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High Fat Diet and
In Utero
Exposure to Maternal Obesity
Disrupts Circadian Rhythm and Leads to Metabolic
Programming of Liver in Rat Offspring
Sarah J. Borengasser
1,2
, Ping Kang
1
, Jennifer Faske
1
, Horacio Gomez-Acevedo
1,2
, Michael L. Blackburn
1
,
Thomas M. Badger
1,2
, Kartik Shankar
1,2
*
1Arkansas Children’s Nutrition Center, Little Rock, Arkansas, United States of America, 2Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock,
Arkansas, United States of America
Abstract
The risk of obesity in adulthood is subject to programming beginning at conception. In animal models, exposure to
maternal obesity and high fat diets influences the risk of obesity in the offspring. Among other long-term changes, offspring
from obese rats develop hyperinsulinemia, hepatic steatosis, and lipogenic gene expression in the liver at weaning.
However, the precise underlying mechanisms leading to metabolic dysregulation in the offspring remains unclear. Using a
rat model of overfeeding-induced obesity, we previously demonstrated that exposure to maternal obesity from pre-
conception to birth, is sufficient to program increased obesity risk in the offspring. Offspring of obese rat dams gain greater
body weight and fat mass when fed high fat diet (HFD) as compared to lean dam. Since, disruptions of diurnal circadian
rhythm are known to detrimentally impact metabolically active tissues such as liver, we examined the hypothesis that
maternal obesity leads to perturbations of core clock components and thus energy metabolism in offspring liver. Offspring
from lean and obese dams were examined at post-natal day 35, following a short (2 wk) HFD challenge. Hepatic mRNA
expression of circadian (CLOCK, BMAL1, REV-ERBa, CRY, PER) and metabolic (PPARa, SIRT1) genes were strongly suppressed
in offspring exposed to both maternal obesity and HFD. Using a mathematical model, we identified two distinct biological
mechanisms that modulate PPARamRNA expression: i) decreased mRNA synthesis rates; and ii) increased non-specific
mRNA degradation rate. Moreover, our findings demonstrate that changes in PPARatranscription were associated with
epigenomic alterations in H3K4me3 and H3K27me3 histone marks near the PPARatranscription start site. Our findings
indicated that offspring from obese rat dams have detrimental alternations to circadian machinery that may contribute to
impaired liver metabolism in response to HFD, specifically via reduced PPARaexpression prior to obesity development.
Citation: Borengasser SJ, Kang P, Faske J, Gomez-Acevedo H, Blackburn ML, et al. (2014) High Fat Diet and In Utero Exposure to Maternal Obesity Disrupts
Circadian Rhythm and Leads to Metabolic Programming of Liver in Rat Offspring. PLoS ONE 9(1): e84209. doi:10.1371/journal.pone.0084209
Editor: Silvia C. Sookoian, Institute of Medical Research A Lanari-IDIM, University of Buenos Aires-National Council of Scientific and Technological Research
(CONICET), Argentina
Received June 19, 2013; Accepted November 21, 2013; Published January 9, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: These studies were supported by the National Institutes of Health-R01-DK084225 (K.S.) and USDA Agriculture Research Service CRIS 6251-51000-007-
04S. The funding agencies had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ShankarKartik@uams.edu
Introduction
The epidemic rise in obesity incidence across the age spectrum
continues to be a daunting public health challenge. Most
concerning is the prevalence of obesity in infants and children as
obesity in childhood tracks strongly into adulthood [1]. It is
estimated that infants in the United States (birth to 2 y) are twice
as likely to be greater than the 95
th
percentile for weight-to-length
measurements, indicating an increasing number of infants weigh
more than is considered healthy [2]. Maternal obesity and
excessive gestational weight gain have been identified as factors
that contribute to increased obesity in the offspring. This is
particularly significant, as over 60% of all pregnancies in the
United States are in women who are either overweight or obese at
conception [3]. Several studies have shown strong associations
between maternal OB and obesity in the offspring, both in
childhood [4–7] and in adulthood [8–10]. A recent study of
37,000 individuals showed greater risk of cardiovascular disease
and pre-mature death in those born to OB women [11]. Likewise,
the diminished risk of obesity in children born to OB women who
lost weight prior to pregnancy, also strongly suggests a role for
developmental programming [12,13]. In animal models, exposure
to maternal obesity unambiguously influences the risk of obesity in
the offspring [14–18]. Collectively, the evidence suggests that
susceptibility to obesity may begin prior to birth. Consequently,
understanding how the intrauterine environment contributes to
offspring obesity development is a pertinent health concern.
The Barker hypothesis offers a framework linking the in utero
environment to long-term health outcomes in offspring [19].
While originally described in low-birth weight infants, it is now
clear that maternal caloric excess and high fat diets also engender
similar long-term programming of offspring obesity risk [20]. Both
epidemiological [12,13,21] and animal [22–25] studies demon-
strate intrauterine exposure to maternal obesity is associated with
increased adiposity and weight gain. We, as well as others, have
reported metabolic programming due to maternal nutrition which
includes insulin resistance [7,22], cardiovascular disease [26,27],
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nonalcoholic fatty liver disease (NAFLD) [28,29], decreased
energy expenditure [30], and impaired ability to utilize fatty acids
for energy in offspring [30]. Notably, our model limits the
exposure of maternal obesity exclusively to the intrauterine
environment through cross-fostering of offspring to lean surrogate
dams at post-natal day (PND) 1 [22]. Our studies have also shown
that impaired energy metabolism in obese dam offspring occurs
prior to alterations in body weight and adiposity [28,30]. Although
many such advances have furthered our understanding of fetal
programming, much of the molecular mechanisms leading to
metabolic dysregulation remain to be elucidated.
