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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