Post on 08-May-2023
Accepted Article Preview: Published ahead of advance online publication
Maternal obesity programs offspring non-alcoholic fatty liver
disease through disruption of 24-hours rhythms in mice
A Mouralidarane, J Soeda, D Sugden, A Bocianowska, RCarter, S Ray, R Saraswati, P Cordero, M Novelli, G Fusai,M Vinciguerra, L Poston, P D Taylor, J A Oben
Cite this article as: A Mouralidarane, J Soeda, D Sugden, A Bocianowska, R
Carter, S Ray, R Saraswati, P Cordero, M Novelli, G Fusai, M Vinciguerra, L
Poston, P D Taylor, J A Oben, Maternal obesity programs offspring non-
alcoholic fatty liver disease through disruption of 24-hours rhythms in mice,
International Journal of Obesity accepted article preview 14 May 2015; doi:
10.1038/ijo.2015.85.
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Accepted article preview online 14 May 2015
© 2015 Macmillan Publishers Limited. All rights reserved.
1
Maternal Obesity Programs Offspring Non-Alcoholic Fatty Liver Disease through
disruption of 24-hours Rhythms in Mice
Angelina Mouralidarane1,2
, Junpei Soeda1, David Sugden
2, Alina Bocianowska
2, Rebeca Carter
1,
Shuvra Ray1, Ruma Saraswati
3, Paul Cordero
1, Marco Novelli
3, Giuseppe Fusai
4, Manlio
Vinciguerra1,5,6*
, Lucilla Poston2, Paul D Taylor
2 & Jude A Oben
1
1 UCL Institute for Liver and Digestive Health, University College London, Royal Free Hospital,
Rowland Hill Street, NW3 2PF, London, UK.
2 Women’s Health Academic Centre, King’s College London, 10
th Floor North Wing, St
Thomas’ Hospital, Westminster Bridge Road, SE1 7EH, London, UK.
3 Histopathology Department, University College Hospital, University College London, 235
Euston Road, NW1 2BU, London, UK.
4 Liver Medicine and Transplant, Sheila Sherlock Liver Centre, University College London,
Royal Free Hospital, Rowland Hill Street, NW3 2PF, London, UK.
5 Gastroenterology Unit, Casa Sollievo della Sofferenza Hospital, viale dei Cappuccini 1, 71013,
San Giovanni Rotondo, Italy.
6 School of Science and Technology, Nottingham Trent University, Nottingham, UK.
Correspondence:
Dr Manlio Vinciguerra, PhD, UCL Institute for Liver and Digestive Health, University College
London, Royal Free Hospital, Rowland Hill Street, NW3 2PF. Email: m.vinciguerra@ucl.ac.uk.
Telephone: +44 (0)20 743-28-74
Short Title: Fatty liver and maternal reprogramming.
© 2015 Macmillan Publishers Limited. All rights reserved.
2
Standard abbreviations
NAFLD: Non-Alcoholic Fatty Liver Disease
SCN: suprachiasmatic nucleus
Per 1:Period 1
Per 2: Period 2
Cry 1: Cryptochrome 1
Cry 2: Cryptochrome 2
OffCon-SC: Offspring of lean mothers, weaned on a chow diet
OffOb-SC: Offspring of obese mothers, weaned on a chow diet
OffCon-OD: Offspring of lean mothers, weaned on an obesogenic diet
OffOb-OD: Offspring of obese mothers, weaned on an obesogenic diet
© 2015 Macmillan Publishers Limited. All rights reserved.
3
ABSTRACT
Background: Maternal obesity increases offspring propensity to metabolic dysfunctions and to
non-alcoholic fatty liver disease (NAFLD), which may lead to cirrhosis or liver cancer. The
circadian clock is a transcriptional/epigenetic molecular machinery synchronising physiological
processes to coordinate energy utilisation within a 24 hour light/dark period. Alterations in
rhythmicity have profound effects on metabolic pathways, which we sought to investigate in
offspring with programmed NAFLD.
Methods: mice were fed a standard or an obesogenic diet, before and throughout pregnancy, and
during lactation. Offspring were weaned onto standard or an obesogenic diet at 3 weeks
postpartum and housed in 12:12 LD conditions. Biochemical and histological indicators of
NAFLD and fibrosis, analysis of canonical clock genes with methylation status, and locomotor
activity were investigated at 6 months.
Results: We show that maternal obesity interacts with an obesogenic post-weaning diet to
promote the development of NAFLD with disruption of canonical metabolic rhythmicity gene
expression in the liver. We demonstrate hyper-methylation of BMAL-1 and Per2 promoter
regions and altered 24-hours rhythmicity of hepatic pro-inflammatory and fibrogenic mediators.
Conclusions: These data implicate disordered circadian rhythms in NAFLD and suggest that
disruption of this system during critical developmental periods may be responsible for the onset
of chronic liver disease in adulthood.
