THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
J Pharmacol Exp Ther 347:126–135, October 2013
Olanzapine Activates Hepatic Mammalian Target of Rapamycin:
New Mechanistic Insight into Metabolic Dysregulation with
Atypical Antipsychotic Drugs
Robin H. Schmidt, Jenny D. Jokinen, Veronica L. Massey, K. Cameron Falkner, Xue Shi,
Xinmin Yin, Xiang Zhang, Juliane I. Beier, and Gavin E. Arteel
Department of Pharmacology and Toxicology (R.H.S., J.D.J., V.L.M., J.I.B., G.E.A.), and Department of Medicine (K.C.F.), Health
Sciences Center, and Department of Chemistry (X.S., X.Y., X.Z.), University of Louisville, Louisville, Kentucky
Received June 28, 2013; accepted August 5, 2013
Olanzapine (OLZ), an effective treatment of schizophrenia and
other disorders, causes weight gain and metabolic syndrome.
Most studies to date have focused on the potential effects of
OLZ on the central nervous system’s mediation of weight;
however, peripheral changes in liver or other key metabolic
organs may also play a role in the systemic effects of OLZ. Thus,
the purpose of this study was to investigate the effects of OLZ
on hepatic metabolism in a mouse model of OLZ exposure.
Female C57Bl/6J mice were administered OLZ (8 mg/kg per day)
or vehicle subcutaneously by osmotic minipumps for 28 days.
Liver and plasma were taken at sacrifice for biochemical
analyses and for comprehensive two-dimensional gas chroma-
tography coupled to time-of-flight mass spectrometry metab-
olomics analysis. OLZ increased body weight, fat pad mass, and
liver-to-body weight ratio without commensurate increase in
food consumption, indicating that OLZ altered energy expendi-
ture. Expression and biochemical analyses indicated that OLZ
induced anaerobic glycolysis and caused a pseudo-fasted state,
which depleted hepatic glycogen reserves. OLZ caused similar
effects in cultured HepG2 cells, as determined by Seahorse
analysis. Metabolomic analysis indicated that OLZ increased
hepatic concentrations of amino acids that can alter metabolism
via the mTOR pathway; indeed, hepatic mTOR signaling was
robustly increased by OLZ. Interestingly, OLZ concomitantly
activated AMP-activated protein kinase (AMPK) signaling. Taken
together, these data suggest that disturbances in glucose and
lipid metabolism caused by OLZ in liver may be mediated, at least
in part, via simultaneous activation of both catabolic (AMPK) and
anabolic(mammalian targetofrapamycin)pathways,which yields
new insight into the metabolic side effects of this drug.
Olanzapine (OLZ) is one of the most effective drug options
for managing the symptoms of schizophrenia and bipolar
disorder (Lee et al., 2002). Unlike the first-generation anti-
psychotic medications (e.g., haloperidol), the risk of tardive
dyskinesia is low with OLZ, and acute idiosyncratic toxicity is
rare. These attributes make OLZ a drug of choice to treat
severe mental illness. Indeed, the lack of severe side effects
has expanded the off-label use of the drug for indications such
as dementia and treatment-resistant anxiety disorders (Maher
and Theodore, 2012). Although OLZ does not share the severe
toxicity of its first-generation predecessors, it does have side
effects that limit its therapeutic potential. Numerous studies
show that OLZ causes substantialweightgain only weeksafter
the start of administration, and that this weight gain persists
throughout treatment (Mathews and Muzina, 2007). These
effects strongly negatively influence patient treatment compli-
ance (Weiden et al., 2004).
OLZ-induced weight gain is not only an issue for patient
compliance, but can also induce sequelae associated with
weight gain/obesity such as glucose intolerance and/or insulin
resistance. Interestingly, the changes induced by OLZ ad-
ministration in carbohydrate and lipid metabolism may in
fact precede weight gain, which suggests a potential direct
effect of the drug on these pathways (Chintoh et al., 2008).
The side effects of OLZ on weight gain and glucose metabolism
are particularly relevant at this time, with obesity increasing
at an alarming rate in the United States and other indus-
trialized countries (Ogden et al., 2006). Moreover, even within
an increasingly overweight general population, obesity
This work was supported in part by the National Institutes of Health
National Institute of General Medical Sciences [Grant GM087735]; and by the
National Institutes of Health National Institute of Environmental Health
Sciences [Grant T32-ES011564] (Predoctoral Fellowship to R.H.S. and V.L.M.).
ABBREVIATIONS: Akt, protein kinase B; AMPK, AMP-activated protein kinase; CNS, central nervous system; 4EBP1, eukaryotic translation
initiation factor 4E-binding protein 1; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GC?GC-TOF-MS, comprehensive two-
dimensional gas chromatography coupled to time-of-flight mass spectrometry; HDL, high-density lipoprotein; LDL, low-density lipoprotein;
MTBSTFA, N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide; mTOR, mammalian target of rapamycin; NEFA, nonesterified fatty acids; OCR,
oxygen consumption rate; OGTT, oral glucose tolerance test; OLZ, olanzapine; p70S6K, 70-kDa ribosomal protein S6 kinase; PAS, periodic acid-
Schiff; PPR, proton production rate; RT-PCR, reverse transcription-polymerase chain reaction; TBDMSCI, tert-butyldimethylchlorosilane; TG,
triglycerides; VLDL, very-low-density lipoprotein.
at Emory University on November 2, 2013
disproportionately affects individuals with severe mental
illnesses (Allison et al., 2009). Therefore, it is necessary to
develop strategies that prevent, minimize, or reverse the
adverse metabolic effects that occur during OLZ treatment,
especially for a population already at risk for obesity and its
sequelae (Heiskanen et al., 2003).
