Olanzapine induced weight gain in the rat: Impact on inflammatory, metabolic and microbiota parameters

Full text

ORIGINAL INVESTIGATION
Gender-dependent consequences of chronic olanzapine
in the rat: effects on body weight, inflammatory, metabolic
and microbiota parameters
Kieran J. Davey &Siobhain M. O’Mahony &Harriet Schellekens &Orla O’Sullivan &
John Bienenstock &Paul D. Cotter &Timothy G. Dinan &John F. Cryan
Received: 29 July 2011 /Accepted: 21 October 2011 / Published online: 11 January 2012
#Springer-Verlag 2012
Abstract
Rationale Atypical antipsychotic drugs (AAPDs) such as
olanzapine have a serious side effect profile including
weight gain and metabolic dysfunction, and a number of
studies have suggested a role for gender in the susceptibility
to these effects. In recent times, the gut microbiota has been
recognised as a major contributor to the regulation of body
weight and metabolism. Thus, we investigated the effects of
olanzapine on body weight, behaviour, gut microbiota and
inflammatory and metabolic markers in both male and
female rats.
Methods Male and female rats received olanzapine (2 or
4 mg/kg/day) or vehicle for 3 weeks. Body weight, food
and water intake were monitored daily. The faecal microbial
content was assessed by 454 pyrosequencing. Plasma cyto-
kines (tumour necrosis alpha,interleukin 8 (IL-8), interleuin-6
and interleukin 1-beta (IL-1β)) as well as expression of genes
including sterol-regulatory element binding protein-1c and
CD68 were analysed.
Results Olanzapine induced significant body weight gain in
the female rats only. Only female rats treated with
olanzapine (2 mg/kg) had elevated plasma levels of IL-8 and
IL-1β, while both males and females had olanzapine-induced
increases in adiposity and evidence of macrophage infiltration
into adipose tissue. Furthermore, an altered microbiota profile
was observed following olanzapine treatment in both genders.
Conclusions This study furthers the theory that gender may
impact on the nature of, and susceptibility to, certain side
effects of antipsychotics. In addition, we demonstrate, what
is to our knowledge the first time, an altered microbiota
associated with chronic olanzapine treatment.
Keywords Olanzapine .Weight gain .Inflammation .
Metabolic syndrome .Gender .Microbiota
Introduction
Atypical antipsychotics (AAPDs) such as olanzapine
represent the mainstay of treatment for schizophrenia and
bipolar disorder. AAPDs are a diverse drug class grouped
Electronic supplementary material The online version of this article
(doi:10.1007/s00213-011-2555-2) contains supplementary material,
which is available to authorized users.
K. J. Davey :S. M. O’Mahony :O. O’Sullivan :P. D. Cotter :
T. G. Dinan :J. F. Cryan
Alimentary Pharmabiotic Centre, Laboratory of
Neurogastroenterology, University College Cork,
Cork, Ireland
K. J. Davey :H. Schellekens
School of Pharmacy, University College Cork,
Cork, Ireland
S. M. O’Mahony (*):J. F. Cryan (*)
Department of Anatomy, University College Cork,
Cork, Ireland
e-mail: somahony@ucc.ie
e-mail: j.cryan@ucc.ie
O. O’Sullivan :P. D. Cotter
Teagasc Food Research Centre, Moorepark,
Fermoy, Ireland
T. G. Dinan
Department of Psychiatry, University College Cork,
Cork, Ireland
H. Schellekens :J. F. Cryan
Food for Health Ireland,
Cork, Ireland
J. Bienenstock
McMaster Brain Body Institute, St. Joseph’s Healthcare,
Hamilton, Ontario, Canada
Psychopharmacology (2012) 221:155–169
DOI 10.1007/s00213-011-2555-2

together based on their lack of extra-pyramidal side effects
associated with typical antipsychotics (De Oliveira and
Juruena 2006). AAPDs however are associated with their
own side effects, most notably weight gain and metabolic
dysfunction (Albaugh et al. 2011; Birkenaes et al. 2008;
Chintoh et al. 2008; Oriot et al. 2008; Perez-Iglesias et al.
2009). Clinically significant weight gain (> 7%) often
occurs in greater than 50% of patients receiving an atypical
antipsychotic (Ahmer et al. 2008; Citrome et al. 2011; Patel
et al. 2009). This has ramifications for the patient in terms
of metabolic and cardiovascular disease co-morbidity as
well treatment compliance (Cohen and Correll 2009;
Correll et al. 2009; Farwell et al. 2004; Nasrallah 2003;
Starrenburg and Bogers 2009).
A number of factors, such as baseline weight (Basson et
al. 2001;Gebhardtetal.2009), therapeutic outcome
(Basson et al. 2001; Meltzer et al. 2003) and gender
(Aichhorn et al. 2007; Haack et al. 2009), have been
proposed to confer susceptibility to the metabolic effects of
antipsychotics. In the majority of cases, females have been
found to have a higher prevalence of AAPD-induced
weight gain (Aichhorn et al. 2007; Haack et al. 2009;
Hakko et al. 2006; Verma et al. 2009). However, exceptions
exist, where men have increased weight gain (Basson et al.
2001) or no gender bias has been apparent (Lee et al. 2004).
Intriguingly, in rat (and, to a lesser extent, mouse)
models of antipsychotic-induced weight gain, there
appears to exist gender-dependent effects with female
rats showing more robust weight gain following treat-
ment compared to males (Albaugh et al. 2006;Choietal.
2007). This has led some to challenge the relevance of
these models (Pouzet et al. 2003). However, recent studies
have demonstrated that male rats do incur a number of
detrimental metabolic effects in the absence of weight gain
(Albaugh et al. 2011; Minet-Ringuet et al. 2006; Victoriano
et al. 2009) and can even incur weight gain with extended
protocols (Shobo et al. 2011). Overall, therefore, gender
differences in animal models may be more relevant to the
clinical setting than previously thought (Weston-Green et al.
2011) and, while current models are not perfect, they are
extremely important in tackling the problems associated with
AAPDs (Boyda et al. 2010).
It is important to note that AAPDs such as olanzapine
can lead to the development of type II diabetes mellitus
with or without the presence of overt weight gain (Kim et
al. 2010; Newcomer 2004). Therefore, direct and indirect
metabolic actions of these drugs are important consider-
ations when assessing their overall metabolic impact.
Inflammation has been proposed as an important factor in
the development of obesity and the metabolic syndrome
(Bastard et al. 2006; Das 2001) and a correlation between
increased cytokine production and AAPD-induced weight
gain has been observed (Kluge et al. 2009).
The gut microbiota comprises the approximate 10
13
–10
14
bacteria which reside within the gastrointestinal tract and
exist in a symbiotic relationship with the host. Recently, a
role for the gut microbiota in body weight, metabolism and
systemic inflammation has begun to be elucidated (Backhed
et al. 2004; Backhed et al. 2007; Bailey et al. 2011;Clarkeet
al. 2010; Ley et al. 2005;Murphyetal.2010; Turnbaugh et
al. 2006). Furthermore, as with many other systems, a
definitive gender divide exists in the composition of the gut
microbiota in both humans and animals (Fushuku and
Fukuda 2008; Mueller et al. 2006). Moreover, the microbiota
can have marked effects on the brain–gut axis (Cryan and
O’Mahony 2011; Bravo et al. 2011). It is currently unclear
whether chronic AAPD treatment can affect microbiota
composition in addition to affecting body weight, metabo-
lism and systemic inflammation.
Thus, we investigated the impact of chronic olanzapine
treatment on metabolic, inflammatory and microbiome
parameters and assessed whether there was a sexually
dimorphic response.
Materials and methods
Animals
Male and female Sprague–Dawley rats, initially weighing
approximately 200 g, were used (Harlan, UK). The animals
were habituated to the animal facility for 1 week. They
were housed at four rats per cage (56×38×17 cm), allowed
access to standard chow and water ad libitum and kept on a
12-h light–dark cycle with lights on at 7:30 am. All
experiments were approved by the Ethical Committee of
University College Cork (#2010/013) and carried out in
accordance with the Cruelty to Animals Act 1876.
Drug administration
Olanzapine (Discovery Fine Chemicals, UK) was dissolved
in a minimal amount of glacial acetic acid (approximately
0.1 ml) and then made to volume with deionised water,
with the pH adjusted to approximately 6.0 with 0.1 M
NaOH. Vehicle consisted of distilled water acidified with
glacial acetic acid and pH was adjusted with 0.1 M NaOH.
All solutions were prepared fresh daily. The treatments
were administered via intra-peritoneal injection B.I.D. with
the first injection between 9:00 am and 10:00 am and the
second injection between 4:00 pm and 5:00 pm.
Treatment groups
Rats (n=8) received vehicle, olanzapine 2 mg/kg/day or
olanzapine 4 mg/kg/day for 21 days. All groups were
156 Psychopharmacology (2012) 221:155–169

