Role of clock genes in a mouse model for developmental origins of the metabolic syndrome

Lead Research Organisation: University of Southampton
Department Name: Development Origin of Health and Disease

Abstract

Daily, or circadian, rhythms are important in all organisms, including humans. Circadian rhythms influence nearly all aspects of physiology and behaviour, including our sleep-wake cycles, activity, feeding behaviour, cardiovascular function, body temperature and metabolism. These rhythms are generated within the body and the mechanisms that control them takes form while the baby is still in the mother's womb. Studies have shown that the diet of the mother during pregnancy is important in maintaining the health of her offspring and may also influence their susceptibility to developing obesity, diabetes and heart disease later in life. If the mother eats a healthy diet during pregnancy, one would expect the development of normal circadian rhythms of the baby's body function and metabolism, enabling the baby to cope with a healthy diet in childhood and adulthood. The risk of the person developing obesity, diabetes and heart disease later in life is very low. However, if the mother eats an unhealthy diet during pregnancy it might result in the abnormal development of circadian rhythms of body function and metabolism. This leads to disruption in sleep and activity patterns and inefficient processing of the food the baby eats. The baby is at risk of becoming heavier and fatter and will have high blood pressure in adulthood. If the baby is not brought up after birth and throughout childhood to eat a healthy diet, but eats instead a diet rich in fat, the baby's already disrupted circadian rhythms of metabolism is now not able to cope with this diet. This person is likely to become obese and risk of adult diabetes and heart disease later in life increases considerably. It is our intention of undertaking this study using a rodent model. Only by choosing an animal model is it possible to study this phenomenon, because it is necessary to change both the pre- and post natal diets and it would be unethical to undertake these changes in pregnant mothers and their babies.

Technical Summary

This proposal aims to identify key molecular clock and clock-controlled mechanisms underlying prenatal induction of phenotypic susceptibility to the metabolic syndrome. We will use a mouse model, where offspring prenatally exposed to a high fat nutritional environment develop the metabolic syndrome phenotype in adulthood following post-weaning high-fat feeding. We are suggesting that a maternal diet during pregnancy that is high in fat can adversely affect the development of the offspring's circadian clock system in the hypothalamic suprachiasmatic nucleus (SCN) and in peripheral tissues, resulting in altered rhythmic expression and function of clock and clock-controlled genes involved in the regulation of adipogenesis, metabolism and cardiovascular function. If these offspring are subsequently fed the same high fat diet, their physiology will be unable to respond appropriately making them particularly susceptible to developing metabolic To address our aim we will look at circadian metabolic, hormonal and activity patterns in mouse offspring from high fat fed dams and examine the effects of a post-weaning high fat feeding. We will then measure the 24h mRNA expression pattern of clock and clock-controlled genes, and other 'candidate' genes linked to both circadian and metabolic/cardiovascular/adipocyte function in neonate and adult offspring SCN and peripheral tissues which are key organelles to the pathogenesis of the metabolic syndrome. We will also examine whether an epigenetic mechanism, i.e. modification of rhythmic histone acetylation of clock-associated genes and RNA polymerase binding, is involved in the aberration and phase-shift in the circadian timing system and maladapted responses to a post-weaning high fat diet. Our proposed study will provide the scientific basis for understanding how an unhealthy diet in early life contributes to metabolic syndrome later in life. Results from this study will have direct relevance to humans.