Key words: fatty liver, chronobiology, maternal transmission.
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4
INTRODUCTION
We have shown previously in a rodent model that maternal obesity programs offspring obesity
and a phenotype with marked similarity to human non-alcoholic fatty liver disease (NAFLD) (1-
5). NAFLD is associated with cirrhosis and liver cancer whilst becoming increasingly prevalent
due to rising rates of obesity (1-4). The mechanisms however, remain unclear. The almost
ubiquitous molecular machinery encoding the biological clock involves a
transcriptional/translational negative feedback loop in which the transactivation of E-box
element containing genes by CLOCK and BMAL1 is inhibited by the repressor genes, Period 1
and 2 (Per 1 and Per 2) and Cryptochrome 1 and 2 (Cry1 and Cry2) (6). Regulatory accessory
pathways involving REV-ERB-α and other members of the nuclear receptor family also stabilise
the circadian clock through repressive and inductive activities on BMAL1 (7).
These cyclical processes occur in the pace-setting suprachiasmatic nucleus (SCN) of the
hypothalamus, and are entrainable by light, nutrients and temperature. These entraining stimuli
together with the synchronisation between the SCN and the peripheral cellular clocks coordinate
multiple behavioural and physiological processes, including activity and feeding, and are
required to achieve metabolic homeostasis (8, 9). The interplay between the molecular daily
clock and food intake has been highlighted in the homozygous CLOCK mutant mouse, which
develops hepatosteatosis secondary to hyperphagia and obesity (10). A high fat diet in rodents
has also been reported to alter locomotor activity and the rhythmic expression of canonical
chronobiology-related genes in the hypothalamus, adipose, muscle and liver tissue. Nuclear
receptors which regulate CLOCK gene transcription factors in peripheral tissues including the
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5
liver are similarly perturbed by a high fat diet (11) and expression of genes encoding lipogenesis,
lipolysis and gluconeogenesis have been shown to possess circadian rhythmicity (12, 13). These
studies indicate an intimate and reciprocal association between feeding behaviour, metabolism
and chronobiology (14-18).
The function of the molecular machinery rhythmicity during foetal development is poorly
understood, however, evidence shows that while foetal hepatic cells possess full functioning
circadian oscillators in vitro, robust rhythms of the murine liver clocks are minimally detected in
utero until late gestation and the immediate post-natal weaning period (19). Therefore, the
maternal hormonal and metabolic environment in mice pregnancy may regulate the circadian
machinery during fetal hepatic development, invoking permanent changes that persist
postpartum (19). Additionally, the pace-setting SCN is still developing in the immediate
postnatal period, and so may also be affected by the maternal milieu. Such disturbances of the
chronobiological system during early ontogenesis in rodents may translate to development of
normal rhythmicity disruption and disease in adulthood (20).
As such there appears to be an association between nutrient status and pathways governing
circadian regulation (21, 22), raising the possibility that a hyper-calorific maternal milieu could
alter the molecular rhythmicity circuitry in the liver, during development and increase offspring
susceptibility to NAFLD. Recently, Borengasser et al., have shown a role for the perturbation of
SIRT1 and PPAR, master regulators of hepatic lipid metabolism, over a 24 hour period, in rat
offspring exposed to maternal obesity (23). A strong correlation between methylation of
rhythmicity-mediated gene bodies and anthropometric parameters such as adiposity and body
mass index has been documented (24). It is likely that the maternal environment can induce
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6
epigenomic and epigenetic changes of key regulatory pathways in the developing offspring
resulting in disease states. However, the mechanism through which this occurs is largely
unknown. We therefore sought to determine a mechanistic role for the hepatic chronobiological
machinery in a pathophysiologically relevant model of NAFLD (5).
MATERIALS AND METHODS
Animal Experimentation
Female C57BL/6J mice (proven breeders with one previous pregnancy, n = 60, Charles River
Laboratories, UK), were fed standard chow RM1 or a highly palatable obesogenic diet consisting
of a semi-synthetic energy-rich high fat diet, (10% simple sugars, 18% animal lard, 4% soya oil,
28%, polysaccharide, 23% protein [w/w], Special Dietary Services, UK diet code: 824053-45%
AFE FAT energy 4.5 kcal/g, n=30) supplemented with fortified sweetened condensed milk
(Nestle, SZ) for 6 weeks ad libitum as previously reported (4). Final dietary composition based
on intake was approximately 16% fat, 33% simple sugars, 15% protein, energy 4.0kcal/g. Mice
on the obesogenic diet entered the breeding protocol after achieving a 30% increase in body
weight, and controls were aged matched. All animals were treated in accordance with The
Animals (Scientific Procedures) Act, UK, 1986 guidelines.
Pregnant dams were maintained on their respective diets throughout pregnancy and lactation.