weight-matched prior to commencing treatment. Doses
were selected on the basis that they reflect therapeutic
concentrations and have been shown to induce weight gain
and metabolic side effects previously (Cooper et al. 2005;
Fell et al. 2005; Kapur et al. 2003).
Daily measurements
Body weight, food intake and water intake were measured
each morning to the nearest 0.01 g using an electronic
balance. This was carried out prior to the first injection.
Locomotor activity
On day 22, animals were allowed 1 h to habituate to the testing
room (13:00–14:00) before being placed into the centre of a
rectangular plastic box (60×50× 40 cm). The behaviour was
recorded via an overhead camera for 30 min (14:00–14:30)
and locomotor activity was analysed using a tracking software
system (Ethovision, Noldus, The Netherlands).
Sample collection
Animals were sacrificed by decapitation and trunk blood was
collected in EDTA-coated tubes and centrifuged for 15 min at
6,000 rpm. Aliquots were derived from the plasma superna-
tant and stored on dry ice. The brain was quickly excised and
dissected and each brain region was initially stored in
RNALater for 24 h. The gonadal, mesenteric and subcutane-
ous fat deposits were carefully excised and weighed to the
nearest 0.0001 g. The gonadal and mesenteric deposits were
added together as a measure of visceral fat. The frontal lobe of
the liver was snap-frozen in isopentane and stored on dry ice.
All samples were frozen at −80°C for later analysis. Faecal
pellets were collected directly from the animals on day 22 on
dry ice and quickly stored at −80°C.
Plasma analysis
Concentrations of the cytokine tumour necrosis factor alpha
(TNF-α), interleukin-8, (IL-8), interleukin-6 (IL-6) and
interleukin-1 beta (IL-1β) were analysed using a commer-
cially available electrochemiluminescence multiplex system
(MSD, Gaithersburg, MD, USA). The highly sensitive
assay has a range of 9.8–40,000 pg/ml. The plates were
analysed on a SECTOR Imager 2400 from Mesoscale
Discovery. A total of 25 μl of plasma was used for each
well and all samples were analysed in duplicate.
Plasma leptin was analysed using a commercially available
enzyme-linked immunosorbent assay kit (Millipore, Billerica,
MA, USA). Theassay range of thisassay is 0.2 to 30 ng/ml. A
total of 10 μl of plasma was used for each well and all samples
were analysed in duplicate.
Plasma ghrelin was measured using a mouse/rat total
ghrelin multi-array assay from Mesoscale discovery (MSD,
Gaithersburg, MD, USA). The assay has a sensitivity of
11–5,000 pg/ml. All samples were measured in duplicate
and analysed on a SECTOR Imager 2400 from Mesoscale
Discovery.
Gene analysis
RNA from brain and liver samples was extracted for gene
analysis using a commercially available kit (Agilent
Technologies, CA, USA). Qiagen RNeasy Lipid Mini Kit
was used for adipose tissue (QIAGEN, Valencia, CA,
USA). mRNA was reverse-transcribed using high-capacity
cDNA reverse transcription kit (Applied Biosystems) in a
G-storm thermocycler (G-storm, Surrey, UK). Gene expres-
sion was analysed using TaqMan Gene Expression Assays
and the AB7300 system (Applied Biosystems). The
expression value of each gene was normalised to that of
β-actin. All samples were analysed in triplicate.
Microbial community composition: pyrosequencing
For analysis of the microbial community composition, total
DNA was extracted from the faecal pellets of two rats per
cage using the QIAamp DNA stool mini-kit according to
the manufacturer’s instructions (Qiagen, West Sussex, UK)
coupled with an initial bead-beating step. Universal 16S
rRNA primers, designed to amplify from highly conserved
regions corresponding to those flanking the V4 region, i.e. the
forward primer F1 (5′-AYTGGGYDTAAAGNG) and a com-
bination of four reverse primers R1 (5′-TACCRGGGTHTC
TAATCC), R2 (5′-TACCAGAGTATCTAATTC), R3 (5′-
CTACDSRGGTMTCTAATC) and R4 (5′-TACNVGGG
TATCTAATC) (RDP's Pyrosequencing Pipeline: http://pyro.
cme.msu.edu/pyro/help.jsp), were used for Taq-based PCR
amplification. Sequencing was performed on a Roche 454
GS-FLX using titanium chemistry by the Teagasc454Sequen-
cingPlatform. Resulting raw sequences reads were quality-
trimmed as previously described (Claesson et al. 2009).
Trimmed FASTA sequences were then BLASTed (Altschul
et al. 1997) against a previously published 16 s rRNA-specific
database (Urich et al. 2008) using default parameters. The
resulting BLAST output was parsed using MEGAN (Huson et
al. 2007a; Huson et al. 2007b). MEGAN assigns reads to
NCBI taxonomies by employing the lowest common ancestor
algorithm. Bit scores were used from within MEGAN for
filtering the results prior to tree construction and summariza-
tion. A bit score of 86 was selected as previously used for 16S
ribosomal sequence data (Urich et al. 2008). Phylum and
family counts for each subject were extracted from MEGAN.
Clustering and alpha diversities were generated with the
MOTHUR software package (Schloss et al. 2009).
Psychopharmacology (2012) 221:155–169 157

Statistical analysis
Two-way repeated measures analysis of variance (ANOVA)
was used to analyse body weight change, food and water
intake and locomotion, with gender and treatment as factors.
Two-way ANOVA was used for gene and cytokine analysis
with gender and treatment as factors. Further analysis was
carried out using Tukey’s post-hoc test. p<0.05 was
considered as statistically significant.
Results
Body weight gain
The effect of olanzapine on body weight gain was signifi-
cantly affected by gender (F(1, 42) = 26.906, p<0.001) and
time (F(2.85, 119.56)=271.878, p<0.001). There was a
significant interaction between gender and treatment (F(2,
42)=4.883, p=0.01). There was also a significant interaction
between gender and time (F(2.85, 119.56)= 34.425,
p<0.001) and a gender×treatment×time (F(5.69, 119.56)=
15.101, p<0.001). The increase in the weight in the female
rats was evident within the first days of treatment, with
significant increases observed in animals given olanzapine
(2 mg/kg) on days 5 to 15 and days 19–23 inclusive (p<0.05
to p<0.001). Females treated with olanzapine (4 mg/kg)
displayed significant weight gain on days 3 to 15
inclusive (p<0.05 to p<0.001) but subsequently showed
a reduction in body weight returning to normal levels such
that the animals receiving olanzapine (2 mg/kg) were
significantly increased compared to those receiving olan-
zapine (4 mg/kg) on days 21–23 inclusive (p<0.05)
(Fig. 1a). In the male rats, no difference between treatment
groups was observed (Fig. 1b).
Food and water intake
Olanzapine treatment significantly affected food intake (F(2,
90)=21.212, p<0.001). There was also a significant effect of
gender (F(1, 90)=530.485, p<0.001) and a significant
time×gender× treatment interaction (F(3.34, 150.66)=4.28,
p<0.01). Hyperphagia was observed in the female rats in
both treatment groups compared to vehicle in week 1
(2 mg/kg, p<0.05; 4 mg/kg, p<0.001) and week 2
(2 mg/kg, p<0.05; 4 mg/kg, p<0.01). The observed
increases in the rats treated with olanzapine (4 mg/kg) were
significantly greater than in those receiving olanzapine
(2 mg/kg) group in week 1 (p<0.05). In week 3, hyperphagia
persisted in the animals treated with olanzapine (2 mg/kg)
(p<0.05) but was significantly reduced in those receiving
olanzapine (4 mg/kg) compared to vehicle-treated rats
(p<0.01) and the olanzapine (2 mg/kg) group (p<0.01). In
the male rats, the olanzapine (4 mg/kg) group displayed a
significant increase in food intake compared to the vehicle
group in week 1 (p<0.05) but not in the subsequent weeks
(Fig. 2a).
Olanzapine treatment also induced significant increases in
water intake (F(2, 90)=6.573, p<0.01) with a further
significant effect of gender (F(1, 90)= 51.397, p<0.001)
and time (F(2, 180)=14.637, p<0.001). There was also a
significant gender×treatment interaction (F(2, 90)=4.895,
p<0.01). The water intake followed a similar pattern to food
intake with increases seen in the female rats treated with
olanzapine (2 and 4 mg/kg) compared to vehicle-treated rats
in week 1 (p<0.05, p<0.01), respectively, and week 2
(p<0.001, p< 0.05). In week 3, only the female rats receiving
olanzapine (2 mg/kg) displayed significant increases versus
vehicle-treated animals (p<0.05). The male rats did not show
differences in any of the weeks (Fig. 2b).
Locomotor activity
In the locomotor activity test, there was a significant effect
of time (F(3.45, 144.68)=221.217, p<0.001) and treatment
(F(2, 42)=7.264, p<0.01). Post-hoc analysis showed that
Fig. 1 Effect of olanzapine (OLZ) (2 and 4 mg/kg) on percentage
body weight gain in afemale rats and bmale rats treated for 21 days
B.I.D. First injection on day 1. Data shown represent mean±SEM.
*p<0.05, **p< 0.01, ***p< 0.001 OLZ (2 mg/kg) significantly
different versus vehicle group.
+
p<0.05,
++
p<0.01,
+++
p<0.001
4 mg/kg significant versus vehicle group.
#
p<0.05, OLZ (2 mg/kg)
significant versus OLZ (4 mg/kg) group
158 Psychopharmacology (2012) 221:155–169

