Theses - Clinical Biochemistry

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  • ItemEmbargo
    Mapping the Feeding Circuitry in the Mouse and Human Brain
    Dowsett, Georgina KC; Dowsett, Georgina [0000-0002-2134-8554]
    The prevalence of obesity and its associated comorbidities has significantly grown over the last 50 years, imposing a great burden on people’s health. Consequently, understanding the mechanisms by which energy intake and bodyweight are controlled is extremely important to help us to further improve prevention and treatment of obesity. Genetic studies highlight the brain as a critical centre for food intake control. In particular, the hypothalamus and the hindbrain play key roles in the integration of signals regarding the body’s energy status and coordinating a response to alter changes in food intake. In particular, the hypothalamic leptin-melanocortin pathway is a well characterised signalling pathway implicated in food intake control, with genetic disruption resulting in severe obesity. Further, glucagon-like peptide 1 (GLP-1) is a hormone secreted from the gut postprandially and acts to potentiate insulin secretion, as well as acting as a satiation signal via activation of GLP-1R in the brain. Current anti-obesity therapeutics include analogues of GLP-1 and have been shown to activate GLP-1R in the hypothalamus and hindbrain to mediate their weight loss effects, through a reduction of food intake. However, most information about the neural circuitry involved in food intake control comes from murine studies, with little information about the hypothalamic cellular architecture in humans. Therefore, this thesis aims to map the feeding circuitry in the mouse and human brain, using transcriptomic approaches. To map the mouse hindbrain, we performed single nucleus RNA sequencing on hindbrain samples from mice fed *ad libitum* and mice subjected to an overnight fast. Here, we profile 16,034 single nuclei, and demonstrate the oligodendrocytes are transcriptionally sensitive to an overnight fast. We characterise cells expressing druggable targets for obesity treatments and highlight their neurotransmitter and neuropeptide properties. Additionally, we discuss the HypoMap, a single cell gene expression atlas of the mouse hypothalamus, created through the integration of 18 single cell, and single nucleus RNA sequencing datasets. Here, we demonstrate the heterogeneity of neurons and non-neuronal cells in the hypothalamus, and profile cells involved in the leptin melanocortin pathway. Through the integration of single nucleus RNA sequencing and spatial transcriptomics, we generate a spatially resolved single cell gene expression atlas of the human hypothalamus, named the HYPOMAP. Here we review non-neuronal cells, and neurons involved in the leptin-melanocortin pathway and identify transcriptionally and spatially distinct populations of POMC neurons, and neurons expressing the MC3R and MC4R. Using the mouse and human hypothalamic atlases, we identify and characterise GLP-1R expressing populations, describing similarities and differences between the mouse and human. We confirm co-expression of *GLP1R* with key transcripts of interest in the human hypothalamus using in situ hybridization. Collectively, this project has characterised the cellular architecture of the mouse and human hypothalamus (and mouse hindbrain) using transcriptomic approaches, profiling cells involved in the neurocircuitry of food intake. We describe similarities and differences between mouse and human appetitive pathways and profile neurons which could be directly modulated by current anti-obesity therapeutics. These datasets will serve as key resources for the identification of novel therapeutic targets for treating obesity.
  • ItemEmbargo
    Exploring INSL5 and RXFP4 in the gut-brain axis and their potential role in feeding behaviour
    Woodward, Orla
    Obesity prevalence has more than tripled in the last four decades with over 750 million people now living with obesity worldwide. The myriad health, social and economic costs of obesity have stimulated research into the physiological mechanisms that govern body weight and feeding behaviour with the aim of developing new treatment strategies. Insulin-like peptide 5 (INSL5), a peptide hormone secreted from the distal gut, and its cognate receptor, relaxin/insulin-like family peptide receptor 4 (RXFP4), have been implicated in feeding in animal models. Administration of INSL5 and chemogenetic manipulation of Rxfp4-expressing cells influence feeding behaviour in mice. As feeding is regulated, in part, by neuroendocrine signalling in the gut-brain axis (GBA), we aimed to examine INSL5/RXFP4 activity at each level of the GBA to determine the potential mechanisms by which this hormone-receptor pair modulate food intake. Using an Rxfp4-Cre mouse model combined with immunohistochemistry and transcriptomic techniques, we identified Rxfp4 expression in enterochromaffin, L and tuft cells in the colon, in neurons of the dorsal root ganglia (DRG) and nodose ganglia (NG), and in multiple brain regions associated with feeding. By combining Rxfp4-Cre mice with a cyclic adenosine monophosphate (cAMP) imaging technique, INSL5 was found to reduce intracellular cAMP levels in Rxfp4-expressing cells in the colon, DRG, NG and the ventromedial hypothalamus (VMH), a known feeding centre in the brain. Transcriptomic analysis of Rxfp4-expressing cells in the hypothalamus revealed enriched expression of multiple feeding-related neuropeptides and receptors. Circuit mapping of hypothalamic Rxfp4-expressing neurons using viral tracing tools indicated that these neurons are part of feeding-, reward- and memory-related neurocircuits. Modulation of Rxfp4-expressing neurons within the VMH using intraparenchymal INSL5 infusions and chemogenetic tools increased and decreased intake of highly palatable meals, respectively. Together, these data suggest that INSL5/RXFP4 signalling within the GBA can regulate feeding behaviour. RXFP4 is therefore a potential target for the development of pharmaceutical treatments for obesity and other feeding-related disorders. Increased understanding of neuroendocrine signalling within the GBA may also aid the development of more successful intervention strategies for obesity management.
