interestingly, several clinical studies showed that metfor-
min prevents olanzapine-induced weight gain (Wu et al.
2008; Baptista et al. 2006). These evidences seem to sup-
port our notion that olanzapine activates hypothalamic
AMPK to induce weight gain and hyperphagia.
In conclusion, we present evidence through this study
that elevation in the phosphorylation levels of hypotha-
lamic AMPK is, at least in part, responsible for weight gain
and hyperphagia induced by chronic treatment of olanza-
pine. Further studies will be required to clarify the precise
mechanisms by which olanzapine activates hypothalamic
AMPK. Based on our findings, AMPK should be consid-
ered to be one of the possible targets for olanzapine-
induced dysregulation of energy homeostasis.
Acknowledgments This study was supported in part by the Grants-
in-Aid for Scientific Research [(B) 17390159], the Grants-in-Aid for
Young Scientists [(Start-up) 18890227 and (Start-up) 20800066], and
the Grants-in-Aid for Young Scientists [(B) 19790199, (B) 21790102,
(B) 21790255, (B) 21790257, and (B) 21790526] from JSPS, Japan,
the Ministry of Health, Labour and Welfare of Japan (H19-nanchi-
ippan-006), the Nakatomi Foundation, Research Foundation ITSUU
Laboratory, and the Kakihara Science and Technology Foundation.
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∗
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