A close relationship exists between metabolism and diurnal
circadian rhythm, in which disruption promotes obesity and
metabolic disease [31–35]. Perturbations in circadian rhythms are
known to impact energetics, particularly in tissues regulating
metabolism (considered peripheral clocks) such as liver, skeletal
muscle, brown adipose, and white adipose [36–38]. Factors such
as feeding, fasting, or type of diet can modulate rhythmicity and
metabolism. More specifically, recent studies suggest that circadian
rhythms are affected by gestational exposure to dietary manipu-
lations such as protein restriction [39] or high fat consumption
[40]. We have previously demonstrated that exposure to maternal
obesity disrupted targets downstream of peroxisome proliferator-
activated receptor (PPAR)aand AMPK which are key regulators
responsible for orchestrating fatty acid oxidation prior to obesity
development [28]. Since, PPARaexpression oscillates in a
circadian fashion throughout the day, and also acts as a circadian
regulator [41–43], it remains unclear whether maternal obesity
impairs PPARasignaling via disruption of circadian rhythm.
In the present study, we examined the hypothesis that
intrauterine exposure to maternal obesity disrupts offspring
circadian rhythm and liver metabolism following a short term
high fat diet (HFD) challenge after weaning. First, we investigated
mRNA expression of core clock components which included
circadian locomotor output cycles kaput (CLOCK), brain and
muscle ARNTL-like protein-1 (BMAL1), REV-ERBa, Crypto-
chromes (CRYs), and Periods (PERs). Second, we applied a
mathematical model to identify distinct mechanisms that contrib-
ute to changes in PPARamRNA expression due to maternal
obesity and HFD. Lastly, we investigated histone modifications in
the PPARapromoter to elucidate epigenetic mechanisms regu-
lating PPARaexpression. Our results demonstrate that offspring
from obese rat dams have detrimental alterations to circadian
machinery that contribute to impaired liver metabolism in
response to high fat feeding.
Materials and Methods
Animals and chemicals
Female Sprague-Dawley rats (150–175 g) were obtained from
Charles River Laboratories (Wilmington, MA). Animals were
housed in an AAALAC-approved animal facility in a temperature
and light controlled room (12 h light-12 h dark cycle). All
experimental protocols were approved by the Institutional Animal
Care and Use Committee at the University of Arkansas for
Medical Sciences (Protocol #2971). Unless specified, all chemicals
were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO).
Experimental protocol
Virgin female Sprague-Dawley rats were intragastrically can-
nulated to receive total enteral nutrition (TEN) at age 8 wk and
allowed to recover for 10 d as previously described [22,44–47].
Rats were fed liquid TEN diets at either 155 kcal/kg
3/4
?d (referred
to as lean dams) or at 220 kcal/kg
3/4
?d (40% excess calories,
referred to as obese dams). We have previously reported body
weights and body compositions of lean and obese dams [22]. TEN
diets met National Research Council (NRC) nutrient recommen-
dations as used previously by our group [22,44,46–50] and
consisted of 20% protein (casein), 75% carbohydrate (dextrose and
maltodextrin), and 5% fat (corn oil) . Infusion of diets was carried
out for 3 wk allowing for precise control of both diet composition
and caloric intake in a low-stress manner. Animals had ad libitum
access to drinking water and body weights were measured three
times per week. Following 3 wk of overfeeding to induce obesity in
the 220 kcal/kg
3/4
?d group, lean and obese rats (N = 15/group)
were allowed to mate for 1 wk. Each female rat was housed with
one lean breeder male and allowed ad libitum access to AIN-93G
diet during this period. After mating all female rats (lean and
obese) received diets at 220 kcal/kg
3/4
?d (NRC recommended
caloric intake for pregnancy in rats). All rats were allowed to give
birth naturally. Numbers and sex of pups, birth weight, and
crown-to-rump and anogenital distance were measured for each
pup on PND1 as previously described [22,28]. On PND1, four
male and four female pups from each litter were cross-fostered to
lean dams that had been previously time-impregnated to give birth
on the same day as the obese dams receiving infusion diets. Cross-
fostered dams were not cannulated and had ad libitum access to
AIN-93G pelleted diets throughout lactation. Using this experi-
mental paradigm, we ensured that offspring’s exposure to any
effects of maternal obesity was limited exclusively to the
intrauterine environment [22]. Female offspring of lean and obese
dams were used for separate experiments, and only data from male
offspring are reported here. At PND21 male offspring were
weaned onto either an AIN-93G (17% kcals fat) or high fat diet
(45% kcals fat) for 2 wk. Offspring were weighed and sedated with
carbon dioxide and euthanized via exsanguination in the fed
condition at PND35 every 4 h over a 24 h period (N = 4/group
for all time points except, N = 5/group for Lean-Con 2AM and
Obese-HFD 6AM and N = 3/group for Lean-Con 10PM and
Obese-Con 10PM, See Figure 1 for detailed offspring grouping).