  • ItemEmbargo
    Maternal Obesity During Pregnancy: Cardiovascular Outcomes for Mother and Baby
    Wilsmore, Phoebe
    The prevalence of obesity has increased significantly over the last 30 years such that it is now more common worldwide to be overweight or obese than normal weight. As a result of this, increasing numbers of women are entering pregnancy overweight or obese. Obesity during pregnancy is associated with an increased risk of numerous negative obstetric outcomes including miscarriage, stillbirth, and neonatal mortality. Moreover, obesity is associated with an increased risk of women developing disorders of pregnancy such as gestational diabetes mellitus (GDM) and pre-eclampsia. These disorders are associated with the development of type 2 diabetes (T2D) and persistently high arterial blood pressure (hypertension) in the years after delivery. Hypertension is the number one risk factor for the development of cardiovascular disease (CVD), and CVD is the leading cause of death across the globe. Therefore, maternal obesity during pregnancy is associated with poor cardiometabolic health both during pregnancy and in later life. Children of obese women are at an increased risk of both becoming obese themselves and developing hypertension and CVD. While some of this increased risk in the general population is attributable to a shared obesogenic postnatal environment, studies in both humans and animals reveal that it is also directly programmed by exposure to maternal obesity *in utero*. This forms part of the Developmental Origins of Health and Disease (DOHaD) hypothesis, which states that exposure to suboptimal environments during development programmes changes in long-term health. Together, this shows that maternal obesity during pregnancy is associated with poor cardiometabolic health in both mother and baby in the short and long-term, which will result in an increasing number of people suffering and dying from CVD. Therefore, it is paramount that interventions are developed in order to break the transgenerational cycle of obesity and cardiovascular dysfunction. Two interventions used in pregnancies complicated by GDM and that are being trialled in glucose-tolerant obese pregnancies are the application of an exercise intervention and the use of the glucose-lowering medication metformin. While evidence of improved maternal cardiometabolic function in response to these interventions has been found in human studies, longitudinal follow-up of children is limited. The Ozanne group uses a well-established mouse model of maternal diet-induced obesity to investigate the effects of maternal obesity and glucose intolerance during pregnancy on the cardiometabolic health of both mother and baby. Using this model, the effects of maternal exercise and metformin intervention during pregnancy on the cardiometabolic health of both mother and offspring can be investigated. Previous studies have found that maternal exercise intervention improves metabolic and cardiac function in young adult male offspring of obese dams but does not prevent the programming of hypertension. This is associated with improved metabolic health in the dam without a significant change in her adiposity. However, how obesity affects maternal cardiovascular function in pregnancy, and whether this is affected by exercise, has not previously been investigated in this model. In Chapter 3 of this thesis, evidence of impaired cardiovascular adaptation to pregnancy was identified in obese dams, although this did not result in overt cardiac dysfunction in late gestation. Maternal obesity also resulted in fetuses being growth-restricted, which is well established to be associated with poor long-term cardiometabolic health. Neither the impaired cardiovascular adaptation nor fetal growth restriction were rescued by maternal exercise. Changes in the autonomic control of the heart, including an increase in sympathetic activity and a decrease in parasympathetic activity, are a common feature of heart failure. Previous studies have suggested that maternal obesity programmes changes in autonomic control of the offspring heart. In Chapter 4 of this thesis, a new protocol was developed to investigate the response of the heart to sympathetic stimulation *in vivo*. This protocol is a powerful tool that can be adapted to investigate the effects of maternal obesity during pregnancy on cardiovascular function in male and female offspring. Metformin administration during obese pregnancy has been found to improve maternal metabolic health in both human and animal studies. However, metformin exposure *in utero* has been found to programme increased adiposity and metabolic dysfunction in male and female offspring of obese dams. No studies have been published investigating the impact of metformin exposure during obese pregnancy on the blood pressure and vascular function of offspring beyond childhood in humans, or at all in animals. Hypertension and CVD are age-related disorders and so the effect of gestational metformin on blood pressure and vascular function must be investigated in aged offspring in order to draw clinically relevant conclusions about its effects. In Chapter 5 of this thesis, it was found that combined exposure to maternal obesity and metformin *in utero* programmed hypertension in aged offspring. This was associated with sex-specific changes in vascular reactivity. This suggests that maternal metformin administration during pregnancy is detrimental to long-term offspring cardiovascular health and highlights the importance of including both male and female offspring in studies of developmental programming. Overall, this thesis investigated the effects of obesity on cardiovascular function in both pregnant mothers and their offspring and developed a new protocol for the investigation of cardiovascular function *in vivo*. It also assessed the effects of exercise intervention on maternal cardiovascular adaptation to pregnancy, and of metformin intervention on long-term cardiovascular health in offspring. Developing effective interventions in obese pregnancy is paramount for improving the cardiometabolic health of the population, both in reducing the incidence of CVD and T2D in overweight and obese mothers, and in preventing the programming of obesity and cardiovascular dysfunction in their offspring.