After spontaneous delivery, litter sizes were standardised to 8 pups per litter (4 males and 4
females). Female offspring born to either lean or obese dams were weaned on to a standard chow
diet (n = 10) (OffCon-SC or OffOb-SC) or the high fat diet (n = 10, Special Dietary Services,
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7
UK, diet code: 824053-45% AFE FAT) (OffCon-OD or OffOb-OD). All offspring were housed
in a 12 hour light/dark cycle in a thermostatically controlled environment (22°C) with lights on
at 07:00. Light intensity was maintained at 30-50 μW/cm2 (PM203, Macam Photometrics Ltd).
For gene expression analysis, a subset of animals was sacrificed at 4-hourly intervals at indicated
zeitgeber timepoints (ZT) over a 24 hour period. Investigation of molecular daily rhythms and
markers implicated in NAFLD pathogenesis was completed at 6 months.
Plasma Analysis
Blood was collected via cardiac puncture under deep terminal anesthesia and plasma assayed for
ALT using an enzymatic colorimetric assay (3) by the Royal Free Hospital, Clinical
Biochemistry Department, London, UK.
Liver Tissue Triglyceride
Whole liver tissue triglyceride was determined by an adaptation of the Folch Method (25) and an
enzymatic colorimetric assay (UNIMATE 5 TRIG, Roche BC1, Sussex, UK).
Gene Expression Analysis
Real Time Polymerase Chain Reaction (RT-PCR) was performed using QuantiTect SYBR Green
PCR System with HotStar Taq DNA Polymerase (Qiagen). The platform used for gene
expression studies was the Rotor-Gene 3000 (Corbett Life Sciences, Qiagen). Gene specific
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8
primers were designed for IL-6, TNF-α, α–Smooth Muscle Actin (α-SMA), transforming growth
factor-β (TGF-β) and collagen Type 1-α2 (Col 1-α2) (Table 1). Quantitect Primer Assays
(Qiagen) using SYBR green based detection were used for CLOCK, BMAL1, Period 1, Period 2,
Cryptochrome 1, Cryptochrome 2 and REV-ERB-α (Table 2). Expression of target genes was
normalised to GAPDH due to its non-time dependent expression. The delta delta CT method
was used for relative quantification and analysed using GraphPad Prism v5.0. Graphs are plotted
using fold change values with standard error of mean.
Gene Methylation Analysis
DNA from liver samples at ZT0 was isolated by using the DNeasy Blood & Tissue Kit (Qiagen
GmbH, Hilden, Germany) and quantified with the NanoDrop 2000 spectrometer (Thermo Fisher
Scientific, Walthman, MA, USA). gDNA methylation percentage was measured by the Epitec
Methyl II PCR primer assay (335112, Qiagen, UK) according to manufacturers’ instructions.
After incubation with restriction enzymes, rt-qPCR was performed by using Rotor-Gene 3000
(Corbett Life Science, Qiagen) and SYBR Green PCR kit (Qiagen). The results were obtained
using Qiagen excel templates for the assay and data were analysed using GraphPad Prism v5.0.
Locomotor Activity
Locomotor activity from 3 mice per experimental group during day (07.00-19.00) and night
(19.00-7.00) was recorded continuously at 10 minute intervals over a 26 day period using a Cage
Rack Photobeam Activity System (San Diego Instruments, San Diego, US). These cages
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9
included horizontal infrared beams around positioned in a height from which gross movements
were recorded. Activity count was presented as actograms (10 min as length of plot) and
periodograms. Data were analyzed using Cage rack Software.
Histology
Offspring liver sections at 6 months of age (n = 5 per experiment group) were formalin (10%)
fixed and paraffin embedded prior to sectioning. One section from each liver was then stained
with haematoxylin and eosin (H&E) and another with Masson’s Trichrome to assess steatosis,
inflammation and fibrosis (26). The Brunt-Kleiner NAFLD Activity Score was used to
quantitatively assess the degree of injury by an expert liver pathologist blinded to the identity of
the groups (27). This score was standardized according to steatosis, lobular inflammation and
ballooning as previously described.
Statistical Analysis
Data is expressed as mean ± SEM unless otherwise stated. Multiple comparisons on data sets
were performed using one-way ANOVA followed by Tukey’s post hoc test. The influence of the
maternal obesity and the post-natal diet and any interaction between them were investigated
using two-way ANOVA (GraphPad Prism v5.0). Rhythmic expression of canonical clock genes
was assessed using cosinor analysis. p ≤ 0.05 was regarded as significant. The statistical unit
was considered the number of dams (n = 5 per group), with 1 offspring studied per litter per time
point.
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10
RESULTS
Maternal obesity and a post-weaning obesogenic diet programs development of offspring
NAFLD at 6 months.
Offspring of obese mice (OffOb) fed an obesogenic diet (OD) post-weaning (OffOb-OD)
developed non-alcoholic steatohepatitis and hepatic fibrosis as evidenced by hepatomegaly, plus
increased ALT, hepatic tissue triglyceride content, NAFLD Activity Score (NAS) and
histological evaluation (Figure 1), with appropriate statistical analysis revealing an independent
effect of maternal obesity (p<0.01).