Publications

10 25 50
 
Description The 24 hour (circadian) patterns of many biological functions such as sleep, activity metabolism and blood pressure are controlled by a key network of genes in the brain called the circadian "clock genes". In the present BBSRC-funded study we have developed a novel use for our mouse model of developmental programming established here in Southampton, where increased dietary fat exposure (45% kcal fat) during early life results in the onset of cardiovascular and metabolic disorder in adulthood. Our data show that offspring from mothers fed a high fat diet exhibit altered patterns of circadian behaviour. Further examination of the offspring's metabolic tissues, including the liver, heart and the brain, has revealed arrhythmic expression of clock genes following exposure to maternal high fat nutrition during fetal development and/or postnatal high fat feeding, which may be linked to poor health outcomes. These changes have deleterious knock on effects for metabolism and cardiovascular function. Therefore, high fat induced changes may result in increased susceptibility to cardiovascular and metabolic diseases.
Results from our work have suggested that proteins called Sirtuin, which regulate clock gene function, and are active in times of metabolic balance, are reduced following high fat exposure, and provide a possible mechanism for developmentally primed metabolic disease and the clock gene network.
We have also shown the impact of an in-utero maternal high fat diet on the ovaries in female offspring. We have novel data showing maternal obesity during pregnancy significantly alters the numbers of maturing eggs in the ovaries of the adult female offspring and this is accompanied by the development of ovarian steatosis ('fatty' ovary) and reduction the in number of mature follicles in the ovaries. This might explain the reduced fertility in these animals, and highlights a possible mechanism explaining the link between human obesity and fertility. We have also shown presence of clock genes in the ovaries and expression levels were altered as a consequence of the in-utero and postnatal HF diet. We were therefore intrigued to find out whether in humans, clock genes are also express in reproductive tissues and whether they are linked to reproductive dysfunction. We have now shown that clock genes are expressed in the human endometrium, which we found to be altered in pathological conditions such in scarred peritoneal mesothelium and chronic pelvic pain.
Data normalisation in real-time RT-PCR is a major step in gene quantification analysis. This involves the use of a stable endogenous control (reference gene) in the assay to correct for sample to sample variations in RT-PCR efficiency and errors in sample quantification that may have large effect on downstream analysis and scientific conclusion. Many studies do not employ sufficient reference selection strategies, and assume that reference expression does not alter with experimental intervention. We now show that tissue type, time of day collection of samples, and nutritional manipulations can impact on stability of reference genes. Thus we have highlighted the need for a strict selection strategy of the most stable reference gene(s) as a key step in RT-PCR quantification analysis.
Our study therefore may provide mechanistic explanation that links obese pregnancy to increasing incidence of obesity and the metabolic syndrome, and fertility problems in future generations. It strengthens the importance of providing good nutrition during pregnancy to benefit both the mother and her offspring.
Exploitation Route Our work has shown the important relationship between nutrition, metabolism and circadian rhythms. Our study therefore may provide mechanistic explanation that links obese pregnancy to increasing incidence of obesity and the metabolic syndrome, and fertility problems in future generations. It strengthens the importance of providing good nutrition during pregnancy to benefit both the mother and her offspring. Industrial collaboration was recently established with Cristal Delivery, a pharmaceutical company developing a new class of nanomedicine based on its proprietary polymeric technologies. We will use our mouse model for pre-clinical testing of the company's CriPec nanoparticles containing small molecules targeted at pathways that can alter or resynchronize clock activity and determine whether this reduces the offspring susceptibility or protects them to developing cardiometabolic pathologies.
We have also been developing therapeutic strategies that are beneficial to both the obese pregnant mothers and the health outcomes in her offspring. We have been funded by Diabetes UK to examine the effect of treating obese diabetic pregnant mouse dams with metformin on the health outcome of the offspring in adulthood. This work is relevant to current MRC studies looking at the effect of metformin treatment to diabetic pregnant women on the health outcomes of her offspring. Our Diabetes UK study investigates possible mechanisms on the effect of metformin that is relevant to both humans and in animals.
We have also linked up with colleagues from our Chemistry Department here at Southampton University, who developed compounds that can potentially regulate clock activity, with the aim of developing chronotherapeutic compounds.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology,Other

 
Description Data normalisation in real-time RT-PCR is a major step in gene quantification analysis. This involves the use of a stable endogenous control (reference gene) in the assay to correct for sample to sample variations in RT-PCR efficiency and errors in sample quantification that may have large effect on downstream analysis and scientific conclusion. Many studies do not employ sufficient reference selection strategies, and assume that reference expression does not alter with experimental intervention. We now show that tissue type, time of day collection of samples, and nutritional manipulations can impact on stability of reference genes, and thus highlighted the need for a strict selection strategy of the most stable reference gene(s) as a key step in RT-PCR quantification analysis.
First Year Of Impact 2011
Sector Education,Environment,Healthcare,Other
Impact Types Societal,Economic

 
Description Can metformin be used to treat pregnant diabetic mothers to reduce future risk to obesity and metabolic syndrome in their offspring?
Amount £87,000 (GBP)
Funding ID 11/0004377 
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2012 
End 08/2015
 
Description The role of maternal high fat in predisposing the offspring to Alzheimer's Disease
Amount £116,153 (GBP)
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2016 
End 03/2017
 