there was a reduction in locomotion in female rats treated
with olanzapine (2 mg/kg) between 15 and 20 min
(p<0.001) and between 20 and 25 min (p<0.05). The
female rats treated with olanzapine (4 mg/kg) only
displayed decreased activity between 15 and 20 min
(p<0.05). The male rats treated with olanzapine (2 and
4 mg/kg) exhibited reduced locomotor activity between the
5- and 10-min time points (p<0.05 and p<0.01, respec-
tively). The male rats treated with olanzapine (2 mg/kg)
further showed reduced movement between 15 and 20 min
and between 20 and 25 min (p<0.05) (Fig. 3).
Adipose tissue
Olanzapine treatment significantly increased visceral fat
mass (F(2, 42)=16.042, p<0.001) and there was a
significant gender×treatment interaction (F(2, 42) = 6.147,
p<0.01). Female rats treated with olanzapine(2 and 4 mg/kg)
had significantly increased visceral fat mass compared to
vehicle-treated animals (p<0.01 and p<0.05, respectively).
The male rats receiving olanzapine (4 mg/kg group) showed
significant increases in visceral fat compared to both the
male vehicle-treated group (p<0.001) and the male olanza-
pine (2 mg/kg)-treated group (p<0.05) (Fig. 4).
Olanzapine treatment also had a significant effect on
CD68 expression (F(2, 38)=8.825, p<0.001) with an
effect of gender (F(1, 38)=16.046, p<0.001). Female rats
treated with olanzapine (4 mg/kg) displayed significantly
increased levels of CD68 mRNA compared to vehicle-
treated rats (p<0.05). In male rats, olanzapine treatment (2
and 4 mg/kg) resulted in increased levels of CD68 mRNA
expression compared to vehicle-treated animals (p< 0.05)
(Fig. 5a).
Interleukin (IL)-6 mRNA expression was significantly
affected by gender (F(1, 37)=26.511, p<0.001) and there
was a significant gender×treatment interaction (F(2, 37)=
3.886, p<0.05). The female animals receiving olanzapine
(4 mg/kg) had increased levels compared to the animals
receiving olanzapine (2 mg/kg) (p<0.05). The male rats
treated with olanzapine did not show significant increases,
though those treated with olanzapine (2 mg/kg) had a
fourfold increase compared to vehicle-treated rats. The
Fig. 3 Effect of olanzapine (OLZ) (2 mg/kg and 4 mg/kg) on
locomotor activity in afemale and bmale rats treated for 21 days B.I.
D. Locomotion measured as distance moved. Data shown represent
mean ± SEM. *p<0.05 versus vehicle
Fig. 4 Effect of olanzapine (OLZ) ( 2 and 4 mg/kg) on the proportion of
visceral fat (gonadal + mesenteric fat deposits) in female and male rats
treated for 21 days B.I.D. Data shown represent mean±SEM. **p<
0.01, ***p<0.001 significantly different versus vehicle group of the
same gender.
#
p<0.05 significantly different versus OLZ (2 mg/kg)
group of the same gender
Fig. 2 Effect of olanzapine (OLZ) (2 and 4 mg/kg) on afood intake
and bwater intake in female and male rats treated for 21 days B.I.D.
Data shown represent mean ± SEM. Food and water intake shown as
amount consumed per cage per day. *p<0.05, **p< 0.01, ***p<0.001
significantly different versus vehicle group of the same gender.
#
p<0.05,
##
p<0.01 versus OLZ (2 mg/kg) group of the same gender
Psychopharmacology (2012) 221:155–169 159

male vehicle-treated rats had significantly lower expression
compared to female vehicle-treated animals (p<0.05)
(Fig. 5b).
Sterol-regulatory element binding protein 1c (SREBP-
1c) expression was significantly affected by olanzapine
treatment (F(2, 34)=4.90, p<0.05). There was also a
significant effect of gender (F(1, 34)= 39.189, p<0.001) and
a significant gender× treatment interaction (F(2, 34) = 3.481,
p<0.05). In female rats, those treated with olanzapine
(4 mg/kg) had a significant reduction in the mRNA
expression of SREBP-1c compared to both the vehicle
(p<0.05) and olanzapine (4 mg/kg) groups (p<0.01). This
reduction was not seen in the male rats, though the male
vehicle group had significantly lower levels than the female
vehicle-treated animals (p<0.05) (Fig. 5c).
Liver
Liver weight as a percentage of body weight was
significantly affected by olanzapine treatment (F(2, 42)=
4.512, p<0.05) and gender (F(1, 42)=26.466, p<0.001).
The female rats treated with olanzapine (2 mg/kg) were
found to have significantly increased liver weight compared
to the vehicle-treated rats (p<0.05). No differences were
observed in the male rats (Fig. 6).
SREBP-1c mRNA expression in the liver was not
affected by treatment but was significantly affected by
gender (F(1, 42)=143.439, p<0.001). The male and
female vehicle-treated groups differed significantly from
one another (p<0.001) (Table 1).
Carbohydrate regulatory element binding protein
(ChREBP) mRNA expression was significantly affected
by gender (F(1, 46)=50.085, p<0.001) and male vehicle-
treated rats were significantly different from female vehicle-
treated animals (Table 1).
TNF mRNA in the liver showed no significant differ-
ences following olanzapine treatment (Table 1).
Plasma cytokines and leptin
Olanzapine treatment had a significant effect on the circulat-
ingplasmalevelsofIL-8(F(2, 41)=3.613, p<0.05). The
female rats treated with olanzapine (2 mg/kg) had increased
levels compared to vehicle-treated rats (p<0.01) (Fig. 7a).
Plasma levels of TNF-αwere significantly affected by
gender (F(1, 41)=102.024, p<0.001) and there was a
significant gender×treatment interaction following olanza-
pine treatment (F(2, 41)=7.049, p<0.01). Male rats treated
with olanzapine (4 mg/kg) showed a reduction in circulating
levels of TNF-α(p<0.05) (Fig. 7b).
Plasma IL-6 levels were significantly affected by
treatment (F(1, 41)=4.005, p<0.05) and gender (F(1,
41)=7.473, p<0.01). There was a significant gender×
treatment interaction (F(2, 41)=5.86, p<0.01). Male animals
treated with olanzapine (2 and 4 mg/kg) had lower levels of
IL-6 compared to the male vehicle-treated rats (p<0.05 and
p<0.01, respectively). The vehicle-treated male animals had
significantly higher levels than female vehicle-treated rats
(p<0.01) (Fig. 7c).
IL-1βplasma levels were significantly affected by
gender (F(1, 41)=5.575, p<0.05) and there was a
significant gender×treatment interaction (F(2, 41)=4.93,
Fig. 6 Effect of olanzapine (OLZ) (2 and 4 mg/kg) on relative liver
weight in female and male rats treated for 21 days B.I.D. Data shown
represent mean ± SEM. *p<0.05 significantly different versus vehicle
group of the same gender
Fig. 5 Effect of olanzapine (OLZ) (2 and 4 mg/kg) on aCD68 mRNA
expression, bIL-6 mRNA expression and cSREBP-1c mRNA
expression in female and male rats treated for 21 days B.I.D. Data
shown represent mean ± SEM. *p<0.05 significantly different versus
vehicle group of the same gender.
#
p<0.05,
##
p<0.01 significantly
different versus OLZ (2 mg/kg) group of the same gender.
$
p<0.05,
$
p<0.01 female vehicle group versus male vehicle group
160 Psychopharmacology (2012) 221:155–169

p<0.05). In the female rats, those receiving olanzapine
(2 mg/kg) had significantly elevated levels compared to
vehicle-treated rats (p<0.05). The male vehicle-treated rats
had significantly higher levels than the female vehicle-
treated rats (p<0.01) (Fig. 7d).
Plasma leptin levels showed a significant effect of
gender (F(1, 42)=12.636, p<0.001). The male vehicle-
treated animals had higher levels than female vehicle-
treated rats (p<0.05) (Fig. 8).
Peripheral and central ghrelin
Olanzapine significantly affected the plasma levels of total
ghrelin (F(2, 42)=3.143, p=0.05). There was also a
significant effect of gender (F(1, 42)=9.109, p<0.01).
The female rats treated with olanzapine (2 mg/kg) had
reduced circulating levels of ghrelin (p<0.05) and those
receiving olanzapine (4 mg/kg) also displayed a trend for
reduced levels (p=0.068). No significant effects were
observed between male groups (Fig. 9).
Hypothalamic expression of the ghrelin 1a receptor
mRNA was significantly affected by gender (F(1, 35)=
13.68, p<0.01) and there was a significant gender×
treatment interaction (F(2, 35)=6.973, p<0.01). The male
rats treated with olanzapine (4 mg/kg) had significantly
higher levels than the vehicle-treated rats (p<0.05)
(Fig. 10).
Gut microbiota
The effects of chronic olanzapine on the microbial
composition of the gut microbiota of the rats was elucidated
through high-throughput pyrosequencing (Roche-454 Tita-
nium) of 16S rRNA (V4) amplicons generated from faecal
DNA obtained at study termination. Species richness,
coverage and diversity estimations were calculated for each
Fig. 7 Effect of olanzapine (OLZ) (2 and 4 mg/kg) on plasma levels
of aIL-8, bTNF-α,cIL-6 and dIL-1βin female and male rats
treated for 21 days B.I.D. Data shown represent mean ± SEM.
*p<0.05, **p< 0.01 significantly different versus vehicle group of
same gender.
$
p<0.05,
$$
p<0.01, significant difference between
female vehicle group and male vehicle group
Table 1 Effect of chronic olanzapine on gene expression in the liver
Gender Female Male
Olanzapine Vehicle 2 mg/kg 4 mg/kg Vehicle 2 mg/kg 4 mg/kg
SREBP-1c 1.0± 0.16 0.73 ± 0.07 0.70 ±0.08 3.26±0.16* 3.57±0.33 3.42± 0.44
ChREBP 1.0± 0.16 1.09 ± 0.17 0.99 ±0.17 2.40±0.21* 2.67±0.26 2.70± 0.47
TNF 1.0± 0.09 0.89 ± 0.14 0.90 ±0.08 1.82±0.42 0.89± 0.20 0.86 ± 0.11
Relative gene expression of SREBP-1c, ChREBP and TNF in liver of olanzapine- and vehicle-treated rats. Values were normalised to female
vehicle group
SREBP sterol-regulating element-binding protein, ChREBP carbohydrate-regulating element-binding protein, TNF tumour necrosis factor
*p<0.01 versus female vehicle group
Psychopharmacology (2012) 221:155–169 161