The beneficial effects of OLZ are assumed to be mediated
most studies into the metabolic side effects of OLZ have also
focused on the CNS (Weston-Green et al., 2012). Although the
CNS clearly plays a key role in regulating food consumption,
et al., 2013), peripheral organs also significantly contribute to
metabolic dysregulation in the intact organism. To date, very
little is understood about the potential effects of OLZ admin-
istration on the peripheral organs critical for metabolic homeo-
stasis. Theliver iskey for overallglucose and lipid homeostasis,
and recent studies have suggested that the effect of OLZ on
the liver may contribute to its metabolic disturbances (Girault
et al., 2012). Therefore, we explored the effects of OLZ on in-
dices of carbohydrate and lipid metabolism in this organ and
determined the underlying mechanisms in a mouse model of
Materials and Methods
Animals and Treatments. Female C57BL/6J mice (8 weeks old)
were purchased from The Jackson Laboratory (Bar Harbor, ME). The
mice were housed in a pathogen-free barrier facility accredited by the
Association for Assessment and Accreditation of Laboratory Animal
Care, and the procedures were approved by the University of
Louisville Institutional Animal Care and Use Committee. Food and
tap water were allowed ad libitum. One week before the initiation of
the experiment, the animals were switched from standard chow to
purified TD.08485 diet (Harlan Laboratories, Madison, WI) to avoid
any batch-to-batch variability in the food content (see Fig. 1A).
Olanzapine (8 mg/kg per day; Kapur et al., 2003) or vehicle (saline)
was given subcutaneously via osmotic minipumps (Alzet, Cupertino,
CA) for 28 days. To avoid concerns of OLZ degradation, the pumps were
replaced after 2 weeks (van der Zwaal et al., 2008). The animals were
weighed on a weekly basis, and their food consumption was recorded
twice per week. After 28 days, the mice were anesthetized with
Body composition was assessed by dual-energy X-ray absorptiometry
(DEXA) using a Lunar PIXImus densitometer (Lunar Corp., Madison,
WI) according to the manufacturer’s instructions. Blood was collected
from the vena cava just before sacrifice by exsanguination, and
citrated plasma was stored at 280°C for further analysis. Portions of
liver tissue were frozen immediately in liquid nitrogen, fixed in 10%
neutral buffered formalin, or frozen-fixed for subsequent sectioning
and mounting on microscope slides. Blood, liver, and gonadal fat pads
were collected for later analysis.
Oral Glucose Tolerance Test. An oral glucose tolerance test
(OGTT) (Andrikopoulos et al., 2008) was performed after 25 days of
OLZ exposure. In brief, mice were transferred to cages that had been
cleared of food and bedding, and they were fasted for 6 hours. Blood
was sampled from the tail vein immediately after the fasting period,
and then 15, 30, 60, 90, and 120 minutes after oral administration of
2 mg/kg D-(1)-glucose (Sigma-Aldrich, St. Louis, MO) in sterile saline
(4 ml/kg) solution. Glucose concentrations were measured using an
Accu-Chek Aviva Plus glucometer and test strips (Roche Diagnostics
Corp., Indianapolis, IN).
Biochemical Analyses and Histology. Plasma levels of alanine
aminotransferase (ALT), aspartate aminotransferase (AST), total
cholesterol, high-density lipoprotein (HDL), low-density lipoprotein
(LDL), very-low-density lipoprotein (VLDL), triglycerides (TG), and
glucose were determined by the Piccolo Lipid Panel Plus Reagent
Disc, used with the Piccolo Xpress Chemistry Analyzer (Abaxis, Inc.,
Union City, CA), according to the manufacturer’s instructions.
Paraffin-embedded sections of liver were stained with hematoxylin
and eosin (H&E) to assess overall hepatic structure. Hepatic lipids—TG
and nonesterified fatty acids (NEFA)—were extracted in chloroform/
methanol (Bligh and Dyer, 1959) and measured as described previously
elsewhere (Bergheim et al., 2006). Results were normalized to the wet
weight of extracted tissue.
For detection of hepatic neutral lipids in tissue, 10-mm frozen
sections were stained with Oil Red O (Sigma-Aldrich) for 10 minutes,
et al., 2006). For hepatic glycogen staining, paraffin sections (5 mm)
were deparaffinized and rehydrated, oxidized in 0.5% periodic acid
solution for 5 minutes, washed, placed in Schiff reagent for 15 minutes,
washed, and counterstained with hematoxylin for 1 minute. Staining
was quantitated by image analysis, as described previously elsewhere
(Bergheim et al., 2006).
Polymerase Chain Reaction. RNA extraction and real-time re-
verse transcription-polymerase chain reaction (RT-PCR) were per-
formed as described previously elsewhere (Bergheim et al., 2006). The
PCR primers and probes were purchased premade from Applied
Biosystems (Carlsbad, CA) and were designed to span introns to avoid
amplification of any contaminating DNA. The comparative Ctmethod
was used to determine fold differences between samples and the
calibrator gene (b-actin), and the purity of PCR products was verified
by gel electrophoresis. The comparative Ctmethod determines the
amount of target, normalized to an endogenous reference (b-actin)
and relative to a calibrator (2DDCt).
Immunoblots. Frozen liver samples were homogenized in radio-
immunoprecipitationassaybuffer (20 mMTris,pH7.4, 150mMNaCl,
1 mM EDTA, 1 mM EGTA, 1 mM b-glycerophosphate, 1 mM Na3VO4,
and 1% w/w Triton X-100) containing protease, tyrosine/phosphatase,
andserine/threonine phosphatase inhibitorcocktails(Sigma-Aldrich).
