Maternal obesity programs offspring nonalcoholic fatty liver disease by innate immune dysfunction in...

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: [email protected].

Telephone: +44 (0)20 743-28-74

Short Title: Fatty liver and maternal reprogramming.


mailto:[email protected]

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


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.


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


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


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,


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


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


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.


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.


11

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).


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,


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).


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).


13

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


14

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.


15

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


16

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.


17

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.

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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


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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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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(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.


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


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


Maternal obesity programs offspring nonalcoholic fatty liver disease by innate immune dysfunction in...

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