data set (Supplementary Table 1). At the 97% similarity
level, the Shannon index, a metric for community diversity,
revealed a high level of overall biodiversity within all
samples with values exceeding 4.2. The Good’s coverage at
the 97% similarity level ranged between 84% and 98% for
all datasets. The Chao1 richness values indicate good
sample richness throughout.
Assessment of the faecal microbiota, in terms of
microbial phyla, revealed that olanzapine treatment in
the female rats seemed to be associated with increased
levels of Firmicutes following olanzapine 2 mg/kg
(72.11% versus 84.06%) and olanzapine 4 mg/kg
(72.11% versus 88.12%), with increases of 11.95% and
15.99% respectively. Olanzapine treatment of 2 and 4 mg/kg
also appeared to reduce diversity compared to vehicle-treated
rats evidenced by reductions in the less represented phyla
Actinobacteria (3.72% versus 0.34% and 0.15%, respectively)
and Proteobacteria (1.60% versus 0.15% and 0.77%, respec-
tively). Animals treated with olanzapine 4 mg/kg also
displayed evidence of reduced Bacteriodetes (17.57% versus
10.88%) (Fig. 11a).
In the male rats, olanzapine treatment (2 mg/kg)
appeared to impact the microbiota minimally with an
apparent reduction in Proteobacteria (3.15% versus
0.94%). Olanzapine treatment of 4 mg/kg, however, seemed
to cause an increase in Firmicutes (82.66% versus 91.63%)
and a reduction on Bacteriodetes of a similar magnitude
(14.08% versus 7.97%) (Fig. 11b).
Correlation analysis
In order to assess the possible relationship between the
main physical alterations induced by olanzapine treatment
in our model and possible biochemical correlates, we
carried out correlation analysis on body weight gain and a
number of biochemical plasma markers. For the female rats,
a significant correlation was found between body weight
gain and plasma leptin (Pearson correlation coefficient=
0.457, r
2
=0 .205, p<0.05) (Fig. 12a). A significant
correlation was also found for body weight gain and plasma
ghrelin (Pearson correlation coefficient=−0.429, r
2
=0.185,
p<0.05) (Fig. 12b). A significant correlation was also
observed for body weight gain and plasma IL-8 levels
(Pearson correlation coefficient= 0.702, r
2
=0.493, p<
0.001) (Fig. 12c). Furthermore, a significant correlation
was found between visceral fat mass and plasma IL-
8 (Pearson correlation coefficient=0.550, r
2
=0.303, p<
0.01) (Fig. 12d).
In the male rats, no significant correlation was observed
between any of the measured physical and biochemical
parameters.
Discussion
Here we show that olanzapine had significant effects on
a number of physiological, inflammatory and microbial
parameters in the rat and that many, but not all of these,
were more pronounced in females compared to males.
Olanzapine induced rapid weight gain in female rats and
not in male rats, which is consistent with previous
reports (Albaugh et al. 2006;Choietal.2007). Both
male and female rats treated with olanzapine did however
exhibit increased visceral fat, though in the males this was
the case only at the higher dose. We also show, to our
knowledge for the first time, specific alterations to the gut
microbiota as a result of antipsychotic treatment, suggest-
ing that microbiota may contribute to AAPD-induced
metabolic dysfunction.
Fig. 10 Effect of olanzapine (OLZ) (2 and 4 mg/kg) on growth
hormone secretagogue 1a receptor mRNA expression in the hypo-
thalamus in female and male rats treated for 21 days. Data shown
represent mean ± SEM. *p<0.05 significantly different versus vehicle
group of the same gender
Fig. 9 Effect of olanzapine (OLZ) (2 and 4 mg/kg) on plasma levels
of total ghrelin in female and male rats treated for 21 days B.I.D. Data
shown represent mean ± SEM. *p<0.05 significantly different versus
vehicle group of the same gender
Fig. 8 Effect of olanzapine (OLZ) (2 and 4 mg/kg) on plasma levels
of leptin in female and male rats treated for 21 days B.I.D. Data
shown represent mean ± SEM. *p<0.05 female vehicle group versus
male vehicle group
162 Psychopharmacology (2012) 221:155–169

The reason for the gender difference in body weight gain
at a pre-clinical level is currently unknown and its
significance to the clinical presentation of AAPD-induced
metabolic alterations is contentious. One reason for this
being that clozapine, an antipsychotic that also causes
considerable weight gain in humans, does not appear to do
so in rats (Cooper et al. 2008). There is considerable
evidence however to suggest that females are more liable to
incur antipsychotic-induced weight gain (Aichhorn et al.
2007; Haack et al. 2009; Hakko et al. 2006), although this
may reflect gender differences in drug pharmacokinetics
(Beierle et al. 1999; Harris et al. 1995). In the present study,
we observed a number of gender differences in baseline
levels of the plasma cytokines IL-1βand IL-6 as well as
local levels of IL-6 in the adipose tissue which may impact
on susceptibility to the effects of AAPDs. Gender dimor-
phism in immune function including cytokine release is
well documented and our findings suggest that these may
have implications for antipsychotic side effects (Bao et al.
2002; Cannon and Pierre 1997; Yokoyama et al. 2005).
The complex nature of body weight regulation may
explain why we did not observe a dose–response relation-
ship in weight gain with olanzapine treatment. This is
supported by clinical findings in which lower doses are not
Fig. 11 Proportional composi-
tion of the faecal microbiota
following 21 days of olanzapine
treatment (2 or 4 mg/kg) in
afemales and bmales. Data
represent the cumulative DNA
of two pellets per cage for
each group
Fig. 12 Correlation analysis
between percent body weight
gain at day 23 and aplasma
leptin, bplasma ghrelin and
cplasma IL-8. dCorrelation
analysis between visceral
fat and plasma IL-8
Psychopharmacology (2012) 221:155–169 163