Title Identification of the most stable reference gene (housekeeping gene) to use in gene expression studies investigating non-alcoholic fatty liver disease (NAFLD) 
Description Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in Western society. Comparative gene expression studies are beginning to elucidate the molecular mechanisms underlying NAFLD progression. We have previously shown that high fat diets during early life can prime non-alcoholic steatohepatitis (NASH) in adulthood, through lipogenesis gene elevation. To generate accurate results in such studies, appropriate housekeeping genes (HKG) which are unaffected by disease processes, are used for data normalisation. However, there is little existing data to show the effects of NAFLD on HKG expression. The aim of the study was to identify the HKG in a mouse model of developmentally primed NAFLD and NASH, which maintains expression stability. We determined the expression stability of six candidates HKG (GAPDH, YWHAZ, B2M, EIF4A2, ACTB and CYC1) in a mouse model of developmentally primed NAFLD in both the day and night, using geNORM qBasePlus software. HKG expression differed across dietary groups and time of day. In the majority of treatment groups and time points the most stable gene was YWHAZ. Following high fat diet interventions CYC1 became notably unstable. Overall the effect of NAFLD and NASH on HKG expression was to maintain stability of YWHAZ, but destabilise CYC1 and EIF4A2. Our data clearly shows that HKG expression is affected by NAFLD severity and time of day sampling, highlighting the importance of suitable HKG gene selection. For comparative gene expression studies investigating NAFLD we would recommend use of YWHAZ as a robust, stably expressed HKG. 
Type Of Material Biological samples 
Year Produced 2012 
Provided To Others? Yes  
Impact Researchers investigating the pathogenesis of diet induced non-alcoholic fatty liver disease (NAFLD) are now made aware that in order to generate useful gene expression data, a constitutively expressed housekeeping gene (HKG) is required as an internal reference control, to correct for small anomalies in sample quantity that may have large effect on downstream analysis and scientific conclusion. Our data clearly shows that HKG expression is affected by NAFLD severity and time of day sampling, highlighting the importance of suitable HKG gene selection. For comparative gene expression studies investigating NAFLD we would recommend use of the gene YWHAZ as a robust, stably expressed HKG 
URL https://www.ncbi.nlm.nih.gov/pubmed/22583519
 
Title Identifying suitable reference genes (Housekeeping genes) for qPCR work on suprachiasmatic nucleus (SCN) tissue samples in mice 
Description The endogenous timing system within the suprachiasmatic nuclei (SCN) of the hypothalamus drives the cyclic expression of the clock molecules across the 24h day-night cycle controlling downstream molecular pathways and physiological processes. The developing fetal clock system is sensitive to the environment and physiology of the pregnant mother and as such disruption of this system could lead to altered physiology in the offspring. Characterizing the gene profiles of the endogenous molecular clock system by quantitative reverse transcription polymerase chain reaction is dependent on normalization by appropriate housekeeping genes (HKGs). However, many HKGs commonly used as internal controls, although stably expressed under control conditions, can vary significantly in their expression under certain experimental conditions. Here we analyzed the expression of 10 classic HKG across the 24h light-dark cycle in the SCN of mouse offspring exposed to normal chow or a high fat diet during early development and in postnatal life. We found that the HKGs glyceraldehyde-3-phosphate dehydrogenase, beta actin and adenosine triphosphate synthase subunit to be the most stably expressed genes in the SCN regardless of diet or time within the 24h light-dark cycle, and are therefore suitable to be used as internal controls. However SCN samples collected during the light and dark periods did show differences in expression and as such the timing of collection should be considered when carrying out gene expression studies. 
Type Of Material Biological samples 
Year Produced 2014 
Provided To Others? Yes  
Impact Researcher are made aware of the importance of validating gene expression studies; that normalizing the gene of interest using the geometric mean of multiple internal control genes increases the quality of the data. Researchers conducting qPCR work using samples of the mouse suprachiasmatic nucleus (SCN) can now use the reference genes that we have idetified to be the most stable regardless of diet or time within the 24 h light-dark cycle. 
URL http://www.sciencedirect.com/science/article/pii/S0006899314007203
 
Title Development of appropriate normalization strategy for real-time PCR 
Description Quantitative real-time PCR provides a sensitive, specific and reproducible means of determining comparative gene mRNA expression profiles. However, an increasing number of reports have shown that RT-PCR data accuracy is highly dependent on appropriate normalization strategies. It is now recognized that expression of classic reference gene (RG) can vary across tissue type and experimental conditions. We therefore analysed the stability in expression of RGs using qBasePLUS software based on geNorm and qBase technology. From this a stability measure (M) was generated by geometric averaging of multiple target genes and mean pair-wise variation of a gene from all other target genes in a given sample. Genes with the lowest M values are deemed the most stable. 
Type Of Material Data analysis technique 
Year Produced 2011 
Provided To Others? Yes  
Impact This method has provided useful information for other researchers carrying out gene expression studies when choosing the appropriate reference genes. It is now widely employed and is beginning to be acknowledged as a requisite in any RT-PCR analysis. 
 