Offspring exposure to maternal obesity and/or a post-weaning obesogenic diet attenuates
the robustness of the rhythm in the activity at 6 months.
Under a 12hr Light:Dark cycle offspring of all groups exhibited a significant rhythm in
locomotor activity with a period close to 24.00hr (Figure 2a). The power of the periodogram
analyses depends of the main peak above the confidence limit line and is associated with the
robustness of the rhythmicity. The greater power for each group (Figure 2b) showed that the 24hr
rhythm of the OffCon-SC and OffOb-SC groups was considerably more robust than the OffCon-
OD and OffOb-OD mice (Figure 2c). These latter groups also show a substantial increase in
activity during the light period.
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Offspring exposure to maternal obesity and a post-weaning obesogenic diet disrupts
rhythmic expression of chronobiological activators in the liver.
Hepatic gene expression analysis revealed profound rhythmic disruption in offspring with
NAFLD compared to controls (OffCon-SC). More specifically, these novel findings showed that
OffOb-OD had attenuated BMAL1 transcription in a 24 hour period with a shift in amplitude
from the light to the dark phase (Figure 3a). Maternal obesity alone (OffOb-SC) induced a
significant increase in both CLOCK and BMAL-1 mRNA expression at ZT16 and ZT20
respectively (Figure 3a and b). Additionally, maternal obesity combined with a post-weaning
obesogenic diet caused a biphasic rhythmic expression of CLOCK with peak levels observed at
ZT12 and ZT20 (Figure 3b).
Offspring exposure to maternal obesity and a post-weaning obesogenic diet disrupts
rhythmic expression of chronobiological repressors in the liver.
Cellular 24-hours rhythmicity was further perturbed by a post weaning obesogenic diet in
OffCon-OD and OffOb-OD as Cry1 transcripts were significantly attenuated at ZT20 with
amplitudes shifted to the dark phase at ZT12, compared to OffCon-SC (Figure 3c). Cry2
however, showed minimal rhythmic variation in transcription in control animals although a
dramatic increase in Cry2 expression was observed at ZT16 in OffOb-OD, and to a lesser extent
at ZT12 in OffOb-SC (Figure 3d). Importantly, an independent main effect of maternal obesity
is attributed to the increased expression levels observed at ZT16 by two-way ANOVA analysis.
Similarly, Per1 expression was increased in OffOb-SC and OffOb-OD at ZT12 and ZT20,
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12
respectively (Figure 3e). Again, an independent main effect of maternal obesity (p ˂ 0.01) and
the postnatal diet (p ˂ 0.001) on Per1 gene expression was observed at ZT20. Additionally,
OffOb-OD displayed increased transcription of Per2 at ZT12 with maternal obesity largely
contributing to the observed phenotype (p < 0.001, post-weaning effect p = 0.93) (Figure 3F).
Per2 expression was in antiphase with BMAL-1 expression as would be expected. REV-ERBα
expression was markedly attenuated in OffCon-OD and OffOb-OD at ZT4 compared to controls
(SC fed offspring) indicating a predominant effect of the postnatal obesogenic diet (p ˂ 0.01) on
REV-ERBα gene expression (Figure 4). Cosinor analysis revealed phase advances in CLOCK,
BMAL1 and Per2 transcription by 3.3, 1.9 and 2 hours respectively, in OffOb-OD compared to
controls (Figure 5).
Offspring exposure to maternal obesity and a post-weaning obesogenic diet disrupts
rhythmic expression of hepatic inflammatory and fibrogenic markers implicated in
NAFLD pathogenesis.
To functionally link hepatic circadian genes and phenotypic data, we now examined the
expression of pro-inflammatory and fibrogenic markers implicated in NAFLD. We show here
for the first time, that interleukin-6 (IL-6) has a rhythmic expression pattern in murine liver with
a biphasic 24-hours expression that is diminished in offspring with phenotypic NAFLD (OffCon-
OD and OffOb-OD) (Figure 6a). In contrast, tumour necrosis factor-α (TNF-α) did not exhibit
rhythmic expression patterns in the liver. Peak expression of this transcript was observed in
OffOb-OD at ZT20 with an independent effect of maternal obesity (p ˂ 0.01) and the postnatal
diet (p ˂ 0.001) (Figure 6b).
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Additionally, alpha smooth muscle actin (α-SMA), transforming growth factor beta (TGF-β) and
collagen, as indicators of induction of hepatic fibrosis, showed disrupted daily expression
patterns in OffOb-OD compared to other groups. Peak expression of α-SMA was observed at
ZT8 and ZT20 with an independent effect of maternal obesity (p ˂ 0.05 at ZT8 and at ZT20). Of
note, peak transcription of these deranged fibrogenic markers were observed predominantly close
to or in the dark phase which is the period of greatest activity in rodents. It is thus plausible that
repair and regeneration that usually best occurs during rest cycles is now compounded in the
activity period to enhance development of fibrosis. Offspring development of fibrosis may
therefore be due to desynchronisation of normal rhythmic expressions of fibrosis inducing genes.