necessarily associated with lower weight gain (Citrome et
al. 2009). The mechanisms by which olanzapine causes
weight gain as in the female rats in this study are unclear
but largely attributed to its diverse pharmacological
receptor profile (Matsui-Sakata et al. 2005; Newcomer
2005; Reynolds et al. 2006; Roth et al. 2003; Silvestre and
Prous 2005). Antagonism of central receptors including
serotonin 5-HT
2C
and histamine H
1
receptors, which play
pivotal roles in appetite regulation as well as long-term
energy balance (Lam et al. 2008; Masaki et al. 2004; Tsuda
et al. 2002), has been particularly implicated in weight gain
associated with antipsychotic treatment (Deng et al. 2010;
Kroeze et al. 2003; Reynolds et al. 2006; Reynolds et al.
2002). Hyperphagia was observed in the female rats and is
believed to drive initial weight gain (Thornton-Jones et al.
2002). This is also seen in clinical studies in which
increased appetite is commonly reported by patients
initiating olanzapine therapy (Basson et al. 2001; Kluge et
al. 2007; Treuer et al. 2009) and in non-psychotic controls
(Fountaine et al. 2010).
Male and female animals treated with olanzapine
displayed a significant accretion of visceral fat. Interestingly,
the female rats treated with olanzapine (4 mg/kg) returned to
control body weight but still had increased visceral fat.
Furthermore, the male rats treated with olanzapine
(4 mg/kg) did not show increases in body weight gain
but did however show increased adiposity. This finding
supports clinical and pre-clinical studies which found
increased adiposity following olanzapine treatment with
(Ader et al. 2008; Raskind et al. 2007; Victoriano et al.
2009) and without weight gain (Victoriano et al. 2009).
Increased visceral mass is considered a key factor in the
development of the metabolic syndrome and in particular
the development of insulin resistance (Bjorntorp 1991;
Demerath et al. 2008). Thus, these data emphasise that the
metabolic threat posed by olanzapine goes beyond merely
increases in body weight gain.
Gonadal adipose tissue of female rats treated with
olanzapine (4 mg/kg) and male rats treated with olanzapine
(2 and 4 mg/kg) displayed increased CD68 expression.
CD68 is a glycoprotein which represents a marker of
macrophage presence. Macrophage infiltration of adipose
tissue is considered a key step in the development of
obesity-related inflammation and subsequent insulin resis-
tance (Xu et al. 2003). Interestingly, CD68 expression did
not mirror weight gain, even in females. Thus, this suggests
that olanzapine can predispose toward a pro-inflammatory
state independent of effects on body weight per se. This
may have important connotations for patient monitoring
following the prescription of AAPDs.
The adipose tissue of female rats treated with olanzapine
(4 mg/kg) also displayed inflammation with increased IL-6
gene expression. Though not significant, the male rats
receiving olanzapine (2 mg/kg) also displayed the same
trend. Like CD68, IL-6 did not follow the pattern of weight
gain. However, sorted cell gene analysis of adipose tissue
has previously suggested that macrophages and adipocytes
secrete roughly equal amounts of IL-6; thus, macrophage
infiltration indicated by elevated CD68 expression likely
led to elevated IL-6 expression (Wisse 2004). Data on in
vitro tests suggest that IL-6 can directly confer insulin
resistance (Rotter et al. 2003) and levels are associated with
increased risk of type II diabetes (Pradhan et al. 2001). This
further suggests therefore that olanzapine can confer risk of
such metabolic abnormalities without overt weight gain and
that IL-6 may be one mediator of this disguised threat.
The female rats further displayed a pro-inflammatory
phenotype with IL-8 and IL-1βbeing significantly elevated
in plasma in the olanzapine (2 mg/kg) group. Increased
circulating levels of each of these cytokines have been
associated with obesity and implicated in insulin resistance
(Kim et al. 2006; Straczkowski et al. 2002). Conversely,
male rats treated with olanzapine displayed an anti-
inflammatory phenotype with reductions in IL-6 and
TNF-αobserved. While this is initially surprising, the
anti-inflammatory effects of antipsychotics have been
recognised for some time (Chedid 1954). Olanzapine has
been shown to suppress TNF-αand Il-6 production in mice
treated with lipopolysaccharide (Sugino et al. 2009). This
discrepancy in circulating cytokines may reflect differences
in their primary source of the cytokines (Trayhurn and
Wood 2004) which could potentially account for the lack of
an observed increase in the males and higher-dose females.
Together these findings imply that systemic inflammation
associated with olanzapine occurs primarily as a result of
body weight gain. Intriguingly, the plasma levels of IL-8 in
the female rats showed significant correlation with body
weight gain and visceral fat mass, implicating this cytokine
in particular as a possible link between inflammation and
body weight gain and vice versa and may potentially be a
biomarker for recognising the induction of AAPD meta-
bolic side effects. This systemic inflammation may also act
to impair metabolism, leading to insulin resistance and
increased risk of metabolic syndrome and diabetes as a
secondary effect. This emphasises the double-edged risk
olanzapine confers on metabolic function with weight gain
inducing systemic inflammation and the direct actions of
the drug impacting on local inflammatory responses, both
of which can converge to induce insulin resistance.
Further disruption to normal metabolic functioning was
evidenced by reductions in sterol-regulatory binding
protein-1c (SREPB-1c) gene expression in the adipose
tissue of female rats treated with 4 mg/kg of olanzapine.
SREBP-1c is a key regulatory transcription factor which
controls a number of genes involved in lipid metabolism
(Ferre and Foufelle 2007). Reduced expression of SREBP-
164 Psychopharmacology (2012) 221:155–169

1c in adipose tissue has been observed in obese patients,
and subsequent weight loss was associated with increased
expression (Kolehmainen et al. 2001). These reductions are
likely to be secondary to insulin resistance as insulin is the
major regulator of SREBP expression. However, antipsy-
chotics have been shown to activate SREBP in vitro (Ferno
et al. 2006; Raeder et al. 2006; Yang et al. 2007) and in a
recent in vivo study of risperidone (Lauressergues et al.
2010). However, a recent study also demonstrated down-
regulation of SREBP-1c following an initial up-regulation
after acute olanzapine treatment (Jassim et al. 2011). A
study of clozapine administration was also associated with
acute increases in SREBP and associated genes followed by
a sustained down-regulation (Ferno et al. 2009). Thus, the
long-term effects of antipsychotics on SREBP system are
not yet clear but seem to involve feedback mechanisms, and
this finding further supports the theory that olanzapine can
directly affect lipid handling in the adipose tissue and thus
directly contribute to fat deposition and dyslipidemia
independent of weight gain (Ferno et al. 2011).
Ghrelin is an orexigenic hormone released from the
stomach and is known as the hunger hormone as it is
involved in meal initiation (Cummings et al. 2001;
Schellekens et al. 2010). We observed reductions in plasma
levels of total ghrelin in the female olanzapine-treated rats.
The effect of antipsychotics on ghrelin has not been
extensively studied though increased levels with prolonged
treatment has been found in human patients (Murashita et
al. 2005; Sentissi et al. 2008). In our studies, negative
feedback may have occurred as a result of hyperphagia
driven centrally. Intriguingly, in humans, higher basal
plasma levels are associated with females (Greenman et
al. 2004). Furthermore, ghrelin levels were found to be
inversely correlated with fat mass and body mass index in
females but not in human males (Makovey et al. 2007). It
must be remembered that ghrelin in vivo exists as
acetylated and non-acetylated forms and only the acetylated
form can cross the blood–brain barrier and activate central
ghrelin receptors. Also, ghrelin displays a circadian rhythm
such that the time of day when the animals were sacrificed
(morning) may have affected the ghrelin levels. Thus, total
plasma ghrelin levels must be interpreted carefully. Hypo-
thalamic ghrelin 1a receptor mRNA was increased in the
male olanzapine (4 mg/kg)-treated animals. Central actions
of ghrelin are associated with fat deposition (Riley et al.
2005). Thus, these results imply that alterations to the
ghrelin system may be one mechanism by which olanza-
pine increases visceral fat and potentially also appetite and
that these effects may be gender sensitive. Moreover, a
significant inverse correlation was found between plasma
ghrelin and body weight gain.
Leptin is a potent anorexigenic hormone with opposing
effects to those of ghrelin. Though not significantly
elevated in the treatment groups, a significant correlation
was found between body weight gain and plasma leptin.
While changes in circulating levels of these hormones
likely represent secondary rather than direct actions of
olanzapine (Baptista and Beaulieu 2002), they may poten-
tially act as important markers for those at risk for sustained
weight gain following commencement of antipsychotic
therapy and are important considerations in the assessment
of antipsychotics’metabolic impact (Sentissi et al. 2008).
The composition of the gut microbiota appeared to be
considerably altered following treatment with olanzapine
in the female rats and also in the male rats receiving
olanzapine (4 mg/kg). In the female and male rats treated
with olanzapine (4 mg/kg), the pooled samples at day 22
show a trend for increased Firmicutes and reduced
Bacteriodetes compared to control animals. There was
also evidence of reduced diversity at the phylum level in
these olanzapine-treated groups with reduced levels of
Proteobacteria in both females and males and reduced
Actinobacteria in the females. The gut microbiota
contributes to metabolism firstly by utilising indigestible
complex polysaccharides via fermentation for their own
energy and thereby producing short-chain fatty acids
whichcanthenbedigestedandusedbythehostfor
energy (Hooper et al. 2002). The microbiota is also
involved in cholesterol reduction and the biosynthesis of
vitamins that can be used by the host. It is estimated that
as much as 10% of our daily energy supply may be
provided in this way (Flint et al. 2008).
Furthermore, in their seminal work, Gordon and
colleagues showed that germ-free mice (mice devoid of
any microbiota) had 40% less body fat than their
conventional littermates. Furthermore, colonisation of the
germ-free mice with the microbiota of lean control mice led
to a significant increase in body fat while colonisation with
the microbiota of genetically obese mice (ob/ob) led to an
even greater level of weight gain (Backhed et al. 2004;
Turnbaugh et al. 2006). Furthermore, germ-free mice are
resistant to diet-induced obesity (Backhed et al. 2007). This
series of experiments also revealed that shifts in the
predominant phyla of the microbiota were associated with
obesity. An increase in the relative abundance of Firmicutes
with a concordant decrease in Bacteriodetes was observed
(Ley et al. 2005; Turnbaugh et al. 2008). This shift was also
found in a human study of obese versus lean twins and in a
study of type II diabetic patients versus non-diabetics,
independent of body weight (Larsen et al. 2010; Turnbaugh
et al. 2009).
Thus, our findings, while preliminary, are extremely
interesting as they are closely in line with the above and
other recent studies investigating the role of the microbiota
in obesity and energy regulation (Cani et al. 2007;
Kalliomaki et al. 2008). However, whether possible alter-
Psychopharmacology (2012) 221:155–169 165