Description A small-molecule inhibitor of AICAR transformylase homodimerization as potential therapeutic treatment for obesity and glucose intolerance in type 2 diabetes 
Organisation University of Southampton
Department Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided expertise in setting up animal experimentations similar to the experimental protocol described in the BBSRC project grant. The obese animals generated were subsequently treated with a small molecule inhibitor that were developed and synthesised by the collaborator, and their metabolism and glucose homeostasis examined.
Collaborator Contribution Funded the synthesis of small molecule inhibitor. Provided funding to set up the animal experimentations and characterisation of the metabolic phenotype of the mice.
Impact 1. This collaborator is multidisciplinary in the area of Chemistry and in Biology. 2. We published a scientific article from data generated for our study in 2015 in Chemical & Biology (vol 22 pp 838-48) 3. The news article released by the University of Southamton generated a lot of media interest including the Washington Post, sciencedaily.com, sciencealert.com, the Daily Mail etc.
Start Year 2014
 
Description Biological Rhythms in Female Reproduction 
Organisation University Hospital Southampton NHS Foundation Trust
Department Princess Anne Hospital
Country United Kingdom 
Sector Hospitals 
PI Contribution Provided expertise and supervision to PhD student undertaking this research work.
Collaborator Contribution Recruited human patients to collect tissue samples and funded the processing and analysis of tissues.
Impact 1. A Clinical PhD student working on this project completed the PhD degree in 2018 2. We published a scientific article from data generated for our study in 2018 in Scientific Reports (Nov 1;7(1):14857) and 1 conference abstracts in Human Reproduction (vol 32 Suppl 1, pp 306)
Start Year 2012
 
Description Developmental Programming of adipogenesis 
Organisation University of Reading
Department School of Biological Sciences Reading
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided aliquots of adipose tissue taken from mice from the BBSRC project grant to generate preliminary data. Consequently, we set up new animal experimentations similar to the experimental protocol described in the BBSRC project grant to generate additional tissue samples.
Collaborator Contribution Funded the processing and analysis of tissues to generate the preliminary data. Provided funding to set up the subsequent animal experimentations and analysis of tissues taken from these mice.
Impact 1. Collaborator used the preliminary data to apply for an MRC New Investigator Research Grant, with me named as collaborator. Subsequently, the collaborator was successful in obtaining funding (£512,486) from the MRC and the project commenced in 2016. 2. We published 2 review article on the subject matter in 2014 in Mammalia Genome (vol 25, pp 413-23) and in Endocrinology (vol 155, pp 1573-1588)
Start Year 2014
 
Description The role of maternal high fat in predisposing the offspring to Alzheimer's Disease 
Organisation University of Southampton
Department Faculty of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided expertise in setting up animal experimentations similar to the experimental protocol described in the BBSRC project grant. The animals generated were subsequently maintained until 1 year of age. The mice were then injected intracerebrally with labelled amyloid and examined for changes in their perivascular pathways for the elimination of amyloid from the ageing brain.
Collaborator Contribution Provided funding to set up the animal experimentations and characterisation of the metabolic phenotype of the mice. Funded the processing of the ageing brains and subsequent analysis for electron microscopy and proteomics.
Impact We obtained grant funding to undertake this project from the Rosetrees Trust for 2016-17 (£108,598)
Start Year 2016
 
Description Expert opinion on a news article 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Dr F Cagampang provided his expert opinion on a news article entitled 'Day to day toll of the night shift' that was published by the Southern Daily Echo.
Year(s) Of Engagement Activity 2014
URL http://www.dailyecho.co.uk/news/11030615.Working_nights_can_damage_health__researchers_find
 
Description Lifelab Southampton 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact LifeLab is an innovative, cross-community educational intervention that develops, delivers and evaluates a number of educational programmes tailored to school students (11-18) that demonstrate the importance of diet, development and impact on Lifelong Health across a number of areas (cardiovascular, diabetes, osteoporosis) based on our world-leading research. By engaging children with the science that shows how lifestyle choices at an early age can affect health and future children's health, we hope to empower them to make healthier choices and reduce their risk of developing chronic diseases later in life.

LifeLab has now established a close partnership with LENScience in Auckland (New Zealand), and this was recently strengthened further through the awarding of funding from the BUPA Foundation to carry out an international evaluation of the intervention in both Southampton and New Zealand.
Year(s) Of Engagement Activity 2010,2011,2012
URL http://www.southampton.ac.uk/medicine/outreach/index.page
 
Description London Technology Network 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Dr K Bruce, the postdoc of the grant was a business fellow for the London Technology Network. During her tenure she liaised with academics throughout the University of Southampton to support their enterprising activities, help identify translatable and exploitable technologies, and establish relationships with industry within the south of England in order to facilitate commercial research collaborations.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012
URL http://laboratorytalk.com/directory/15944/london-technology-network
 
Description Media interest following the publication of our paper in the British Journal of Nutrition in 2012 on the role of the circadian clock system in nutrition and metabolism 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Provide scientific explanation for something intuitively, that is structured eating patterns are beneficial for health and well being.

A lot of comments generated from this news article.
Year(s) Of Engagement Activity 2012
URL http://blogs.webmd.com/food-and-nutrition/2012/08/how-your-brain-wants-you-to-eat.html