Offspring with programmed NAFLD displayed marked attenuation of BMAL-1 and significant
up-regulation of PER-2 transcription compared to all other groups. These distinct and closely
paired associations instigated investigation of methylation statuses in an attempt to ascertain a
causative relationship.
BMAL1 and Per2 hypermethylation is observed in offspring with programmed NAFLD.
Both BMAL1 and Per2 were found to display differences, with hyper-methylated promoter
regions identified in offspring with programmed liver disease all harvested at the same time
point, ZT0 (Figure 7). In OffOb-OD, hypermethylation of BMAL-1 was paralleled by marked
reduction of BMAL-1 expression over 24hrs with ablation of its peak expression at ZT20 as
compared to controls. An independent main effect of maternal obesity and the post-weaning diet
was observed with significant interactions between the two variables. In the same animals,
hypermethylation of Per2 correlated with a marked increase in total gene expression with an
© 2015 Macmillan Publishers Limited. All rights reserved.
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exaggerated peak expression at ZT12. Again, an independent main effect of maternal obesity on
Per2 methylation was observed (Figure 7).
DISCUSSION
Disruption of central and peripheral rhythms in humans and rodents has been shown to cause
metabolic disorders including impaired glucose homeostasis and hyperlipidaemia (11, 17, 28).
This suggests a causal association between rhythmic daily transcription and translation of
canonical clock genes and metabolism. We report here, that nutrient excess or a hyper-calorific
milieu during in utero development interacts with the post-natal obesogenic environment to
induce permanent changes in the hepatic molecular chronobiological network.
In this pathophysiologically relevant model of programmed NAFLD in offspring, it is shown
here for the first time that exposure to maternal obesity increased temporal expression of
CLOCK mRNA transcripts, peaking at ZT16 or in the dark phase. Moreover, additional insult
with continued exposure of a hyper-calorific diet in post-natal life induced a biphasic rhythmicity
pattern of expression with peaks observed in both the light and dark phases. Rhythmic disruption
of CLOCK transcription in the liver may therefore be partially responsible for the observed
dysmetabolic and fatty liver phenotype. Such a causal association is supported by earlier reports
of adiposity, impaired lipid metabolism and insulin resistance in CLOCK mutant mice (10, 14).
Although a high fat diet has been shown to alter rhythmic expression of CLOCK in adipose and
hepatic tissue, it is clear that these present observations are not phenomenological as offspring
exposed to only an obesogenic diet displayed no alteration in time-dependent transcription of
CLOCK compared to controls.
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The rhythmic 24-hours expression of BMAL1, a co-activator of the circadian molecular network,
was similarly disrupted following exposure to maternal obesity and a post-weaning obesogenic
diet. Observations of arrhythmic BMAL1 transcription are in keeping with previous reports of
impaired adipogenesis and hepatic carbohydrate metabolism in rodents with BMAL1 ablation
(29, 30).
Moreover, profound perturbation of rhythmic expression of CLOCK and BMAL1 in offspring
exposed to maternal obesity and an obesogenic diet post-weaning was evidenced by phase
advances in transcription of approximately 3.3 and 1.9 hours, respectively. Additionally, a higher
baseline constitutive expression of CLOCK and BMAL1 was observed from greater than control
values for minima as determined by cosinor analysis.
In this present study, exposure to maternal obesity induced a significant increase in Cry2
transcription in offspring which was further enhanced in the context of a post-weaning
obesogenic diet in the dark phase. Therefore and most importantly, offspring exposure to
maternal obesity significantly induces arrhythmic expression of Cry2 and as such may be in part
responsible for the observed NAFLD phenotype. This is further supported by reports of
cryptochrome mediated regulation of hepatic gluconeogenesis and cAMP signalling, re-affirming
the association between core clock genes and their control of metabolic pathways (31).
Per2 is an important intermediary between metabolic and circadian pathways as it has been
shown to control lipid metabolism through regulation of PPARγ. Importantly, it has been
reported that Per2 deficient mice display profound reduction of triacylglycerol and non-esterified
fatty acids (32) and so Per2 acts to inhibit lipid metabolism, potentiating hepatosteatosis as
observed in offspring with programmed NAFLD, here, corroborating our findings of increased
© 2015 Macmillan Publishers Limited. All rights reserved.