ations to the gut microbiota are a direct result of olanzapine
treatment or secondary to other effects is unclear. A direct
effect of the drug on intestinal bacteria cannot be ruled out,
and a more quantitative analysis of gut flora following
olanzapine treatment in the future may provide clarity on
this. It is however, tempting to speculate that olanzapine
may have influenced the gut microbiota via as yet unknown
mechanisms and these changes could well contribute to or
exacerbate metabolic dysfunction induced by AAPDs—in
particular, fat accumulation. If this is the case, modulation
of the gut flora by antibiotic, prebiotic or indeed probiotic
therapy may represent a useful adjunctive therapy for
olanzapine-induced weight gain in the future.
In this study, systemic inflammation occurred in a
gender-dependent fashion and was only observed in female
rats and as such likely occurred secondary to weight gain
which was also only seen in the females. Importantly,
however, both the female and male rats did incur a number
of physiologically relevant changes including increased
adipose tissue, local inflammation and alterations to the gut
microbiota. Thus, this study brings into focus the need to
consider the side effects of antipsychotics as a double threat
involving not only weight gain but also independent
metabolic effects which may include modulation of the
gut microbiota. Furthermore, appreciating differences between
the sexes may have important clinical implications in not only
prescribing the treatment but also monitoring of patients in the
future (Seeman 2004).
Acknowledgements The authors would like to thank the staff of the
biological services unit for their assistance with animal maintenance.
Thanks also go to Lisa Quigley, Declan McKernan and Patrick
Fitzgerald for technical assistance and advice. The Alimentary
Pharmabiotic Centre is a research centre funded by Science Founda-
tion Ireland (SFI) through the Irish Government’s National Develop-
ment Plan. The authors and their work were supported by SFI (grant
nos. 02/CE/B124 and 07/CE/B1368). The centre is also funded by
GlaxoSmithKline.
Conflicts of interest The authors declare no conflicts of interest.
References
Ader M, Garvey WT, Phillips LS, Nemeroff CB, Gharabawi G,
Mahmoud R, Greenspan A, Berry SA, Musselman DL, Morein J,
Zhu Y, Mao L, Bergman RN (2008) Ethnic heterogeneity in
glucoregulatory function during treatment with atypical antipsy-
chotics in patients with schizophrenia. J Psychiatr Res 42:1076–85
Ahmer S, Khan RA, Iqbal SP (2008) Association between anti-
psychotics and weight gain among psychiatric outpatients in
Pakistan: a retrospective cohort study. Ann Gen Psychiatry 7:12
Aichhorn W, Whitworth AB, Weiss EM, Hinterhuber H, Marksteiner J
(2007) Differences between men and women in side effects of
second-generation antipsychotics. Nervenarzt 78:45–52
Albaugh VL, Henry CR, Bello NT, Hajnal A, Lynch SL, Halle B,
Lynch CJ (2006) Hormonal and metabolic effects of olanzapine
and clozapine related to body weight in rodents. Obesity (Silver
Spring) 14:36–51
Albaugh VL, Judson JG, She P, Lang CH, Maresca KP, Joyal JL,
Lynch CJ (2011) Olanzapine promotes fat accumulation in male
rats by decreasing physical activity, repartitioning energy and
increasing adipose tissue lipogenesis while impairing lipolysis.
Mol Psychiatry 16:569–581
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,
Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids
Res 25:3389–402
Backhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A,
Semenkovich CF, Gordon JI (2004) The gut microbiota as an
environmental factor that regulates fat storage. Proc Natl Acad
Sci U S A 101:15718–15723
Backhed F, Manchester JK, Semenkovich CF, Gordon JI (2007)
Mechanisms underlying the resistance to diet-induced obesity in
germ-free mice. Proc Natl Acad Sci U S A 104:979–984
Bailey MT, Dowd SE, Galley JD, Hufnagle AR, Allen RG, Lyte M
(2011) Exposure to a social stressor alters the structure of the
intestinal microbiota: implications for stressor-induced immuno-
modulation. Brain Behav Immun 25:397–407
Bao M, Yang Y, Jun HS, Yoon JW (2002) Molecular mechanisms for
gender differences in susceptibility to T cell-mediated autoimmune
diabetes in nonobese diabetic mice. J Immunol 168:5369–5375
Baptista T, Beaulieu S (2002) Are leptin and cytokines involved in
body weight gain during treatment with antipsychotic drugs? Can
J Psychiatry 47:742–9
Basson BR, Kinon BJ, Taylor CC, Szymanski KA, Gilmore JA,
Tollefson GD (2001) Factors influencing acute weight change in
patients with schizophrenia treated with olanzapine, haloperidol,
or risperidone. J Clin Psychiatry 62:231–238
Bastard JP, Maachi M, Lagathu C, Kim MJ, Caron M,Vidal H, Capeau J,
Feve B (2006) Recent advances in the relationship between obesity,
inflammation, and insulin resistance. Eur Cytokine Netw 17:4–12
Beierle I, Meibohm B, Derendorf H (1999) Gender differences in
pharmacokinetics and pharmacodynamics. Int J Clin Pharmacol
Ther 37:529–547
Birkenaes AB, Birkeland KI, Engh JA, Faerden A, Jonsdottir H, Ringen
PA, Friis S, Opjordsmoen S, Andreassen OA (2008) Dyslipidemia
independent of body mass in antipsychotic-treated patients under
real-life conditions. J Clin Psychopharmacol 28:132–7
Bjorntorp P (1991) Metabolic implications of body-fat distribution.
Diabetes Care 14:1132–1143
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
Bravo JA, Forsythe P, Chew MV, Escaravage E, Savignac HM, Dinan
TG, Bienenstock J, Cryan JF (2011) Ingestion of Lactobacillus
strain regulates emotional behavior and central GABA receptor
expression in a mouse via the vagus nerve. Proc Natl Acad Sci
USA 108(38):16050–16055
Cani PD, Neyrinck AM, Fava F, Knauf C, Burcelin RG, Tuohy KM,
Gibson GR, Delzenne NM (2007) Selective increases of
bifidobacteria in gut microflora improve high-fat-diet-induced
diabetes in mice through a mechanism associated with endotox-
aemia. Diabetologia 50:2374–2383
Cannon JG, Pierre BAS (1997) Gender differences in host defense
mechanisms. J Psychiatr Res 31:99–113
Chedid L (1954) *Actions Comparees De La Promethazine, De La
Chlorpromazine Et De La Cortisone Chez La Souris Recevant Des
Doses Mortelles Dune Endotoxine Bacterienne. Comptes Rendus Des
Seances De La Societe De Biologie Et De Ses Filiales 148:1039–1043
Chintoh AF, Mann SW, Lam TK, Giacca A, Remington G (2008)
Insulin resistance following continuous, chronic olanzapine
treatment: an animal model. Schizophr Res 104:23–30
166 Psychopharmacology (2012) 221:155–169