Each offspring group represents 2–4 distinct biological litters. At
sacrifice, liver was weighed and immediately frozen in liquid
nitrogen and stored at 270uC for later analyses. Serum was
obtained by centrifugation of blood samples and stored at 220uC.
Animal characteristics and serum parameters are shown in Table
S2 in File S1 and Figure S1 in File S1.
Real-time RT-PCR
Total RNA was isolated from liver of offspring at PND35
(N = 3–5 rats per group per time point) using RNeasy mini
columns (QIAGEN, Valencia, CA) including on-column DNase
digestion. One microgram of total RNA was reverse transcribed
using iScript cDNA synthesis kit (BioRad, Hercules, CA). Real-
time PCR analysis was performed as described previously using an
ABI Prism 7500 Fast instrument (Carlsbad, CA) [50,51]. Area
under the curve (AUC) was calculated using the trapezoidal rule.
Gene specific primers were designed using Primer Express
Software (Table S1 in File S1). Relative amounts of mRNA
were quantified using a standard curve and normalized to the
expression of SRP14 mRNA.
Mathematical Modeling
The mammalian circadian cycle has been computationally
modeled by Leloup and Golbeter under normal physiological
conditions [52,53]. Here, we extended their model to analyze the
effect of maternal obesity and HFD on PPARamRNA expression.
The relative mRNA expression level of PPARawas analyzed with
a differential equation that has the form:
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dX (t)
dt ~
np(B(tztp))np
Knp
pz(B(tztp))np{wp(X(t))mp
Kmp
qz(X(t))mp{dpX(t)
where X(t) represented the relative gene expression level of
PPARa. A more detailed description of the model is described in
Table S3 in File S1. Parameter values were obtained by Monte
Carlo simulations (n = 10,000) followed by a conjugate-gradient
method implemented in Mathematica 8 (Wolfram Research Inc,
Champaign, IL) as shown in Table S4 in File S1. To assess the
reliability and robustness of the model, we performed a sensitivity
analysis using Partial Rank Correlation Coefficient (PRCC)
method at two switching points of the dark/light cycle that are
critical for the circadian cycle, namely 6AM and 6PM with sample
size set to N = 1000 [54].
Chromatin Immunoprecipitation (ChIP)
Histone modifications on the PPARapromoter were assessed
using ChIP. Samples were processed according to a previously
established protocol [55] and using a ChIP-IT enzymatic kit
(Active Motif, Carlsbad, CA) with minor modifications as
described previously [56]. Briefly, pools of liver samples (each
pool representing 4–5 separate animals) at 6AM and 6PM, from
offspring of lean or obese dams fed either control or HFD, were
used for analyses. Samples were minced and fixed in a 1%
formaldehyde solution and dounce homogenized. Nuclear isola-
tion was performed and chromatin was sheared using a Covaris
S220 focused-ultrasonicator (Woburn, MA). MagnaChIP protein
A/G beads (Millipore, Billerica, MA) and chromatin were pre-
blocked with 0.5% BSA and used to pre-clear the chromatin.
Immunoprecipitation was performed using 2.5 mg of ChIP grade
antibodies for H3K4me3 (Active Motif, Carlsbad, CA),
H3K27me3 (Millipore, Billerica, MA), or matched nonspecific
IgG (Millipore, Billerica, MA). Target binding regions on PPARa
promoter were amplified via qPCR using an Eco Real-Time PCR
System (Illumina, San Diego, CA), 6500 base pairs from the
transcription start site (TSS) (See Table S1 in File S1 for
sequences).
Statistical Analysis
Data are expressed as means 6SEM, significance was set at
p,0.05. Differences in mRNA expression between offspring of
lean and obese dams at each time point at PND35 were
determined using two-tailed Student’s t-test. Differences between
offspring of lean and obese dams at PND35 fed AIN-93G or HFD
for all other measured parameters were analyzed using two-way
analysis of variance (ANOVA). Significant interactions identified
by two-way ANOVA were followed by a one-way ANOVA and all
pair-wise comparisons by Student-Newman-Keuls. Statistical
analyses were performed using SigmaPlot 12.5 software (Systat
Software Inc., San Jose, CA).
Figure 1. Schematic of experimental design, interventions and outcomes.
doi:10.1371/journal.pone.0084209.g001
Maternal Obesity and Circadian Rhythm
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Results
Maternal obesity and high fat diet disrupt hepatic mRNA
amplitude of clock machinery in offspring
We examined mRNA expression of the core clock components
(CLOCK, BMAL1, REV-ERBa, Per1, Per2, Per3, Cry1, and
Cry2) to evaluate if maternal obesity and/or exposure to HFD
altered circadian rhythm over a 24 h period (Figure 2). The clock
machinery forms a transcriptional-translational loop (TTL) where
BMAL1 dimerizes with CLOCK forming a heterodimer that
binds to E-box promoter elements to positively regulate CLOCK
and clock controlled genes [32]. As expected, mRNA expression of
CLOCK and BMAL1 resembled each other in their cyclic
oscillations in which both peaked at 10AM and steadily declined
until 6PM, when the lowest levels of expression occurred as
depicted in Figure 2A and 2B. Maternal obesity did not alter
rhythmicity or levels of mRNA expression of either CLOCK or
BMAL1 in control-diet fed offspring of lean and obese dams.