Lysates were sonicated and subsequently centrifuged for 5 minutes at
16,000g. The protein concentration of the supernatants was de-
termined with the Bio-Rad DC Protein Assay (Bio-Rad Laboratories,
Hercules, CA); 100 mg of total protein were mixed with 4? sample
loading buffer (250 mM Tris, pH 7.4, 10% SDS, 20% 2-mercaptoetha-
nol, 40% glycerol, and 0.01% w/v bromphenol blue) and incubated at
95°C for 5 minutes. The samples were loaded onto SDS-polyacrylamide
gels (Bio-Rad Laboratories), followed by electrophoresis and Western
Biosciences, Piscataway, NJ). Antibodies against AMP-activated protein
kinase a (AMPKa), phospho-AMPKa (p-AMPKa), protein kinase B
(Akt), phospho-Akt (p-Akt), mammalian target of rapamycin (mTOR),
phospho-mTOR (p-mTOR), 70-kDa ribosomal protein S6 kinase
(p70S6K) (Bethyl Laboratories, Montgomery, TX), phospho-p70S6K
(p-p70S6K), eukaryotic translation initiation factor 4E-binding protein
1 (4EBP1) (Bethyl Laboratories), and phospho-4EBP1 (p-4EBP1) were
used at the dilutions recommended by the suppliers (Cell Signaling
Technology, Danvers, MA; unless otherwise indicated). Horseradish
peroxidase-coupled secondary antibodies and chemiluminescence de-
tection reagents were obtained from Pierce (Rockford, IL). The signals
were detected using Classic Blue autoradiography film BX (MIDSCI,
St. Louis, MO). Densitometric quantitation was performed with
UN-SCAN IT analysis software (Silk Scientific, Orem, UT), as
described previously elsewhere (Bergheim et al., 2006).
surements were made using an XF96 Extracellular Flux Analyzer
(Seahorse Biosciences, Billerica, MA). HepG2 cells (American Type
Culture Collection, Manassas, VA) were plated at 10,000 cells per
well and grown for 24 hours in Dulbecco’s modified Eagle’s medium
(Invitrogen, Carlsbad, CA) containing either glucose (25 mM) or
galactose (10 mM) and 6 mM pyruvate. Immortalized cells preferen-
tially use anaerobic glycolysis to meet energy demands, even in the
Olanzapine and Hepatic Metabolic Dysfunction
presence of adequate oxygen (Warburg et al., 1967). Anaerobic
glycolysis of galactose yields no net ATP and forces cells to rely on
oxidative phosphorylation (Marroquin et al., 2007). Cells were then
treated with graded concentrations of olanzapine from 0 to 25 mM.
One hour before the commencement of measurements, the medium
was changed to unbuffered Dulbecco’s modified Eagle’s medium (Sea-
horse Biosciences) containing the same concentrations of sugars,
pyruvate, or olanzapine. The XF96 Extracellular Flux analyzer measures
the OCR (oxygen consumption rate) and PPR (proton production rate) in
a small transient chamber in specialized plates. Seeding density was
optimized under the cell culture conditions described to ensure linear
oxygen consumption and adequate mixing during the re-equilibration
phases. Coupled and uncoupled OCR and PPR measurements were
made by addition of oligomycin (1 mg/ml) and carbonyl cyanide
sequential injections from ports in the Seahorse Flux Pak cartridges.
Metabolite Sample Preparation. A sample of liver tissue was
weighed and homogenized for 2 minutes after adding water at a
concentration of 100 mg/ml. The homogenized sample was then stored
at –80°C until use. A 100-ml aliquot of the homogenized liver sample
and 400 ml of ice-cold methanol were mixed and vortexed for 2
minutes and then incubated for 10 minutes on ice followed by another
2-minute vortex. The mixtures were centrifuged at 4°C for 10 minutes
tube, which was then dried overnight in a centrifugal evaporator
(SpeedVac; Thermo-Fisher, Waltham, MA). The extracted metabolites
were then dissolved in 40 ml of acetonitrile. After adding 40 ml of N-(tert-
butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) mixed
with 1% tert-butyldimethylchlorosilane (TBDMSCI), the mixture was
sonicated for 3 hours followed by overnight derivatization at room
temperature. The samples were then transferred to gas chromatog-
raphy vials for analysis. The derivatization was performed just before
comprehensive two-dimensional gas chromatography coupled to time-
of-flight mass spectrometry (GC?GC-TOF-MS) analysis.
GC3GC-TOF-MS Analysis. The LECO Pegasus 4D GC?GC-
TOF-MS instrument was equipped with an Agilent 6890 gas chromato-
graph and a Gerstel MPS2 autosampler (GERSTEL, Inc., Linthicum,
MD), featuring a LECO two-stage cryogenic modulator and secondary
oven.Theprimarycolumnwasa60m? 0.25mm1dc? 0.25mm1df,DB-
5ms GC capillary column [phenyl arylene polymer virtually equivalent
to (5%-phenyl)-methylpolysiloxane]. A second GC column of 1 m ? 0.25
mm1dc? 0.25 mm2df, DB17ms [(50% phenyl)-methylpolysiloxane]
wasplacedinsidethe secondaryGC ovenafterthethermal modulator.
Both columns were obtained from Agilent Technologies (Santa
The helium carrier gas (99.999% purity) flow rate was set to 2.0
ml/min at a corrected constant flow via pressure ramps. The inlet
temperature was set at 280°C. The primary column temperature was
programmed with an initial temperature of 60°C for 0.5 minutes and
then was ramped at 5°C per minute to 270°C and kept for 15 minutes.
The secondary column temperature program was set to an initial
temperature gradient used in the first column to 280°C accordingly.
The thermal modulator was set to 115°C relativeto the primary oven,
and a modulation time of PM5 2 second was used. The mass range
was set as 292800 m/z with an acquisition rate of 200 mass spectra
per second. The ion source chamber was set at 230°C with the transfer
line temperature set to 280°C, and the detector voltage was 1450 V
Fig. 1. Effect of OLZ on body weight gain and
adiposity. (A) Schematic of the experimental design.
(B) Average weekly food consumption and weight
gain. (C) Fat pad and total body fat. (D) Represen-
tative dual-energy X-ray absorptiometry (DEXA)
scan images. Low-density areas depict soft matter
(i.e., body fat). *P , 0.05 compared with controls.
Schmidt et al.
with electron energy of 70 eV. The acceleration voltage was turned on
after a solvent delay of 674 second. The split ratio was set at 25:1.
GC3GC–TOF MS Data Analysis. The GC?GC–TOF MS data
were processed using LECO’s instrument control software (Chroma-
TOF) for peak picking and tentative metabolite identification, followed
by retention index matching, peak merging, peak list alignment,
normalization, and statistical significance test using MetPP software
(Wei et al., 2013). For metabolite identification using ChromaTOF, each
chromatographic peak was tentatively assigned to a metabolite if its
experimental mass spectrum and a database spectrum have a spectral
was performed using the iMatch method with the P value threshold set
as P # 0.001 (Zhang et al., 2011). The pairwise two-tail t test was used to
determine whether a metabolite had a statistically significant abun-
dance difference between sample groups by setting the threshold of false
discovery rate q # 0.3.
