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, R Carter, 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 nonalcoholic 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. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Accepted article preview online 14 May 2015
©
2015 Macmillan Publishers Limited. All rights reserved.
Maternal Obesity Programs Offspring Non-Alcoholic Fatty Liver Disease through disruption of 24-hours Rhythms in Mice Angelina Mouralidarane1,2, Junpei Soeda1, David Sugden2, Alina Bocianowska2, Rebeca Carter1, Shuvra Ray1, Ruma Saraswati3, Paul Cordero1, Marco Novelli3, Giuseppe Fusai4, Manlio Vinciguerra1,5,6*, Lucilla Poston2, Paul D Taylor2 & Jude A Oben1 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, 10th 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.
1
©
2015 Macmillan Publishers Limited. All rights reserved.
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
2
©
2015 Macmillan Publishers Limited. All rights reserved.
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. 3
©
2015 Macmillan Publishers Limited. All rights reserved.
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) (15). 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 4
©
2015 Macmillan Publishers Limited. All rights reserved.
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 5
©
2015 Macmillan Publishers Limited. All rights reserved.
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, 6
©
2015 Macmillan Publishers Limited. All rights reserved.
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 7
©
2015 Macmillan Publishers Limited. All rights reserved.
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 8
©
2015 Macmillan Publishers Limited. All rights reserved.
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. 9
©
2015 Macmillan Publishers Limited. All rights reserved.
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
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 nonalcoholic 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. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Accepted article preview online 14 May 2015
©
2015 Macmillan Publishers Limited. All rights reserved.
Maternal Obesity Programs Offspring Non-Alcoholic Fatty Liver Disease through disruption of 24-hours Rhythms in Mice Angelina Mouralidarane1,2, Junpei Soeda1, David Sugden2, Alina Bocianowska2, Rebeca Carter1, Shuvra Ray1, Ruma Saraswati3, Paul Cordero1, Marco Novelli3, Giuseppe Fusai4, Manlio Vinciguerra1,5,6*, Lucilla Poston2, Paul D Taylor2 & Jude A Oben1 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, 10th 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.
1
©
2015 Macmillan Publishers Limited. All rights reserved.
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
2
©
2015 Macmillan Publishers Limited. All rights reserved.
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. 3
©
2015 Macmillan Publishers Limited. All rights reserved.
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) (15). 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 4
©
2015 Macmillan Publishers Limited. All rights reserved.
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 5
©
2015 Macmillan Publishers Limited. All rights reserved.
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, 6
©
2015 Macmillan Publishers Limited. All rights reserved.
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 7
©
2015 Macmillan Publishers Limited. All rights reserved.
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 8
©
2015 Macmillan Publishers Limited. All rights reserved.
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. 9
©
2015 Macmillan Publishers Limited. All rights reserved.
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