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Per2 transcription in offspring with the most severe phenotype. A corollary of the
aforementioned findings is asynchronisation in the liver induced by offspring exposure to
maternal obesity in the context of a post-weaning obesogenic diet. It is evident from previous
reports and present findings that these canonical clock genes affect metabolic physiology. It is
now shown here, that these circadian-related genes regulate hepatic inflammatory and fibrogenic
pathways implicated in NAFLD pathogenesis. It is tempting to speculate that the resulting
asynchrony not only affects metabolic homeostasis as demonstrated here, but also identifies
hepatic pro-inflammatory and pro–fibrogenic pathways involved in NAFLD as possible clock
output pathways.
Hypermethylation in the promoters of two core canonical clock genes, BMAL1 and Per2,
suggest that epigenetic programming via maternal obesity could occur through DNA
methylation, as supported by a direct association between an individual’s adiposity and
methylation status at CpG sites of core canonical clock genes (24). Our findings are corroborated
by an earlier report that DNA methylation might contribute to the developmental expression of
clock genes in mice (33). A diabetic uterus environment, as in the streptozotocin (STZ)-induced
hyperglycemic mouse model, significantly altered the methylation of imprinted genes Peg3 and
H19 (34), as a further example.
Similarly, a “fat” uterus environment may alter physiological functioning of the hepatic
molecular circadian network during developmental plasticity in offspring, causing rhythmic
disruption and increased expression of inflammatory and fibrogenic markers implicated in
NAFLD pathogenesis. The implications of these experimental findings can be extrapolated to the
clinical presentations of increasing maternal obesity.
© 2015 Macmillan Publishers Limited. All rights reserved.
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ACKNOWLEDGEMENTS
We thank Jahm Persaud, Department of Clinical Biochemistry Royal Free Hospital, UCLH/UCL
Comprehensive Biomedical Research Centre, for his invaluable help. We thank also Joaquim
Pombo and Paul Seed, Women’s Health Academic Centre, King’s College London, for technical
assistance.
FUNDING
Wellcome Trust and Fiorina Elliot Fellowship to JAO. My First AIRC Grant to MV (n.13419).
CONFLICT OF INTEREST
The authors do not have any competing financial interests in relation to the work described.
REFERENCES
1. Bohinc BN, Diehl AM. Mechanisms of disease progression in NASH: new paradigms. Clin Liver Dis. 2012; 16: 549-565.
2. Diehl AM. Neighborhood watch orchestrates liver regeneration. Nat Med. 2012; 18: 497-499.
3. Oben JA, Mouralidarane A, Samuelsson AM, Matthews PJ, Morgan ML, McKee C, et al. Maternal obesity during pregnancy and lactation programs the development of offspring non-alcoholic fatty liver disease in mice. J Hepatol. 2010; 52: 913-920.
© 2015 Macmillan Publishers Limited. All rights reserved.
18
4. Samuelsson AM, Matthews PA, Argenton M, Christie MR, McConnell JM, Jansen EH, et al. Diet-induced obesity in female mice leads to offspring hyperphagia, adiposity, hypertension, and insulin resistance: a novel murine model of developmental programming. Hypertension. 2008; 51: 383-392.
5. Mouralidarane A, Soeda J, Visconti-Pugmire C, Samuelsson AM, Pombo J, Maragkoudaki X, et al. Maternal obesity programs offspring nonalcoholic fatty liver disease by innate immune dysfunction in mice. Hepatology. 2013; 58: 128-138.
6. Bass J, Takahashi JS. Circadian integration of metabolism and energetics. Science. 2010; 330: 1349-1354.
7. Cho H, Zhao X, Hatori M, Yu RT, Barish GD, Lam MT, et al. Regulation of circadian behaviour and metabolism by REV-ERB-alpha and REV-ERB-beta. Nature. 2012; 485: 123-127.
8. Kohsaka A, Bass J. A sense of time: how molecular clocks organize metabolism. Trends Endocrinol Metab. 2007; 18: 4-11.
9. Schibler U, Ripperger J, Brown SA. Peripheral circadian oscillators in mammals: time and food. J Biol Rhythms. 2003; 18: 250-260.
10. Turek FW, Joshu C, Kohsaka A, Lin E, Ivanova G, McDearmon E, et al. Obesity and metabolic syndrome in circadian Clock mutant mice. Science. 2005; 308: 1043-1045.
11. Kohsaka A, Laposky AD, Ramsey KM, Estrada C, Joshu C, Kobayashi Y, et al. High-fat diet disrupts behavioral and molecular circadian rhythms in mice. Cell Metab. 2007; 6: 414-421.
12. Liu S, Brown JD, Stanya KJ, Homan E, Leidl M, Inouye K, et al. A diurnal serum lipid integrates hepatic lipogenesis and peripheral fatty acid use. Nature. 2013; 502: 550-554.
13. Tong X, Yin L. Circadian rhythms in liver physiology and liver diseases. Compr Physiol. 2013; 3: 917-940.
14. Oishi K, Miyazaki K, Kadota K, Kikuno R, Nagase T, Atsumi G, et al. Genome-wide expression analysis of mouse liver reveals CLOCK-regulated circadian output genes. J Biol Chem. 2003; 278: 41519-41527.
15. Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, et al. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell. 2002; 109: 307-320.
© 2015 Macmillan Publishers Limited. All rights reserved.
19
16. Yang X, Downes M, Yu RT, Bookout AL, He W, Straume M, et al. Nuclear receptor expression links the circadian clock to metabolism. Cell. 2006; 126: 801-810.
17. Zvonic S, Ptitsyn AA, Conrad SA, Scott LK, Floyd ZE, Kilroy G, et al. Characterization of peripheral circadian clocks in adipose tissues. Diabetes. 2006; 55: 962-970.
18. Tevy MF, Giebultowicz J, Pincus Z, Mazzoccoli G, Vinciguerra M. Aging signaling pathways and circadian clock-dependent metabolic derangements. Trends Endocrinol Metab. 2013; 24: 229-237.
19. Dolatshad H, Cary AJ, Davis FC. Differential expression of the circadian clock in maternal and embryonic tissues of mice. PLoS One. 2010; 5: e9855.
20. Sumova A, Sladek M, Polidarova L, Novakova M, Houdek P. Circadian system from conception till adulthood. Prog Brain Res. 2012; 199: 83-103.
21. Eckel-Mahan K, Sassone-Corsi P. Metabolism control by the circadian clock and vice versa. Nat Struct Mol Biol. 2009; 16: 462-467.
22. Mazzoccoli G, Pazienza V, Vinciguerra M. Clock genes and clock-controlled genes in the regulation of metabolic rhythms. Chronobiol Int. 2012; 29: 227-251.
23. Borengasser SJ, Kang P, Faske J, Gomez-Acevedo H, Blackburn ML, Badger TM, et al. 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. 2014; 9: e84209.
24. Milagro FI, Gomez-Abellan P, Campion J, Martinez JA, Ordovas JM, Garaulet M. CLOCK, PER2 and BMAL1 DNA methylation: association with obesity and metabolic syndrome characteristics and monounsaturated fat intake. Chronobiol Int. 2012; 29: 1180-1194.
25. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957; 226: 497-509.
26. Rappa F, Greco A, Podrini C, Cappello F, Foti M, Bourgoin L, et al. Immunopositivity for histone macroH2A1 isoforms marks steatosis-associated hepatocellular carcinoma. PLoS One. 2013; 8: e54458.
27. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005; 41: 1313-1321.
© 2015 Macmillan Publishers Limited. All rights reserved.
20
28. Orozco-Solis R, Matos RJ, Guzman-Quevedo O, Lopes de Souza S, Bihouee A, Houlgatte R, et al. Nutritional programming in the rat is linked to long-lasting changes in nutrient sensing and energy homeostasis in the hypothalamus. PLoS One. 2010; 5: e13537.
29. Rudic RD, McNamara P, Curtis AM, Boston RC, Panda S, Hogenesch JB, et al. BMAL1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis. PLoS Biol. 2004; 2: e377.
30. Shimba S, Ishii N, Ohta Y, Ohno T, Watabe Y, Hayashi M, et al. Brain and muscle Arnt-like protein-1 (BMAL1), a component of the molecular clock, regulates adipogenesis. Proc Natl Acad Sci U S A. 2005; 102: 12071-12076.
31. Zhang EE, Liu Y, Dentin R, Pongsawakul PY, Liu AC, Hirota T, et al. Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis. Nat Med. 2010; 16: 1152-1156.
32. Grimaldi B, Bellet MM, Katada S, Astarita G, Hirayama J, Amin RH, et al. PER2 controls lipid metabolism by direct regulation of PPARgamma. Cell Metab. 2010; 12: 509-520.
33. Ji Y, Qin Y, Shu H, Li X. Methylation analyses on promoters of mPer1, mPer2, and mCry1 during perinatal development. Biochem Biophys Res Commun. 2010; 391: 1742-1747.
34. Ge ZJ, Liang QX, Luo SM, Wei YC, Han ZM, Schatten H, et al. Diabetic uterus environment may play a key role in alterations of DNA methylation of several imprinted genes at mid-gestation in mice. Reprod Biol Endocrinol. 2013; 11: 119.
FIGURE LEGENDS
Figure 1. Biochemical and Histological Evidence of Hepatic Injury and Fibrosis at 6
months: (A) Liver Weight, (B) Serum ALT, (C) Hepatic Triglyceride Content (D) NAFLD
Activity Score, (E) Representative H&E sections and (F) Representative Masson’s Trichrome
sections. n = 5 per group, values shown are mean ± SEM, one-way ANOVA. *Mean values
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21
significantly greater than OffCon-SC (p < 0.05). #Mean values significantly greater than
OffCon-OD (p < 0.05). §Mean values significantly greater than OffOb-SC (p < 0.05). Tables
represent two-way ANOVA analysis.