Functional clustering, enrichment and pathway analysis of the
regulated metabolites was performed using the Metacore (Thomson
Reuters, http://www.genego.com/metacore.php) platform, according
to the site’s instructions. Through this platform, metabolites were
analyzed using the map folders, pathway maps, process networks,
and metabolic networks enrichment analysis. Final networks were
transferred to Pathway Map Creator tool (Thomson Reuters) to create
a signaling map.
Statistical Analysis. Results are reported as mean 6 S.E.M. (n 5
4–8). Student’s t test or repeated measures analysis of variance was
used for to determine the statistical significance between control and
OLZ groups, as appropriate. P , 0.05 was considered statistically
OLZ Administration Increases Weight and Adiposity.
Average body weight at the beginning of the study was 19.4 6
0.1 g, and did not significantly differ between the groups. All
animals gained weight during the course of the study, and
there was no mortality or morbidity in any group. OLZ
administration did not significantly increase food consump-
tion compared with the animals administered vehicle (con-
trol). Despite no apparent increase in food consumption, OLZ
administration significantly increased body weight gain by
Fig. 2. OLZ promotes hepatic lipid accumula-
tion. (A) Representative photomicrographs
depicting general histology (H&E; upper pan-
els) and lipid accumulation (Oil Red O; lower
panels). (B) Hepatic triglyceride (TG) and
nonesterified fatty acid (NEFA) contents. (C)
Liver weight measurements taken at sacrifice
expressed as a percentage of total body weight.
*P , 0.05 compared with controls.
Olanzapine and Hepatic Metabolic Dysfunction
previously by other groups (Cope et al., 2005). An increase
in the body fat percentage and gonadal fat pad mass
accompanied the weight gain (Fig. 1, C and D) in OLZ-exposed
OLZ Promotes Hepatic Lipid Accumulation. Obesity
and/or metabolic syndrome commonly cause lipids to accu-
mulate in the liver (i.e., steatosis). As OLZ administration
increased the body weight and total body fat (Fig. 1), the effect
of OLZon hepatic lipid accumulation wasdetermined. A crude
index of hepatic lipid accumulation is liver size. OLZ
increased liver weight as indicated by the elevation in liver
weight to body weight ratio (Fig. 2C). Four weeks of OLZ
treatment also increased hepatic fat, as indicated by the
presence of macrovesicular and microvesicular lipid droplets
in H&E staining (Fig. 2A, top). This effect of OLZ on hepatic
lipid accumulation was confirmed by Oil Red O staining (Fig.
OLZ administration significantly elevated hepatic TG
content ∼2-fold. Interestingly, this increase in TG was not
coupled with a concomitant increase in hepatic NEFA. This
increase in hepatic lipids was paralleled by a statistically
Effect of OLZ on plasma variables
Animals and treatments are described under Materials and Methods. Data are mean 6
S.E.M. (n = 4–8) and are reported as indicated in the individual rows.
Biochemical Analyses ControlOLZ
196 6 6
42 6 2
50 6 6
34 6 4
8 6 2
9 6 1
33 6 9
56 6 2
329 6 13*
80 6 7*
62 6 3
42 6 4
4 6 2
16 6 1*
76 6 9*
80 6 6*
ALT, alanine aminotransferase; AST, aspartate aminotransferase; HDL, high-
density lipoprotein; LDL, low-density lipoprotein; OLZ, olanzapine; VLDL, very-low-
*P , 0.05 compared with controls.
Fig. 3. Effects of OLZ on hepatic expression
of metabolism-regulating genes and in vitro
metabolism of OLZ-treated HepG2 cells. (A)
Hepatic mRNA expression of genes for lipid
and carbohydrate metabolism determined by
quantitative RT-PCR. (B) OCR and PPR rate
data (Seahorse) for all time points in HepG2
cells treated with 25 mM OLZ or control. (C)
Effect of increasing concentrations of OLZ on
steady-state PPR/OCR. *P , 0.05 compared
Schmidt et al.
significant increase in plasma TG and VLDL (Table 1). These
effects of OLZ were also accompanied by liver injury, as
indicated by significant elevations of plasma alanine amino-
transferase (ALT) (∼2-fold) and aspartate aminotransferase
(AST) (∼1.5-fold; Table 1).
OLZ Modifies Hepatic Expression of Metabolism-
Regulating Genes. Hepatic steatosis is often mediated via
direct alterations in the expression of genes key to lipid and
carbohydrate metabolism. To further explore the effects of
OLZ on hepatic energy metabolism, mRNA expression of
genes that are key in regulating the synthesis and catabolism
of carbohydrates and lipids was examined by quantitative
RT-PCR (Fig. 3A). Four weeks of OLZ administration signif-
icantly downregulated the expression of a number of genes
involved in lipid biosynthesis, including sterol regulatory
element-binding protein 1 (Srebf1), fatty acid synthase (Fasn),
and ATP citrate lyase (Acly). Similarly, OLZ exposure upregu-
lated expression of glycogen synthase kinase-3b (Gsk3b), a pro-
tein that suppresses glycogen synthesis. These changes in
expression of anabolism-regulating genes were accompanied by
a significant (2.5-fold) increase in expression of glucokinase
(Gck), a key rate-limiting enzyme in glycolysis. Expression of
carnitine palmitoyltransferase 1a (Cpt1a), which encodes the
rate-limiting enzyme in fatty acid b-oxidation, was unchanged,
as were phosphoenolpyruvate carboxykinase 1 (Pck1; gluconeo-
genesis) and glucose transporter type 1 and glucose transporter
type 4 (Glut1 and Glut4; basal and insulin-mediated glucose
Effects of OLZ on Mitochondrial Respiration. The
mRNA expression data suggest that OLZ administration
favors glycolysis (Fig. 3). Previous studies with isolated brain
mitochondria have suggested that OLZ partially inhibits
mitochondrial respiration [e.g., (Hroudova and Fisar, 2010)].