However, in HFD-fed offspring AUC for CLOCK and BMAL1
mRNA were lowest in offspring from obese dams indicated by a
significant interaction of maternal obesity and HFD consumption
(p,0.001) (Table 1).
Similar to CLOCK/BMAL1, Cry and Per also form a
heterodimer, which acts as a negative regulator by impairing the
action of CLOCK/BMAL1 to complete the TTL. As anticipated,
Cry2 and Per 1, 2, and 3 all exhibited mRNA expression patterns
that were antiphasic to that of CLOCK/BMAL1 as these targets
peaked at 6PM and reached their lowest levels at 10AM
(Figure 2D–G). Per2 mRNA expression of control-fed lean and
obese dam offspring displayed slightly shifted expression, peaking
occurred at 10PM as opposed to 6PM (Figure 2F). Similar to
CLOCK/BMAL1, maternal obesity by itself did not alter mRNA
expression of Cry (1, 2) and Per (1,2,3), but only in combination
with HFD did maternal obesity show an effect of further reducing
expression levels of Cry and Per (Table 1). Cry1 did not follow an
anti-phasic pattern of mRNA expression to CLOCK/BMAL1,
instead Cry1 was highest at 6AM and 2AM and was lowest at
2PM as shown in Figure 2C.
Two nuclear receptors are central to the regulation of the above
mentioned clock components via control of BMAL1 transcription.
Retinoic acid receptor-related orphan receptor (ROR)ais thought
Figure 2. Hepatic mRNA expression of core circadian genes. mRNA expression was assessed in lean and obese dam offspring (A–H) fed either
control or high fat diets (HFD) from weaning through PND35 (N =3–5 animals per group, Lean-Con 2AM and Obese-HFD 6AM (N =5 per group), Lean-
Con 10AM and Obese-Con 10PM (N = 3 per group), and all the remaining groups (N = 4)). Gene expression was assessed via real-time RT-PCR. All
genes were normalized to SRP14 and expressed relative to Lean-Con at 6AM. Data are presented as mean 6SEM Statistical differences were
determined using a Student’s ttest. * denotes significance, P,0.05.
doi:10.1371/journal.pone.0084209.g002
Maternal Obesity and Circadian Rhythm
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Table 1. 24 h mRNA Expression Area Under the Curve (AUC) (N = 3–5/group).
Target Gene
Normalized to SRP14
P
Values
Offspring of Lean Dams Offspring of Obese Dams Maternal Obesity x
Postweaning Diet
Effect of Maternal
Obesity
Effect of Postweaning
HFD
Control HFD Control HFD
Core Clock Machinery
CLOCK 23.760.6
a
24.060.8
a
23.760.6
a
16.060.3
b
,
0.001 0.002
,
0.001
BMAL1 15.260.9
a
15.760.8
a
17.860.8
a
9.460.3
b
,
0.001 0.686 0.001
Reverba11886116 396627 1043683 251637 0.885 0.050
,
0.001
Per1 53.761.9
a
61.562.8
a
62.665.9
a
30.461.7
b
,
0.001 0.239 0.012
Per2 47.061.4
a
47.963.6
a
48.763.7
a
30.461.4
b
0.019 0.150 0.036
Per3 550659 422623 585635 450645 0.982 0.489 0.027
Cry1 18.161.3
a
13.660.6
b
17.361.2
a
8.160.4
c
0.040 0.036
,
0.001
Cry2 37.361.7
a
33.761.4
a
35.961.3
a
15.960.7
b
,
0.001
,
0.001
,
0.001
Metabolic and Epigenetic Regulators
PPARa60.562.4
a
24.662.2
b
59.762.3
a
12.160.7
c
0.024
,
0.049
,
0.001
EZH2 26.962.1
a
29.466.3
a
27.662.0
a
56.066.5
b
0.001 0.002 0.001
SIRT1 30.361.2
a
25.760.6
b
31.561.2
a
17.260.6
c
,
0.001 0.047
,
0.001
Area under the curve was assessed using the trapezoidal rule. Statistical differences were determined using a two-way ANOVA examining the effects of maternal obesity and post-weaning HFD. Significant interactions identified by
two-way ANOVA were followed by a one-way ANOVA and all pair-wise comparisons by Student-Newman-Keuls. Data are expressed as mean 6SEM, bold values represent significant main effects and interactions. Values with
different letters (
a,b,c
) are significantly different from each other (P,0.05).
doi:10.1371/journal.pone.0084209.t001
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to activate BMAL1 transcription, while REV-ERBasuppresses it.
In the present study, we found that RORamRNA expression did
not differ between groups (data not shown). However, REV-ERBa
mRNA expression was robustly induced at 2 PM in lean-CON
(,114-fold) and obese-CON (,95-fold) relative to 6AM
(Figure 2H). However, there effects were severely blunted in
high fat-fed offspring via a decrease of ,81-fold in lean dam
offspring and ,75-fold in obese dam offspring in mRNA
expression amplitude as compared to control-fed counterparts
(Figure 2H). This dramatic HFD-induced reduction of REV-
ERBamRNA expression did not coincide with substantial
changes in BMAL1 transcription in HFD-fed offspring suggesting
BMAL1 transcription may be regulated via other mechanisms.