Choi S, DiSilvio B, Unangst J, Fernstrom JD (2007) Effect of chronic
infusion of olanzapine and clozapine on food intake and body
weight gain in male and female rats. Life Sci 81:1024–30
Citrome L, Holt RIG, Walker DJ, Hoffmann VP (2011) Weight gain
and changes in metabolic variables following olanzapine treat-
ment in schizophrenia and bipolar disorder. Clin Drug Invest
31:455–482. doi:10.2165/11589060-000000000-00000
Citrome L, Stauffer VL, Chen L, Kinon BJ, Kurtz DL, Jacobson JG,
Bergstrom RF (2009) Olanzapine plasma concentrations after
treatment with 10, 20, and 40 mg/d in patients with schizophrenia:
an analysis of correlations with efficacy, weight gain, and prolactin
concentration. J Clin Psychopharmacol 29:278–283
Claesson MJ, O’Sullivan O, Wang Q, Nikkila J, Marchesi JR, Smidt
H, de Vos WM, Ross RP, O’Toole PW (2009) Comparative
analysis of pyrosequencing and a phylogenetic microarray for
exploring microbial community structures in the human distal
intestine. 4:e6669
Clarke TB, Davis KM, Lysenko ES, Zhou AY, Yu YM, Weiser JN (2010)
Recognition of peptidoglycan from the microbiota by Nod1
enhances systemic innate immunity. Nat Med 16:228–U137
Cohen D, Correll CU (2009) Second-generation antipsychotic-associated
diabetes mellitus and diabetic ketoacidosis: mechanisms, predictors,
and screening need. J Clin Psychiatry 70:765–6
Cooper GD, Harrold JA, Halford JCG, 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
Neuropsychopharmacology Biol Psychiatry 32:428–436
Cooper GD, Pickavance LC, Wilding JP, Halford JC, Goudie AJ
(2005) A parametric analysis of olanzapine-induced weight gain
in female rats. Psychopharmacology (Berl) 181:80–9
Correll CU, Manu P, Olshanskiy V, Napolitano B, Kane JM, Malhotra
AK (2009) Cardiometabolic risk of second-generation antipsychotic
medications during first-time use in children and adolescents.
JAMA 302:1765–73
Cryan JF, O’Mahony SM (2011) The microbiome-gut-brain axis: from
bowel to behavior. Neurogastroenterology and Motility 23
(3):187–192
Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE,
Weigle DS (2001) A preprandial rise in plasma ghrelin levels
suggests a role in meal initiation in humans. Diabetes 50:1714–
1719
Das UN (2001) Is obesity an inflammatory condition? Nutrition
17:953–966
De Oliveira IR, Juruena MF (2006) Treatment of psychosis: 30 years
of progress. J Clin Pharm Ther 31:523–534
Demerath EW, Reed D, Rogers N, Sun SS, Lee M, Choh AC, Couch
W, Czerwinski SA, Chumlea WC, Siervogel RM, Towne B
(2008) Visceral adiposity and its anatomical distribution as
predictors of the metabolic syndrome and cardiometabolic risk
factor levels. Am J Clin Nutr 88:1263–71
Deng C, Weston-Green K, Huang XF (2010) The role of histaminergic
H1 and H3 receptors in food intake: a mechanism for atypical
antipsychotic-induced weight gain? Prog Neuropsychopharmacol
Biol Psychiatry 34:1–4
Farwell WR, Stump TE, Wang J, Tafesse E, L'Italien G, Tierney WM
(2004) Weight gain and new onset diabetes associated with
olanzapine and risperidone. J Gen Intern Med 19:1200–5
Fell M, Neill J, Rao C, Marshall K (2005) Effects of sub-chronic
antipsychotic drug treatment on body weight and reproductive
function in juvenile female rats. Psychopharmacology 182:499–
507
Ferno J, Skrede S, Vik-Mo AO, Havik B, Steen VM (2006) Drug-
induced activation of SREBP-controlled lipogenic gene expres-
sion in CNS-related cell lines: marked differences between
various antipsychotic drugs. BMC Neurosci 7:69
Ferno J, Skrede S, Vik-Mo AO, Jassim G, Le Hellard S, Steen VM
(2011) Lipogenic effects of psychotropic drugs: focus on the
SREBP system. Front Biosci (Landmark) 16:49–60
Ferno J, Vik-Mo AO, Jassim G, Havik B, Berge K, Skrede S,
Gudbrandsen OA, Waage J, Lunder N, Mork S, Berge RK,
Jorgensen HA, Steen VM (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
Ferre P, Foufelle F (2007) SREBP-1c transcription factor and lipid
homeostasis: clinical perspective. Horm Res 68:72–82
Flint HJ, Bayer EA, Rincon MT, Lamed R, White BA (2008)
Polysaccharide utilization by gut bacteria: potential for new
insights from genomic analysis. Nat Rev Microbiol 6:121–131
Fountaine RJ, Taylor AE, Mancuso JP, Greenway FL, Byerley LO,
Smith SR, Most MM, Fryburg DA (2010) Increased food intake
and energy expenditure following administration of olanzapine to
healthy men. Obesity 18:1646–1651
Fushuku S, Fukuda K (2008) Gender difference in the composition of
fecal flora in laboratory mice, as detected by denaturing gradient
gel electrophoresis (DGGE). Exp Anim 57:489–493
Gebhardt S, Haberhausen M, Heinzel-Gutenbrunner M, Gebhardt N,
Remschmidt H, Krieg J-C, Hebebrand J, Theisen FM (2009)
Antipsychotic-induced body weight gain: predictors and a
systematic categorization of the long-term weight course. J
Psychiatr Res 43:620–626
Greenman Y, Golani N, Gilad S, Yaron M, Limor R, Stern N (2004)
Ghrelin secretion is modulated in a nutrient- and gender-specific
manner. Clin Endocrinol 60:382–388
Haack S, Seeringer A, Thürmann PA, Becker T, Kirchheiner J
(2009) Sex-specific differences in side effects of psychotropic
drugs: genes or gender?, pp 1511–1526
Hakko H, Komulainen MT, Koponen H, Saari K, Laitinen J, Järvelin
M-R, Lindeman S (2006) Are females at special risk of obesity if
they become psychotic? The longitudinal Northern Finland 1966
Birth Cohort Study. Schizophr Res 84:15–19
Harris RZ, Benet LZ, Schwartz JB (1995) Gender effects in
pharmacokinetics and pharmacodynamics. Drugs 50:222–239
Hooper LV, Midtvedt T, Gordon JI (2002) How host–microbial
interactions shape the nutrient environment of the mammalian
intestine. Annu Rev Nutr 22:283–307
Huson D, Auch A, Qi J, Schuster S (2007a) MEGAN analysis of
metagenomic data. Genome Res 17:377–386
Huson DH, Auch AF, Qi J, Schuster SC (2007b) MEGAN analysis of
metagenomic data. Genome Res 17:377–86
Jassim G, Skrede S, Vázquez M, Wergedal H, Vik-Mo A, Lunder N,
Diéguez C,Vidal-Puig A, Berge R, López M, Steen V, Fernø J
(2011) Acute effects of orexigenic antipsychotic drugs on lipid and
carbohydrate metabolism in rat. Psychopharmacology: 1–12
Kalliomaki M, Collado MC, Salminen S, Isolauri E (2008) Early
differences in fecal microbiota composition in children may
predict overweight. Am J Clin Nutr 87:534–538
Kapur S, Vanderspek SC, Brownlee BA, Nobrega JN (2003)
Antipsychotic dosing in preclinical models is often unrepresen-
tative of the clinical condition: a suggested solution based on in
vivo occupancy. J Pharmacol Exp Ther 305:625–631
Kim CS, Park HS, Kawada T, Kim JH, Lim D, Hubbard NE, Kwon
BS, Erickson KL, Yu R (2006) Circulating levels of MCP-1 and
IL-8 are elevated in human obese subjects and associated with
obesity-related parameters. Int J Obes 30:1347–1355
Kim SH, Nikolics L, Abbasi F, Lamendola C, Link J, Reaven GM,
Lindley S (2010) Relationship between body mass index and
insulin resistance in patients treated with second generation
antipsychotic agents. J Psychiatr Res 44:493–498
Kluge M, Schuld A, Himmerich H, Dalal M, Schacht A, Wehmeier
PM, Hinze-Selch D, Kraus T, Dittmann RW, Pollmacher T
Psychopharmacology (2012) 221:155–169 167