The effect of OLZ exposure on the balance between glycolysis
and mitochondrial respiration was therefore determined in
containing media (see Materials and Methods). Under these
conditions, the PPR is used as an index of glycolysis, and the
OCR is used as an index of mitochondrial function (Fig. 3, B
and C). As expected, inhibition of mitochondrial respiration
with oligomycin, which forced the cells to rely on anaerobic
glycolysis, decreased the rate of OCR while simultaneously
increasing the rateof PPR,thereby greatly increasing the PPR/
OCR ratio. Uncoupling the mitochondria with FCCP increased
the OCR while still maintaining an elevated PPR, causing the
PPR/OCR ratio to decrease relative to oligomycin but still remain
OLZ had no apparent effect on OCR or PPR at any concentration.
When the Warburg/Crabtree effect was overcome by incubating
HepG2 cells in galactose-containing media (Warburg et al.,
1967), the ratio of PPR/OCR was dramatically decreased, as
the cells were forced to rely on oxidative phosphorylation
caused a dose-dependent increase in PPR without significantly
affecting OCR; these effects of OLZ statistically significantly
increased the PPR/OCR ratio (Fig. 3, B and C).
Effects of OLZ and Glucose and Glycogen Expendi-
ture. OLZ increased basal (unfasted) plasma glucose, as
indicated by plasma taken at sacrifice (Table 1). Previous
studies have indicated that OLZ administration may cause
insulin resistance and/or glucose intolerance (Coccurello and
Moles, 2010). To determine whether OLZ administration
under the current conditions affected glucose tolerance, fasted
mice were subjected to the OGTT at 25 days of vehicle or drug
administration (see Materials and Methods, and Fig. 4A). OLZ
did not affect the area under the curve of the OGTT during the
testing period. However, OLZ exposure significantly in-
creased the plasma glucose concentrations 15 minutes after
bolus gavage (Fig. 4A), although the values were similar to
controls at all subsequent time points.
As mentioned earlier, the expression of a key inhibitor of
glycogen storage (glycogen synthase kinase-3b) was statisti-
cally significantly increased in livers of OLZ-exposed mice
(see Fig. 3A). The effect of OLZ administration on hepatic
glycogen storage was therefore assessed by periodic acid-
Schiff (PAS) staining (Fig. 4, B and C, representative photo-
micrographs and quantitative image analysis, respectively).
OLZ administration statistically significantly decreased the
amount of glycogen stores in unfasted liver by 2-fold compared
OLZ Effects on the Hepatic Metabolome, mTOR, and
AMPK. Examining metabolites onan individual basis is time
consuming, and the smaller amounts of data produced may
unnecessarily narrow the scope of a study. Metabolomic analysis
was therefore used to simultaneously characterize several effects
of OLZ on liver. Metabolites that varied significantly between the
OLZ group and the control group were then analyzed with
Fig. 4. Effects of OLZ and glucose and glycogen expenditure. (A) Effect of
OLZ on oral glucose tolerance (OGTT). (B) Representative photomicro-
graphs stained for glycogen content (PAS). (C) Summary of quantitative
image-analysis of PAS staining. *P , 0.05 compared with controls by
repeated measures analysis of variance (A) or t test (C).
Olanzapine and Hepatic Metabolic Dysfunction
pathway analysis software, which predicted and mapped the
pathways that may be relevant to OLZ treatment (Fig. 5, A
and B). Among other findings, amino acids were significantly
affected by OLZ: L-glutamine, a putative mediator of mTOR
activation (Nicklin et al., 2009), was increased in animals
administered OLZ. OLZ also caused a decrease in L-leucine
and a parallel increase in L-glutamate, which can act as
nitrogen donor and nitrogen acceptor, respectively, in the trans-
amination reaction that regulates the glutamate-glutamine cycle
(Islam et al., 2010). Network analysis further predicted the
involvement of amino acid transporters Slc7a5 (LAT1) and
Slc38a2 (SNAT2), which were recently shown to function as
regulators of mTOR signaling, and excitatory amino acid trans-
porter 3 (EAAT3), which is regulated by mTOR activation
(Almilaji et al., 2012).
Given the key role of mTOR in mediating carbohydrate
and lipid metabolism (Tsang et al., 2007) and the effects of
OLZ on these pathways (see Figs. 3–5), the effect of OLZ
administration on the activation of mTOR and dependent
signaling cascades was determined by Western blot anal-
ysis (Fig. 6). OLZ administration increased mTOR activa-
tion, as indicated by a statistically significant increase in
phosphorylation at Ser248. OLZ administration also in-
creased phosphorylation of p70S6K at Thr389 and 4EBP1
at Thr37/46, indicative of mTORC1 activation (Hara et al.,
1997), as well as Akt phosphorylation at Ser473, indicative
of mTORC2 activation (Guertin et al., 2006). Interestingly,
OLZ treatment also concomitantly increased AMPK acti-
vation, as indicated by an increase in phosphorylation at
Fig. 5. OLZ effects on hepatic metabolite
profile. (A) Metabolites determined by
GC?GC-TOF-MS and evaluated with
map. (B) Abundance data for selected
metabolites. *P , 0.05 compared with
Schmidt et al.