Disruption of circadian machinery alters PPARain obese
dam offspring
PPARaoscillates in a circadian manner regulating multiple
pathways relating to hepatic lipid metabolism. Similar to the gene
expression patterns of the clock components, PPARadid not differ
between lean and obese dam offspring fed control diet
(Figure 3A). However, AUC values for PPARasuggested that
offspring of obese dams have an exacerbated response when fed
HFD by displaying the lowest AUC values among groups, similar
to the mRNA expression of core clock machinery (Table 1).
These findings were consistent with our previous reports showing
decreased activation of PPARatargets in offspring of obese dams
[28].
Mathematical modeling of PPARatime changes
We utilized mathematical modeling of PPARamRNA expres-
sion to identify mechanistic parameters that drive alterations in
PPARarhythm [52,53]. The model consisted of a differential
equation with eight parameters that could contribute to the
expression or regulation of PPARaand are defined in Table S3
in File S1. The fitted model for PPARarelative mRNA
expression is shown in Figure 3C and 3D. An optimal value
for each parameter was determined by Monte Carlo simulations
(n = 10,000) followed by a conjugated gradient method (Table S4
in File S1). Partial Rank Correlation Coefficients (PRCC) was
performed to determine the predominant contribution of each
parameter in the model.
PRCC revealed three parameters were consistently significant
for the PPARamodel (see Table S5 in File S1 for the PRCC
values), which included v
p
,K
p
, and d
p
(p,0.001 for all parameters).
Figure 3E and 3F show the estimated values for v
p
and d
p
where
v
p
represented the maximum rate of mRNA synthesis and d
p
non-
specific rates of mRNA degradation. Decreased n
p
values, shown
in Figure 3E, indicated that the maximum rate of PPARa
mRNA synthesis is decreased due to maternal obesity. Figure 3F
showed a stepwise increase of d
p
, which suggests that obese-HFD
offspring have the highest levels of non-specific PPARamRNA
degradation. K
p
represented the activation constant for enhance-
ment of gene expression by BMAL1 (Table S5 in File S1).
Histone modifications near the PPARatranscription start
site
The TTL, as mentioned previously, involves dynamic tran-
scription factor binding to gene promoters of core clock machinery
and metabolism-related targets over the course of the day.
Assessing chromatin states offers insight into whether gene
transcription was activated or repressed. Here, we focused on
PPARatranscription at the start of the light (6AM) and dark
(6PM) cycles. Accordingly, ChIP was performed on the PPARa
promoter to assess whether the effects of maternal obesity and
HFD on PPARamRNA expression were related to epigenetic
changes, specifically histone modifications. ChIP was performed
for an active gene transcription mark (H3K4me3) and a silencing
transcription mark (H3K27me3) in liver at 6AM and 6PM.
Quantitative RT-PCR was performed 6500 base pairs from the
PPARaTSS to assess the histone modification status of H3K4me3
and H3K27me3 as these two histone marks are bivalently located
near the TSS as shown in Figure 4 and Figure 5.
H3K4me3 showed a divergent pattern in control-fed offspring
of lean and obese dams, displaying higher enrichment occurring at
6AM and lower at 6PM, both upstream (2500 base pairs) and
downstream (+500 base pairs) from the PPARaTSS (Figure 4).
Lean and obese dam offspring fed HFD lacked differential
H3K4me3 enrichment differences between 6AM and 6PM
upstream from the TSS (Figure 4A). The downstream region
from the TSS was similar to the upstream region as lean and obese
dam offspring fed the control diet displayed differential enrich-
ment (Figure 4A and 4B). These findings would suggest that
control-fed offspring of lean and obese dams would demonstrate
more rhythmic PPARamRNA expression while HFD-fed groups
would lack oscillatory expression. In fact, PPARamRNA
expression was reflective of these histone changes as control-fed
offspring of lean and obese dams showed rhythmic expression that
was substantially blunted in HFD-fed offspring of lean and obese
dams (Figure 3A and 3B).
We also assessed the repressive histone mark, H3K27me3.
There was a significant effect due to maternal obesity at 6PM +500
base pairs from the TSS, but this effect was driven by the obese-
HFD group as shown in Figure 5B. Promoter chromatin
enrichment of H3K27me3 was significantly higher in HFD-fed
offspring of lean and obese dams at 6AM (Figure 5A). At 6PM,
the obese-HFD group had higher enrichment levels as compared
to all other groups (p,0.001) as shown in Figure 5B. In addition,
at 6AM, enrichment of H3K27me3 at both 6500 base pairs from
the PPARaTSS was increased in lean and obese dam offspring
fed HFD (Figure 5A and 5B). This would suggest that obese-
HFD should display the lowest PPARamRNA expression as
enrichment of H3K27me3 as elevated at both 6AM and 6PM and
at both TSS locations which is concordant with the PPARa
mRNA expression shown in Figure 3A and 3B.
Enhancer of zeste homolog (EZH2) is recognized as the primary
histone methyltransferase within the polycomb repressive complex
(PRC)-2 that mediates H3K27 trimethylation and has also been
shown to co-immunoprecipitate with CLOCK and BMAL1 in
mouse liver [57]. EZH2 mRNA expression is shown in Figure 6A
and 6B. There were no differences in levels of expression between
lean-con, obese-con, or lean-HFD. However, there was an
increase in EZH2 expression at nearly every time point in
obese-HFD offspring. AUC values in Table 1 further support that
obese-HFD had the highest EZH2 levels. In addition, EZH2 has
been shown to be associated with sirtuin 1 (SIRT1) [58,59];
SIRT1 deletion leads to increased EZH2 expression levels. Our
data coincided with this relationship as SIRT1 levels were reduced
(Figure 6C and 6D) and EZH2 levels were increased (Figure 6A
and 6B). Notably, EZH2 mRNA expression was also reflective of
H3K27me3 at 6PM (Figure 5B) and was consistent with PPARa
mRNA expression (Figure 3A and 3B).