(2007) Clozapine and olanzapine are associated with food
craving and binge eating—results from a randomized double-
blind study. J Clin Psychopharmacol 27:662–666
Kluge M, Schuld A, Schacht A, Himmerich H, Dalal MA, Wehmeier
PM, Hinze-Selch D, Kraus T, Dittmann RW, Pollmächer T
(2009) Effects of clozapine and olanzapine on cytokine systems
are closely linked to weight gain and drug-induced fever.
Psychoneuroendocrinology 34:118–128
Kolehmainen M, Vidal H, Alhava E, Uusitupa MIJ (2001) Sterol
regulatory element binding protein 1c (SREBP-1c) expression in
human obesity. Obes Res 9:706–712
Kroeze WK, Hufeisen SJ, Popadak BA, Renock SM, Steinberg S,
Ernsberger P, Jayathilake K, Meltzer HY, Roth BL (2003) H1-
histamine receptor affinity predicts short-term weight gain for typical
and atypical antipsychotic drugs. Neuropsychopharmacology
28:519–526
Lam DD, Przydzial MJ, Ridley SH, Yeo GS, Rochford JJ, O'Rahilly
S, Heisler LK (2008) Serotonin 5-HT2C receptor agonist
promotes hypophagia via downstream activation of melanocortin
4 receptors. Endocrinology 149:1323–8
Larsen N, Vogensen FK, van den Berg FWJ, Nielsen DS, Andreasen
AS, Pedersen BK, Abu Al-Soud W, Sorensen SJ, Hansen LH,
Jakobsen M (2010) Gut microbiota in human adults with type 2
diabetes differs from non-diabetic adults. PLoS One 5:10
Lauressergues E, Martin F, Helleboid A, Bouchaert E, Cussac D,
Bordet R, Hum D, Luc G, Majd Z, Staels B, Duriez P (2010)
Overweight induced by chronic risperidone exposure is correlated
with overexpression of the SREBP-1c and FAS genes in mouse
liver. Naunyn Schmiedebergs Arch Pharmacol 383:423–436
Lee E, Leung CM, Wong E (2004) Atypical antipsychotics and weight
gain in Chinese patients: a comparison of olanzapine and
risperidone. J Clin Psychiatry 65:864–6
Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon
JI (2005) Obesity alters gut microbial ecology. Proc Natl Acad
Sci U S A 102:11070–11075
Makovey J, Naganathan V, Seibel M, Sambrook P (2007) Gender
differences in plasma ghrelin and its relations to body composition
and bone—an opposite-sex twin study. Clin Endocrinol 66:530–537
Masaki T, Chiba S, Yasuda T, Noguchi H, Kakuma T, Watanabe T,
Sakata T, Yoshimatsu H (2004) Involvement of hypothalamic
histamine H1 receptor in the regulation of feeding rhythm and
obesity. Diabetes 53:2250–60
Matsui-Sakata A, Ohtani H, Sawada Y (2005) Receptor occupancy-
based analysis of the contributions of various receptors to
antipsychotics-induced weight gain and diabetes mellitus. Drug
Metab Pharmacokinet 20:368–78
Meltzer HY, Perry E, Jayathilake K (2003) Clozapine-induced weight gain
predicts improvement in psychopathology. Schizophr Res 59:19–27
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 46:254–62
Mueller S, Saunier K, Hanisch C, Norin E, Alm L, Midtvedt T, Cresci
A, Silvi S, Orpianesi C, Verdenelli MC, Clavel T, Koebnick C,
Zunft HJF, Dore J, Blaut M (2006) Differences in fecal
microbiota in different European study populations in relation
to age, gender, and country: a cross-sectional study. Appl
Environ Microbiol 72:1027–1033
Murashita M, Kusumi I, Inoue T, Takahashi Y, Hosoda H, Kangawa K,
Koyama T (2005) Olanzapine increases plasma ghrelin level in
patients with schizophrenia. Psychoneuroendocrinology 30:106–110
Murphy EF, Cotter PD, Healy S, Marques TM, O'Sullivan O, Fouhy F,
Clarke SF, O'Toole PW, Quigley EM, Stanton C, Ross PR,
O'Doherty RM, Shanahan F (2010) Composition and energy
harvesting capacity of the gut microbiota: relationship to diet,
obesity and time in mouse models. Gut 59:1635–1642
Nasrallah H (2003) A review of the effect of atypical antipsychotics
on weight. Psychoneuroendocrinology 28(Suppl 1):83–96
Newcomer JW (2004) Metabolic risk during antipsychotic treatment.
Clin Ther 26:1936–46
Newcomer JW (2005) Second-generation (atypical) antipsychotics
and metabolic effects: a comprehensive literature review. CNS
Drugs 19(Suppl 1):1–93
Oriot P, Feys JL, Mertens de Wilmars S, Misson A, Ayache L, Fagnart
O, Gruson D, Luts A, Jamart J, Hermans MP, Buysschaert M
(2008) Insulin sensitivity, adjusted beta-cell function and adipo-
nectinaemiaamong lean drug-naive schizophrenic patients
treated with atypical antipsychotic drugs: a nine-month prospec-
tive study. Diabetes Metab 34:490–6
Patel JK, Buckley PF, Woolson S, Hamer RM, McEvoy JP, Perkins
DO, Lieberman JA, Investigators C (2009) Metabolic profiles of
second-generation antipsychotics in early psychosis: findings
from the CAFE study. Schizophr Res 111:9–16
Perez-Iglesias R, Mata I, Pelayo-Teran JM, Amado JA, Garcia-
Unzueta MT, Berja A, Martinez-Garcia O, Vazquez-Barquero
JL, Crespo-Facorro B (2009) Glucose and lipid disturbances after
1 year of antipsychotic treatment in a drug-naive population.
Schizophr Res 107:115–21
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 75:133–40
Pradhan AD, Manson JE, Rifai N, Buring JE, Ridker PM (2001) C-
reactive protein, interleukin 6, and risk of developing type 2
diabetes mellitus. JAMA 286:327–334
Raeder MB, Ferno J, Vik-Mo AO, Steen VM (2006) SREBP
activation by antipsychotic- and antidepressant-drugs in cultured
human liver cells: relevance for metabolic side-effects? Mol Cell
Biochem 289:167–173
Raskind MA, Burke BL, Crites NJ, Tapp AM, Rasmussen DD (2007)
Olanzapine-induced weight gain and increased visceral adiposity
is blocked by melatonin replacement therapy in rats. Neuro-
psychopharmacology 32:284–8
Reynolds GP, Hill MJ, Kirk SL (2006) The 5-HT2C receptor and
antipsychotic-induced weight gain—mechanisms and genetics. J
Psychopharmacol 20:15–8
Reynolds GP, Zhang ZJ, Zhang XB (2002) Association of antipsy-
chotic drug-induced weight gain with a 5-HT2C receptor gene
polymorphism. Lancet 359:2086–7
Riley LG, Fox BK, Kaiya H, Hirano T, Grau EG (2005) Long-term
treatment of ghrelin stimulates feeding, fat deposition, and alters
the GH/IGF-I axis in the tilapia, Oreochromis mossambicus. Gen
Comp Endocrinol 142:234–240
Roth BL, Sheffler D, Potkin SG (2003) Atypical antipsychotic drug
actions: unitary or multiple mechanisms for "atypicality"? Clin
Neurosci Res 3:108–117
Rotter V, Nagaev I, Smith U (2003) Interleukin-6 (IL-6) induces
insulin resistance in 3 T3-L1 adipocytes and is, like IL-8 and
tumor necrosis factor-alpha, overexpressed in human fat cells
from insulin-resistant subjects. J Biol Chem 278:45777–45784
Schellekens H, Dinan TG, Cryan JF (2010) Lean mean fat reducing
"ghrelin" machine: hypothalamic ghrelin and ghrelin receptors as
therapeutic targets in obesity. Neuropharmacology 58:2–16
Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister
EB, Lesniewski RA, Oakley BB, Parks DH, RobinsonCJ, Sahl JW,
Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing
mothur: Open-Source, Platform-Independent, Community-Sup-
ported Software for Describing and Comparing Microbial Commu-
nities. Appl Environ Microbiol 75(23):7537–7541
Seeman MV (2004) Gender differences in the prescribing of
antipsychotic drugs. Am J Psychiatry 161:1324–1333
Sentissi O, Epelbaum J, Olie J-P, Poirier M-F (2008) Leptin and
ghrelin levels in patients with schizophrenia during different
168 Psychopharmacology (2012) 221:155–169

antipsychotics treatment: a review. Schizophr Bull 34:1189–
1199
Shobo M, Yamada H, Mihara T, Kondo Y, Irie M, Harada K, Ni K,
Matsuoka N, Kayama Y (2011) Two models for weight gain and
hyperphagia as side effects of atypical antipsychotics in male
rats: validation with olanzapine and ziprasidone. Behav Brain
Res 216:561–568
Silvestre JS, Prous J (2005) Research on adverse drug events. I.
Muscarinic M3 receptor binding affinity could predict the risk of
antipsychotics to induce type 2 diabetes. Methods Find Exp Clin
Pharmacol 27:289–304
Starrenburg FC, Bogers JP (2009) How can antipsychotics cause diabetes
mellitus? Insights based on receptor-binding profiles, humoral
factors and transporter proteins. Eur Psychiatry 24:164–70
Straczkowski M, Dzienis-Straczkowska S, Stepien A, Kowalska I,
Szelachowska M, Kinalska I (2002) Plasma interleukin-8 concen-
trations are increased in obese subjects and related to fat mass
and tumor necrosis factor-{alpha} system. J Clin Endocrinol
Metab 87:4602–4606
Sugino H, Futamura T, Mitsumoto Y, Maeda K, Marunaka Y (2009)
Atypical antipsychotics suppress production of proinflammatory
cytokines and up-regulate interleukin-10 in lipopolysaccharide-
treated mice. Prog Neuropsychopharmacol Biol Psychiatry
33:303–307
Thornton-Jones Z, Neill JC, Reynolds GP (2002) The atypical
antipsychotic olanzapine enhances ingestive behaviour in the
rat: a preliminary study. J Psychopharmacol 16:35–37
Trayhurn P, Wood IS (2004) Adipokines: inflammation and the
pleiotropic role of white adipose tissue. Br J Nutr 92:347–355
Treuer T, Hoffmann VP, Chen AKP, Irimia V, Ocampo M, Wang G, Singh
P, Holt S (2009) Factors associated with weight gain during
olanzapine treatment in patients with schizophrenia or bipolar
disorder: Results from a six-month prospective, multinational,
observational study. World J Biol Psychiatry 10:729–740
Tsuda K, Yoshimatsu H, Niijima A, Chiba S, Okeda T, Sakata T
(2002) Hypothalamic histamine neurons activate lipolysis in rat
adipose tissue. Exp Biol Med (Maywood) 227:208–213
Turnbaugh PJ, Baeckhed F, Fulton L, Gordon JI (2008) Diet-induced
obesity is linked to marked but reversible alterations in the mouse
distal gut microbiome. Cell Host Microbe 3:213–223
Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A,
Ley RE, Sogin ML, Jones WJ, Roe BA, Affourtit JP, Egholm M,
Henrissat B, Heath AC, Knight R, Gordon JI (2009) A core gut
microbiome in obese and lean twins. Nature 457:480–U7
Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER,
Gordon JI (2006) An obesity-associated gut microbiome with
increased capacity for energy harvest. Nature 444:1027–1031
Urich T, Lanzen A, Qi J, Huson DH, Schleper C, Schuster SC (2008)
Simultaneous assessment of soil microbial community structure
and function through analysis of the meta-transcriptome. PLoS
One 3:e2527
Verma S, Liew A, Subramaniam M, Poon LY (2009) Effect of
treatment on weight gain and metabolic abnormalities in patients
with first-episode psychosis. Aust NZ J Psychiatry 43:812–7
Victoriano M, Hermier D, Even PC, Fromentin G, Huneau JF, Tome
D, de Beaurepaire R (2009) Early perturbation in feeding
behaviour and energy homeostasy in olanzapine-treated rats.
Psychopharmacology (Berl) 206:167–76
Weston-Green K, Huang XF, Deng C (2011) Sensitivity of the female
rat to olanzapine-induced weight gain—far from the clinic?
Schizophr Res 116:299–300
Wisse BE (2004) The inflammatory syndrome: the role of adipose
tissue cytokines in metabolic disorders linked to obesity. J Am
Soc Nephrol 15:2792–800
Xu HY, Barnes GT, Yang Q, Tan Q, Yang DS, Chou CJ, Sole J,
Nichols A, Ross JS, Tartaglia LA, Chen H (2003) Chronic
inflammation in fat plays a crucial role in the development of
obesity-related insulin resistance. J Clin Invest 112:1821–1830
Yang LH, Chen TM, Yu ST, Chen YH (2007) Olanzapine induces
SREBP-1-related adipogenesis in 3 T3-L1 cells. Pharmacol Res
56:202–208
Yokoyama Y, Nimura Y, Nagino M, Bland KI, Chaudry IH (2005)
Current understanding of gender dimorphism in hepatic patho-
physiology. J Surg Res 128:147–156
Psychopharmacology (2012) 221:155–169 169

Reproducedwith permission of the copyright owner. Further reproductionprohibited without permission.