OLZ is an effective drug for treating several psychiatric
illnesses and is the drug of choice for schizophrenia and bipolar
disorder, but its benefits of OLZ are countered by side effects
such as weight gain, glucose intolerance, and dyslipidemia,
which impact not only treatment compliance but also increase
the health risks to patients. An improved understanding of OLZ
actions in the peripheral tissue may therefore identify new
approaches to increase the long-term safety and utility of this
As has been observed in other studies, OLZ administration
increases body weight and adiposity in mice (Fig. 1). Interest-
ingly, this effect of OLZ was not concurrent with a detectable
increase in food consumption (Fig. 1), which suggests that OLZ
administration may directly alter energy usage in vivo. In
support of this hypothesis, previous experiments have shown
that both acute and chronic OLZ administration dysregulates
peripheral carbohydrate and lipid metabolism (Chintoh et al.,
2008; Coccurello and Moles, 2010; Girault et al., 2012) and
directly increases the expression of lipid biosynthetic genes in
both liver and adipose tissue (Skrede et al., 2012). In our study,
OLZ administration caused a complex phenotypic alteration in
hepatic carbohydrate metabolism. OLZ administration increased
glycolysis without an apparent increase in mitochondrial res-
piration (Figs. 3 and 4) and favored glycogen depletion (Figs. 4
OLZ administration also caused a slight but significant
increase in glucose intolerance (Fig. 4) and unfasted plasma
glucose (Table 1). A shift to an elevated but incomplete
(i.e., anaerobic) glycolysis would favor ATP generation from
alternate sources such as lipid oxidation. In support of this
hypothesis, OLZ administration increases plasma lactic acid
levels in humans (Glavina et al., 2011), which is indicative of
increased anaerobic glycolysis. Furthermore, OLZ adminis-
tration lowers the respiratory exchange ratio in rats (Albaugh
et al., 2012), which is indicative of a preferential shift to
NEFA over carbohydrates as fuel. These results are in line
with the observation here that hepatic and plasma triglycer-
ides were elevated by OLZ administration without a commen-
surate increase in NEFA (Fig. 2; Table 1).
Within the cell, carbohydrate and lipid metabolism are
usually under tight control by the protein kinases AMPK and
Fig. 6. OLZ mediates signaling through mTOR pathways.
Liver homogenates were prepared as described under
Materials and Methods. (A) Representative immunoblots.
(B) Ratio of phosphorylated to total protein via quantitative
analysis. (C) Proposed mechanisms by which OLZ admin-
istration activates mTOR. OLZ promotes flux of glutamine
(Gln) and leucine (Leu) through solute carrier 38a2
(SLC38A2 or SNAT2) and solute carrier 7a5 (SLC7A5 or
LAT1). Increased concentrations of Gln and Leu induce
mTOR signaling. Excess intracellular Leu acts as a nitrogen
donor in the branched chain aminotransferase (BCAT)
reaction that forms glutamate (Glu). Leu and Glu also
contribute metabolic intermediates (e.g., acetyl CoA) to the
Krebs cycle. Interestingly, AMPK was simultaneously
activated under these conditions (see Discussion for addi-
tional information). *P , 0.05 compared with controls.
Olanzapine and Hepatic Metabolic Dysfunction
mTOR. Both AMPK and mTOR are known to act as “sensors”
of cellular energy status and to help maintain homeostasis
(Hardie, 2007; Guertin and Sabatini, 2009). In general, the
downstream effects of AMPK activation are considered
catabolic and favor ATP generation during energy depletion.
Glycolysis, for example, is enhanced by AMPK. Signaling
downstream of AMPK also inhibits ATP-consuming processes
(Krause et al., 2002). In contrast to AMPK, mTOR is activated
during times of high nutrient availability and favors storage
of excess nutrients (e.g., as triglycerides). Activation of the
mTOR pathway promotes ATP-consuming processes such as
protein and lipid synthesis through its downstream targets
p70S6K and 4EBP1.
Previous studies have demonstrated that AMPK is acti-
vated in the CNS by OLZ administration (Kim et al., 2007).
Furthermore, OLZ increases p70S6K1 and 4EBP1 phosphor-
ylation in cultured hepatocytes, indicative of mTOR activa-
tion (Oh et al., 2011). The experiments here demonstrate, for
thefirst time, that OLZ in vivoconcomitantly activates AMPK
and mTOR pathways in the liver. AMPK and mTOR are
generally differentially activated and mediate opposing
cellular functions (Kimball, 2006). The outcomes of OLZ admin-
istration on hepatic metabolism may reflect these contradictory
inputs. These data suggest that treatment with OLZ results
but cannot be efficiently used.
The reason for OLZ’s concurrent activation of AMPK and
mTOR is not immediately clear. Should OLZ indeed induce a
pseudo-fasted state, the activation of AMPK may be, in
principle, via the “energy sensing” activation of this pathway.
As mentioned earlier, OLZ increases peripheral concentra-
tions of glutamate and its metabolites (Fig. 5) (Goff et al.,
2002; Yudkoff et al., 2005). The increase in these molecules in
the liver may directly activate mTOR (Tato et al., 2011;
Laplante and Sabatini, 2012).
Previous studies have indicated that activating AMPK with
metformin (a classicapproach totreat the metabolicsyndrome)
the metabolic effects of OLZ (Ehret et al., 2010). This lack of
effectiveness may be due to the fact that OLZ administration
itself activates AMPK. Alternatively, it has been suggested
that “normalizing” mTOR overactivation may be beneficial in
obesity and diabetes (Laplante and Sabatini, 2012). It may be
that such an approach would be more effective in the context of
OLZ administration. The glutamatergic model of mental
illness proposes that psychotic disorders are disorders of
excitatory amino acid metabolism (Tsai and Coyle, 2002).
Therefore, inhibiting mTOR would be arguably downstream of
the assumed therapeutic mechanisms of OLZ in psychiatric
disorders, and may be a beneficial treatment approach to
prevent this drug’s metabolic side effects OLZ.
Another important aspect of this work is that OLZ adminis-
tration significantly increased plasma indices of liver injury in
mice (Table 1). Elevation of liver enzymes is described as a
common reaction with OLZ administration, and generally
results in drug discontinuation (Dominguez-Jimenez et al.,
2012). The pathologic changes observed in liver here (Fig. 2) are
analogous to those caused by nonalcoholic fatty liver disease
(NAFLD). As mentioned earlier, obesity and its sequelae (e.g.,
NAFLD) have been reaching alarmingly high levels in de-
veloped countries. NAFLD is a spectrum of liver diseases,
ranging from the relatively benign simple steatosis to active
to hepatocellular carcinoma (Ratziu et al., 2002). Although the
prevalence of simple steatosis in individuals at risk for NAFLD
can be very high (i.e., .90%), the prevalence of the more severe
factors emphasize that the risk for developing more severe
stages of NAFLD is not based solely on obesity or other primary
and James, 1998). It is therefore possible that the metabolic
effects of OLZ administration could serve as such a second hit in
Participated in research design: Schmidt, Zhang, Arteel.