Discussion
The maintenance of diurnal circadian rhythm is necessary for
normal metabolic function. Misalignment of activities of the
molecular clock such as oscillatory disruption in the period, phase,
Maternal Obesity and Circadian Rhythm
PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e84209
or amplitude of core clock components are associated with the
development of metabolic diseases, including obesity, diabetes,
and cardiovascular dysfunction [37,60]. Moreover, alterations in
diet composition (high fat diets), timing of eating (night eating), or
life-style factors (sleep deprivation or night-shift work) are known
to alter circadian rhythms and impair metabolism. However, the
influence of gestational experiences (such as maternal obesity) on
circadian rhythm in the offspring remains poorly understood.
Since, maternal diet and obesity during pregnancy have been
shown to influence offspring metabolism, appetite, and adiposity;
altered circadian rhythms could be causal mediators.
Although circadian disruptions in periodicity or phase were not
apparent in the present report, our findings unequivocally
demonstrated that oscillatory amplitude of both core clock
regulators and circadian metabolic regulators were altered in
offspring when challenged with HFD that was exacerbated in
offspring of obese dams. In particular, exposure to both maternal
obesity and 2 week post-weaning HFD challenge resulted in the
most impairment of gene transcription of core clock machinery
and metabolic and epigenetic targets. Changes in PPARamRNA
expression were specifically linked to decreased rates of mRNA
synthesis and increased rates of degradation as determined by
mathematical modeling. Additionally, epigenomic changes were
Figure 3. Circadian expression of PPAR-amRNA. Hepatic PPARamRNA was assessed in lean and obese dam offspring fed A) control or B) HFD
at PND35 (N =3–5 animals per group, Lean-Con 2AM and Obese-HFD 6AM (N = 5 per group), Lean-Con 10AM and Obese-Con 10PM (N =3 per group),
and all the remaining groups (N = 4)). Statistical differences were determined using a Student’s ttest. * denotes significance, P,0.05. Mathematical
model derived from relative PPARamRNA expression of offspring of lean and obese dams fed C) control diet or D) HFD at PND35. Model fitting
incorporated eight parameters representing distinct biological mechanisms. E) Optimal values for the parameter n
p
which represents rates of PPARa
mRNA synthesis and F) d
p
which represents rates of PPARamRNA degradation were determined by Monte Carlo simulations (n = 10,000) followed by
a conjugate-gradient method implemented in Mathematica.
doi:10.1371/journal.pone.0084209.g003
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PLOS ONE | www.plosone.org 7 January 2014 | Volume 9 | Issue 1 | e84209
evident via differences in enrichment of H3K4me3 and
H3K27me3 histone marks on the PPARapromoter. In agreement
with mRNA expression, the coupling of maternal obesity and
HFD exposure led to the most dramatic changes in our
mathematical model and histone mark enrichment.
In addition to its central role as a pleiotropic regulator of lipid
metabolism, PPARadirectly interacts with core clock components
in a circadian fashion. Dietary challenges such as fasting and HFD
lead to increased PPARaas a normal adaptive response aimed at
shunting lipids toward oxidation and away from storage. In an
ongoing study, where offspring were fasted for 24 h (at PND22),
we observed an attenuated induction in PPARamRNA and
nuclear protein expression in obese dam offspring compared to
lean dam counterparts (data not shown). These findings clearly
indicate that maternal obesity impairs the underlying mechanisms
regulating transcriptional induction of PPARa. Moreover, we
have previously demonstrated that exposure to maternal obesity
leads to reduced mRNA expression of PPARatarget genes prior
to the development of obesity or adiposity gains [28]. Conse-
quently, offspring of obese dams are unable to adequately mount a
response to metabolic demands that require mobilization of lipids
(viz. fasting and high fat diets); specifically, via an inability to
induce PPARaand its downstream targets. We have previously
shown that another critical regulator of hepatic fatty acid
oxidation, SIRT3, was reduced at both the transcript and
mitochondrial protein levels in offspring of obese dams [30].
Hence, our studies indicate that complementary pathways
involved in hepatic lipid metabolism (PPARaand SIRT3) are
impaired by maternal obesity. These may, in concert, contribute
to increased offspring susceptibility to nonalcoholic fatty liver
disease at weaning [28,30] and development of obesity in later life
[22,61]. Nevertheless, our previous work did not investigate
mechanisms related to changes in PPARatranscription. Indeed,
there are limited studies regarding control of PPARatranscription
Figure 4. H3K4me3 enrichment on the PPARapromoter. Chromatin immunoprecipitation for H3K4me3 was carried out and A) upstream
(2500 base pairs) and B) downstream (+500 base pairs from TSS) regions of the PPAR-agene were amplified in pools of liver samples (each pool
represents 3–5 separate animals) from offspring of lean or obese dams fed either control or HFD (run in triplicate). Enrichment was determined by real
time RT-PCR and normalized to input levels. Data are presented as mean 6SEM. Statistical differences were determined using a two-way ANOVA to
examine the effects of maternal obesity and post-weaning HFD. Significant interactions were followed by one way ANOVA and Student-Newman-
Keuls post hoc analyses (P,0.05). Bold values represent significant main effects and interactions and values with different letter superscripts are
significantly different from each other (P,0.05).
doi:10.1371/journal.pone.0084209.g004
Maternal Obesity and Circadian Rhythm
PLOS ONE | www.plosone.org 8 January 2014 | Volume 9 | Issue 1 | e84209
and most research has focused on PPARa-mediated transactiva-
tion of downstream targets.