Figure 2. Offspring Locomotor Activity at 6 months: (A) Representative actograms of mouse
locomotor activity (black bars measured in 10 min bins) in mice from each group during day
(07.00-19.00) and night (19.00-07.00). (B) Representative periodogram analysis of mice from
each group. Y-axis indicates the power at periods from 6-48hrs. The horizontal line represents
significant period lengths at p=0.05. (C) Graph of the percentage of locomotor activity occurring
during the Light period in each group (mean ± SEM, from 26 days of recording in 3 mice per
group). Tables represent two-way ANOVA analysis.
Figure 3. Hepatic 24-hours gene expression rhythmicity is disrupted in offspring with
programmed NAFLD: (A) BMAL1 mRNA, (B) CLOCK mRNA, (C) Cry1 mRNA, (D) Cry2
mRNA, (E) Per1 mRNA and (F) Per2 mRNA. n = 5 per group, values shown are mean ± SEM,
one-way ANOVA. *Mean values significantly greater than OffCon-SC (p < 0.05). #Mean values
significantly greater than OffCon-OD (p < 0.05). §Mean values significantly greater than
OffOb-SC (p < 0.05).
Figure 4. Hepatic 24-hours gene expression pattern of Rev-erb-α is disrupted in offspring
exposed to a post-weaning obesogenic diet: n = 5 per group, values shown are mean ± SEM,
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22
one-way ANOVA. *Mean values significantly greater than OffCon-SC (p < 0.05). #Mean values
significantly greater than OffCon-OD (p < 0.05). §Mean values significantly greater than OffOb-
SC (p < 0.05).
Figure 5. Maternal obesity and a post-weaning obesogenic diet induce phase shifts of core
canonical clock genes in offspring with programmed NAFLD: (A) CLOCK mRNA, (B)
BMAL1 mRNA and (C) Per2 mRNA. n = 5 per group, values shown are mean ± SEM, one-way
ANOVA. *Mean values significantly greater than OffCon-SC (p < 0.05). #Mean values
significantly greater than OffCon-OD (p < 0.05). §Mean values significantly greater than
OffOb-SC (p < 0.05).
Figure 6. Maternal obesity and a post-weaning obesogenic diet disrupts rhythmic
expression of hepatic inflammatory and fibrogenic markers in offspring: (A) IL-6 mRNA,
(B) TNF-α mRNA, (C) α-SMA mRNA, (D) TGF-b mRNA and (E) Collagen mRNA. n = 5 per
group, values shown are mean ± SEM, one-way ANOVA. *Mean values significantly greater
than OffCon-SC (p < 0.05). #Mean values significantly greater than OffCon-OD (p < 0.05).
§Mean values significantly greater than OffOb-SC (p < 0.05).
Figure 7. Maternal obesity and a post-weaning obesogenic diet induces hyper-methylation
of BMAL1 and Per2 promotor regions: (A) BMAL1 and (B) Per2. n = 5 per group, values
shown are mean ± SEM, one-way ANOVA. *Mean values significantly greater than OffCon-SC
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23
(p < 0.05). #Mean values significantly greater than OffCon-OD (p < 0.05). §Mean values
significantly greater than OffOb-SC (p < 0.05). Tables represent two-way ANOVA analysis.
© 2015 Macmillan Publishers Limited. All rights reserved.
Table 1: Liver Injury and Fibrosis Gene Specific Primers
Gene Primer Sequence
Expected
weight
Annealing
Temp
IL-6
F: 5’-TTCACAGAGGATACCACTCC-3’
R: 5’-GTTTGGTAGCATCCATCATT-3’
203bp 55˚C
TNF-α
F: 5’-TCCAGCTGACTAAACATCCT-3’
R: 5’-CCCTTCATCTTCCTCCTTAT-3’
220bp 55˚C
ASMA
F: 5’-ATCTGGCACCACTCTTTCTA-3’
R: 5’-GTACGTCCAGAGGCATAGAG-3’
191bp 59˚C
TGF-β
F: 5’-AAAATCAAGTGTGGAGCAAC-3’
R: 5’-CCACGTGGAGTTTGTTATCT-3’
224bp 59˚C
Collagen 1-α2
F: 5’-GAACGGTCCACGATTGCATG-3’
R: 5’-GGCATGTTGCTAGGCACGAAG-3’
167bp 55˚C
© 2015 Macmillan Publishers Limited. All rights reserved.
Table 2: Circadian Gene Primer Assays
Primer Assay Reference Annealing Temp
Circadian Locomotor Output Cycles
Kaput (CLOCK)
QT00197547 55°C
Brain and Muscle Arnt Like-1
(Bmal-1)
QT00101647 55°C
Period 1 (Per 1) QT00113337 55°C
Period 2 (Per 2) QT00198366 55°C
Cryptochrome 1 (Cry 1) QT00117012 55°C
Cryptochrome 2 (Cry 2) QT00168868 55°C
REV-ERBα QT00164556 55°C
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