Conducted experiments: Schmidt, Jokinen, Massey, Falkner, Shi,
Contributed new reagents or analytic tools: Zhang.
Performed data analysis: Schmidt, Zhang, Shi, Yin, Beier, Arteel.
Wrote or contributed to the writing of the manuscript: Schmidt, Shi,
Albaugh VL, Vary TC, Ilkayeva O, Wenner BR, Maresca KP, Joyal JL, Breazeale S,
Elich TD, Lang CH, and Lynch CJ (2012) Atypical antipsychotics rapidly and
inappropriately switch peripheral fuel utilization to lipids, impairing metabolic
flexibility in rodents. Schizophr Bull 38:153–166.
Allison DB, Newcomer JW, Dunn AL, Blumenthal JA, Fabricatore AN, Daumit GL,
Cope MB, Riley WT, Vreeland B, and Hibbeln JR, et al. (2009) Obesity among those
with mental disorders: a National Institute of Mental Health meeting report. Am J
Prev Med 36:341–350.
Almilaji A, Pakladok T, Guo A, Munoz C, Föller M, and Lang F (2012) Regulation of
the glutamate transporter EAAT3 by mammalian target of rapamycin mTOR.
Biochem Biophys Res Commun 421:159–163.
Andrikopoulos S, Blair AR, Deluca N, Fam BC, and Proietto J (2008) Evaluating the
glucose tolerance test in mice. Am J Physiol Endocrinol Metab 295:E1323–E1332.
Bergheim I, Guo L, Davis MA, Lambert JC, Beier JI, Duveau I, Luyendyk JP, Roth
RA, and Arteel GE (2006) Metformin prevents alcohol-induced liver injury in the
mouse: critical role of plasminogen activator inhibitor-1. Gastroenterology 130:
Bligh EG and Dyer WJ (1959) A rapid method of total lipid extraction and purifi-
cation. Can J Biochem Physiol 37:911–917.
Chintoh AF, Mann SW, Lam L, Lam C, Cohn TA, Fletcher PJ, Nobrega JN, Giacca A,
and Remington G (2008) Insulin resistance and decreased glucose-stimulated insulin
secretion after acute olanzapine administration. J Clin Psychopharmacol 28:494–499.
Coccurello R and Moles A (2010) Potential mechanisms of atypical antipsychotic-
induced metabolic derangement: clues for understanding obesity and novel drug
design. Pharmacol Ther 127:210–251.
Cope MB, Nagy TR, Fernández JR, Geary N, Casey DE, and Allison DB (2005)
Antipsychotic drug-induced weight gain: development of an animal model. Int J
Obes (Lond) 29:607–614.
Day CP and James OF (1998) Steatohepatitis: a tale of two “hits”? Gastroenterology
Domínguez-Jiménez JL, Puente-Gutiérrez JJ, Pelado-García EM, Cuesta-Cubillas D,
and García-Moreno AM (2012) Liver toxicity due to olanzapine. Rev Esp Enferm
Ehret M, Goethe J, Lanosa M, and Coleman CI (2010) The effect of metformin on
anthropometrics and insulin resistance in patients receiving atypical antipsychotic
agents: a meta-analysis. J Clin Psychiatry 71:1286–1292.
Girault EM, Alkemade A, Foppen E, Ackermans MT, Fliers E, and Kalsbeek A (2012)
Acute peripheral but not central administration of olanzapine induces hypergly-
cemia associated with hepatic and extra-hepatic insulin resistance. PLoS ONE 7:
Glavina T, Mrass D, Dodig T, Glavina G, Prani? c S, and Ugle? si? c B (2011) Blood lactate
levels in patients receiving first- or second- generation antipsychotics. Croat Med J
Goff DC, Hennen J, Lyoo IK, Tsai G, Wald LL, Evins AE, Yurgelun-Todd DA,
and Renshaw PF (2002) Modulation of brain and serum glutamatergic concen-
trations following a switch from conventional neuroleptics to olanzapine. Biol
Grayson BE, Seeley RJ, and Sandoval DA (2013) Wired on sugar: the role of the CNS
in the regulation of glucose homeostasis. Nat Rev Neurosci 14:24–37.
Guertin DA and Sabatini DM (2009) The pharmacology of mTOR inhibition. Sci
Guertin DA, Stevens DM, Thoreen CC, Burds AA, Kalaany NY, Moffat J, Brown M,
Fitzgerald KJ, and Sabatini DM (2006) Ablation in mice of the mTORC components
raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-
FOXO and PKCalpha, but not S6K1. Dev Cell 11:859–871.
Hara K, Yonezawa K, Kozlowski MT, Sugimoto T, Andrabi K, Weng QP, Kasuga M,
Nishimoto I, and Avruch J (1997) Regulation of eIF-4E BP1 phosphorylation by
mTOR. J Biol Chem 272:26457–26463.
Hardie DG (2007) AMP-activated/SNF1 protein kinases: conserved guardians of
cellular energy. Nat Rev Mol Cell Biol 8:774–785.
Schmidt et al.
Heiskanen T, Niskanen L, Lyytikäinen R, Saarinen PI, and Hintikka J (2003) Met-
abolic syndrome in patients with schizophrenia. J Clin Psychiatry 64:575–579.
Hroudova J and Fisar Z (2010) Activities of respiratory chain complexes and citrate
synthase influenced by pharmacologically different antidepressants and mood
stabilizers. Neuroendocrinol Lett 31:336–342.
Islam MM, Nautiyal M, Wynn RM, Mobley JA, Chuang DT, and Hutson SM (2010)
Branched-chain amino acid metabolon: interaction of glutamate dehydrogenase with the
mitochondrial branched-chain aminotransferase (BCATm). J Biol Chem 285:265–276.