The use of a mathematical model offered valuable insight into
the magnitude that distinct biological mechanisms played in
modifying PPARamRNA expression. We identified that mRNA
synthesis and degradation rates were mechanisms involved in
modulating mRNA expression of PPARa(Figure 3E and 3F).
Notably, obese-HFD offspring had the highest rates of degradation
and the lowest PPARamRNA expression. These findings suggest
an imbalance between transcription and mRNA degradation.
There are specific mechanisms associated with the coupling of
initiation and decay of mRNA which include mRNA imprinting,
alternative transcription start site, alternative splicing, alternative
poly-adenylation, and promoter-regulated decay as recently
described in a review by Haimovich et al. (2013) [62]. Each
mechanism has different factor(s) that regulate mRNA stability
and mRNA decay according to location, binding sites, and timing.
It is likely that one or more of these specialized mechanisms are
influenced by maternal obesity. The mathematical model was
limited to the eight parameters that were chosen a priori; it is likely
that other mechanisms, not included in the model, may have also
contributed to changes in PPARamRNA expression. Neverthe-
less, this model was chosen due to its capacity to detect parameters
contributing to mRNA expression due to alterations in circadian
rhythmicity. A limitation of the study, but not the model, was that
we did not experimentally evaluate rates of PPARamRNA
synthesis and decay. While identifying the precise machinery
involved with mRNA stability and degradation was outside of the
scope of the current study, these certainly warrant further
investigation.
The prevailing hypothesis in developmental programming is
that gestational events alter epigenetic and epigenomic regulation
Figure 5. H3K27me3 enrichment on the PPARapromoter. Chromatin immunoprecipitation for H3K27me3 was carried out and A) upstream
(2500 base pairs) and B) downstream (+500 base pairs from TSS) regions of the PPAR-agene were amplified in pools of liver samples (each pool
represents 3–5 separate animals) from offspring of lean or obese dams fed either control or HFD (run in triplicate). Enrichment was determined by real
time RT-PCR and normalized to input levels. Data are presented as mean 6SEM. Statistical differences were determined using a two-way ANOVA to
examine the effects of maternal obesity and post-weaning HFD (P,0.05). Significant interactions were followed by one way ANOVA and Student-
Newman-Keuls post hoc analyses (P,0.05). Bold values represent significant main effects and interactions and values with different letter superscripts
are significantly different from each other (P,0.05).
doi:10.1371/journal.pone.0084209.g005
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PLOS ONE | www.plosone.org 9 January 2014 | Volume 9 | Issue 1 | e84209
of key genes [63–66]. While the initiating signals whereby
maternal obesity influences epigenetic regulation of genes remains
unclear, increasing information about epigenetically regulated
metabolic targets genes is forthcoming. Embryonic development
and differentiation are associated with large changes in the
epigenomic landscape [67]. In fact, recent evidence suggests that
key physiological patterns such as circadian rhythms are associated
with highly coordinated alterations in epigenetic histone marks
and transcription factors [60,68–70]. Consequently, via ChIP, we
assessed if maternal obesity and HFD led to differences in histone
demarcation (H3K4me3 and H3K27me3) on the PPARa
promoter that were associated with changes in gene expression.
H3K4me3 is known to follow a rhythmic pattern of enrichment at
TSSs and a lag time between H3K4me3 and permissive gene
transcription has been reported [68] and our findings supported
this notion (Figures 4A and 4B). Although our H3K4me3
findings offer valuable insight into the HFD-induced reduction in
PPARamRNA amplitude, they do not fully explain the
exacerbated suppression of mRNA expression in the obese-HFD
group. It has been reported that H3K4me3 may be more strongly
correlated with increasing gene expression in low CpG promoters
as compared to high CpG promoters [71]. PPARais considered a
high CpG promoter which may be a possible explanation for the
reason H3K4me3 was not as indicative of HFD-induced changes
in PPARamRNA expression. However, the rhythmic pattern of
PPARamRNA expression can, in part, be explained by
H3K4me3 enrichment near the TSS.
Polycomb repressive complexes (PRC)1 and PRC2 mediate the
developmentally important repressive mark H3K27me3. PRC1
and PRC2 have crucial roles in pattern specification, organ
development, and cellular proliferation and differentiation [72–
74]. The histone methyltransferase EZH2 is part of PRC2 and the
sole enzyme that methylates H3K27me3, but it has also been
shown to be involved in the maintenance of circadian clock
machinery. Knockdown of EZH2 disrupts circadian rhythm and
has been linked to changes in H3K27me3 binding to clock gene
promoters [57]. More recently, there is evidence that reduced
SIRT1 leads to increased stability and expression of EZH2 [59].