Kapur S, VanderSpek SC, Brownlee BA, and Nobrega JN (2003) Antipsychotic dosing
in preclinical models is often unrepresentative of the clinical condition: a suggested
solution based on in vivo occupancy. J Pharmacol Exp Ther 305:625–631.
Kim SF, Huang AS, Snowman AM, Teuscher C, and Snyder SH (2007) From the cover:
Antipsychotic drug-induced weight gain mediated by histamine H1 receptor-linked
activation of hypothalamic AMP-kinase. Proc Natl Acad Sci USA 104:3456–3459.
Kimball SR (2006) Interaction between the AMP-activated protein kinase and mTOR
signaling pathways. Med Sci Sports Exerc 38:1958–1964.
Krause U, Bertrand L, and Hue L (2002) Control of p70 ribosomal protein S6 kinase
and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phos-
phatases in isolated hepatocytes. Eur J Biochem 269:3751–3759.
Laplante M and Sabatini DM (2012) mTOR signaling in growth control and disease.
Lee CT, Conde BJ, Mazlan M, Visanuyothin T, Wang A, Wong MM, Walker DJ,
Roychowdhury SM, Wang H, and Tran PV (2002) Switching to olanzapine from
previous antipsychotics: a regional collaborative multicenter trial assessing 2
switching techniques in Asia Pacific. J Clin Psychiatry 63:569–576.
Machado M, Marques-Vidal P, and Cortez-Pinto H (2006) Hepatic histology in obese
patients undergoing bariatric surgery. J Hepatol 45:600–606.
Maher AR and Theodore G (2012) Summary of the comparative effectiveness review on
off-label use of atypical antipsychotics. J Manag Care Pharm 18(5, Suppl B)S1–S20.
Marroquin LD, Hynes J, Dykens JA, Jamieson JD, and Will Y (2007) Circumventing
the Crabtree effect: replacing media glucose with galactose increases susceptibility
of HepG2 cells to mitochondrial toxicants. Toxicol Sci 97:539–547.
Mathews M and Muzina DJ (2007) Atypical antipsychotics: new drugs, new chal-
lenges. Cleve Clin J Med 74:597–606.
Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B, Yang H, Hild
M, Kung C, and Wilson C, et al. (2009) Bidirectional transport of amino acids
regulates mTOR and autophagy. Cell 136:521–534.
Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, and Flegal KM (2006) Prev-
alence of overweight and obesity in the United States, 1999–2004. JAMA 295:1549–1555.
Oh KJ, Park J, Lee SY, Hwang I, Kim JB, Park TS, Lee HJ, and Koo SH (2011)
Atypical antipsychotic drugs perturb AMPK-dependent regulation of hepatic lipid
metabolism. Am J Physiol Endocrinol Metab 300:E624–E632.
Ratziu V, Bonyhay L, Di Martino V, Charlotte F, Cavallaro L, Sayegh-Tainturier
MH, Giral P, Grimaldi A, Opolon P, and Poynard T (2002) Survival, liver failure,
and hepatocellular carcinoma in obesity-related cryptogenic cirrhosis. Hepatology
Sandoval DA, Obici S, and Seeley RJ (2009) Targeting the CNS to treat type 2
diabetes. Nat Rev Drug Discov 8:386–398.
Skrede S, Fernø J, Vázquez MJ, Fjær S, Pavlin T, Lunder N, Vidal-Puig A,
Diéguez C, Berge RK, and López M, et al. (2012) Olanzapine, but not aripi-
prazole, weight-independently elevates serum triglycerides and activates
lipogenic gene expression in female rats. Int J Neuropsychopharmacol 15:
Tato I, Bartrons R, Ventura F, and Rosa JL (2011) Amino acids activate mammalian
target of rapamycin complex 2 (mTORC2) via PI3K/Akt signaling. J Biol Chem
Tsai G and Coyle JT (2002) Glutamatergic mechanisms in schizophrenia. Annu Rev
Pharmacol Toxicol 42:165–179.
Tsang CK, Qi H, Liu LF, and Zheng XF (2007) Targeting mammalian target of
rapamycin (mTOR) for health and diseases. Drug Discov Today 12:112–124.
van der Zwaal EM, Luijendijk MCM, Adan RAH, and la Fleur SE (2008) Olanzapine-
induced weight gain: chronic infusion using osmotic minipumps does not result in
stable plasma levels due to degradation of olanzapine in solution. Eur J Pharmacol
Warburg O, Geissler AW, and Lorenz S (1967) Uber Wachstum von Krebszellen in
Medien, deren Glucose durch Galaktose ersetzt ist. Hoppe Seylers Z Physiol Chem
Wei X, Shi X, Koo I, Kim S, Schmidt RH, Arteel GE, Watson WH, McClain C,
and Zhang X (2013) MetPP: a computational platform for comprehensive two-
dimensional gas chromatography time-of-flight mass spectrometry-based metab-
olomics. Bioinformatics 29:1786–1792.
Weiden PJ, Mackell JA, and McDonnell DD (2004) Obesity as a risk factor for an-
tipsychotic noncompliance. Schizophr Res 66:51–57.
Weston-Green K, Huang XF, and Deng C (2012) Alterations to melanocortinergic,
GABAergic and cannabinoid neurotransmission associated with olanzapine-
induced weight gain. PLoS ONE 7:e33548.
Yudkoff M, Daikhin Y, Nissim I, Horyn O, Luhovyy B, Lazarow A, and Nissim I
(2005) Brain amino acid requirements and toxicity: the example of leucine. J Nutr
Zhang J, Fang A, Wang B, Kim SH, Bogdanov B, Zhou Z, McClain C, and Zhang X
(2011) iMatch: a retention index tool for analysis of gas chromatography-mass
spectrometry data. J Chromatogr A 1218:6522–6530.
Address correspondence to: Dr. Gavin E. Arteel, Department of Pharma-
cology and Toxicology, University of Louisville Health Sciences Center,
Louisville, KY 40292. E-mail: email@example.com
Olanzapine and Hepatic Metabolic Dysfunction