Our data are consistent with this model, where in response to
HFD, a decline in SIRT1 was associated with increased expression
of EZH2 and thus increased H3K27me3.
Figure 6. Circadian expression of EZH2 and SIRT1 mRNA. Hepatic mRNA expression of EZH2 in A) Control-fed and B) HFD-fed offspring of lean
and obese dams at PND35 (N = 3–5 animals per group, Lean-Con 2AM and Obese-HFD 6AM (N = 5 per group), Lean-Con 10AM and Obese-Con 10PM
(N = 3 per group), and all the remaining groups (N = 4)). C) Hepatic mRNA expression of SIRT1 in Control-fed and D) HFD-fed offspring of lean and
obese dams at PND35 (N = 3–5 animals per group, Lean-Con 2AM and Obese-HFD 6AM (N = 5 per group), Lean-Con 10AM and Obese-Con 10PM
(N = 3 per group), and all the remaining groups (N = 4)). Data are presented as mean 6SEM. Statistical differences were determined using a Student’s
ttest. * denotes significance, P,0.05.
doi:10.1371/journal.pone.0084209.g006
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Here we showed, H3K27me3 enrichment directly correspond-
ed to the highly suppressive HFD effect on PPARamRNA
expression in both lean-HFD and obese-HFD offspring
(Figure 5A and 5B). Interestingly, obese-HFD groups showed
high levels of enrichment at both 6AM and 6PM as opposed to the
lean-HFD rats which only had increased enrichment at 6AM
(Figure 5A and 5B) suggesting PPARamRNA expression may
be more suppressed by HFD in obese-dam offspring. Indeed,
PPARamRNA expression was lowest in the obese-HFD group
(Figure 3B and Table 1). These findings suggest that maternal
obesity and HFD are more strongly related to epigenetic changes
associated with gene silencing, rather than the repression of gene
activation. These results are also in agreement with our modeling
data which indicated increased rates of mRNA degradation. In
physiological terms, this relationship makes sense as well, as
increased EZH2 is associated with adipogenesis and reduced
SIRT1 is linked to decreased energy expenditure. Although we did
not measure markers of adipogenesis or energy expenditure in this
study, we have recently demonstrated increased adipogenesis in
white adipose tissue [61] and decreased energy expenditure in
obese dam offspring [30]. Our epigenetic changes are in
agreement with other reports that found maternal HFD [40,75]
and maternal protein restrictions [76,77] led to histone modifica-
tions.
There are a few limitations to the current study. We did not
monitor feeding behavior or cage activity which are both known to
be associated with circadian rhythmicity [78–81]. We have
previously shown that exposure to maternal obesity does not alter
food intake in offspring on either a control or HFD [22]; however,
it is possible that the timing of food intake or activity is altered
which is associated with circadian disruption. In addition, the
present studies focused solely on the effects of maternal obesity or
HFD on offspring liver and did not examine effects on the central
clock. Each of these certainly warrants further investigation in
future studies. It is also worthy to note that recent studies have
examined the influence of maternal HFD-induced obesity on gene
regulation of circadian rhythms. These studies conducted in non-
human primates also found a robust effect of maternal and post-
natal HFD on expression of the Clock paralog, Npas2 [40] and
SIRT1 expression and acetylation [82]. In contrast to our findings,
these effects were mainly driven by maternal HFD and not by
maternal obesity per se, as revealed by diet-reversal during
gestation. While the precise reasons are unclear, differences in
diet composition to produce obesity, degree of maternal adiposity,
exposure to maternal diets during lactation, and species differences
may certainly contribute to differences in findings to those
observed in our model.
Collectively, our results indicated that the combination of
maternal obesity and HFD led to the greatest disruption of core
clock machinery and reductions of PPARamRNA expression.
Mathematical modeling revealed that exposure to maternal
obesity led to decreased mRNA synthesis rates and a stepwise
increase in mRNA degradation, with obese-HFD displaying the
highest rates of mRNA degradation. Epigenomic changes in
H3K4me3 appeared to contribute to the rhythmic expression of
PPARain control-fed groups and blunted rhythmic expression in
HFD-fed groups while H3K27me3 appeared to play a greater role
in HFD-induced effects in the presence or absence of maternal
obesity. In conclusion, exposure to maternal obesity in utero and
post-weaning HFD appear to ‘‘poise’’ offspring to be hyper-
responsive to HFD promoting development of obesity in later life.
Supporting Information
File S1 SUPPLEMENTARY MATERIALS AND METH-
ODS. FIGURE S1. Serum Parameters at 6AM. TABLE
S1. Primers Sequences for Real-time RT-PCR Analyses.
TABLE S2. Animal Characteristics of Offspring of Lean
and Obese Dam Offspring at 6AM. TABLE S3. Model
Parameters and Definitions. TABLE S4. Estimated
PPARaParameter Values. TABLE S5. Most Highly
Correlated Model Parameter PRCC Values.
(DOCX)
Acknowledgments
We thank Matt Ferguson, Hoy Pittman, Bobby Fay and other members of
the ACNC-Animal Research Core Facility for their assistance with animal
studies.
Author Contributions
Conceived and designed the experiments: SJB TMB KS. Performed the
experiments: SJB PK JF . Analyzed the data: SJB HGA MLB. Wrote the
paper: SJB HGA MLB TMB KS.
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