  • ItemOpen Access
    Quantitation of enteroendocrine hormones as metabolic disease biomarkers in human biological matrices by liquid chromatography tandem mass spectrometry
    Foreman, Rachel
    Polypeptides are widely present in biology and many of them act as biomarkers, both for disease and system regulation. Many gut hormone peptides are secreted by enteroendocrine cells (EECs) and some, known as incretins, are released after a meal to control blood glucose and stimulate neuronal satiety. These are being studied as novel targets for treatment of metabolic diseases such as obesity. Traditionally quantitative analysis of these hormones is performed by enzyme-linked immunosorbent assays (ELISAs). The development of these immunological techniques can take many years and is not always cost effective, so new biomarker assay approaches are being explored in research. Liquid Chromatography Mass Spectrometry (LC-MS/MS) methods are becoming an alternative approach and analytical technique combines the physical separation abilities of liquid chromatography with specific mass analysis of mass spectrometry. The methods described in this thesis are traditional bioanalytical extractions used to isolate small molecule analytes from biological matrices. Assay development can be a challenging process, especially when the target biomarker is expected to be in the low (picomolar) ranges. To improve sensitivity of the methods a number of different extraction procedures were investigated and the optimal conditions for each peptide target were identified. Additionally the mass spectrometry hardware systems were assessed and modified, working to find a balance between sensitive detection limits and reducing sample analysis time to maintain high throughput methods for clinical application. Where applicable, the optimised LC-MS/MS methods were validated by commercial immunoassays and the strong correlation in concentrations confirmed the accuracy of the developed assay. An additional advantage of mass spectrometry is the ability to monitor multiple analytes in a single sample, so additional data on all possible isoforms of the target analyte are available, as opposed to the ‘total’ response usually measured by antibody capture techniques. This advantage is clearly important in the instance of prohormone intermediate species (as discovered in Chapter 3) and enzymatically cleaved and inactive forms peptides (Chapter 4 and Chapter 5). Plasma is one of the most accessible biological matrices, and the majority of the methods were found to be suitable for analysis of circulatory concentrations of peptide hormones. With support from clinicians, the final methods were applied to plasma samples from participants who had had specific meal tests, to gauge the postprandial response of insulin and C-peptide (Chapter 3 and Chapter 4), motilin and gastric inhibitory peptide (Chapter 4), and cholecystokinin (Chapter 5) following nutrient stimulation. As intestinal tissue is a more complex and invasive matrix to collect, organoid models have been developed for *in vitro* studies of specific cell activity. An extraction method for organoid secretion supernatants was optimised (in collaboration with Emily Miedzybrodzka), to be able to quantify the target analytes in direct response to stimuli. Analysis of organoid cultures was utilised to understand the secretory response of motilin (Chapter 4), cholecystokinin (Chapter 5) and glucagon-like peptide 2 (Chapter 6), respectively. To summarise, this thesis describes the development of analytical methods for metabolic gut hormone peptides, for clinical applications. The manuscript is separated into chapters based on specific gut hormone analytes, as multiple high throughput methods were developed on UPLC liquid chromatography system and triple quadrupole mass spectrometer. Each chapter in this thesis describes the method development, validation and application of the optimised methods to biological samples, with the results providing some insightful and novel discoveries to aid in metabolic disease biomarker quantitation.
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    Investigation of hypothalamic molecular mechanisms underlying narcolepsy and obesity through DNMT1 and leptin receptor signalling​
    Chisowa, Tendai
    Sleep disorders and obesity are common diseases that can reduce the length or quality of life for millions of people. These diseases result in part from the dysfunction of cell populations in the hypothalamus that regulate the sleep/wake cycle and energy balance, respectively. There has been increased knowledge in recent decades regarding the cellular and molecular basis of sleep disorders and obesity, but much remains to be discovered to convert current understanding into effective therapies. This work details efforts to identify molecular mechanisms of two diseases. The first project explored whether mutations in the DNMT1 gene that cause autosomal dominant neurodegenerative disease in humans, which preferentially affects the hypocretin neurons that regulate sleep, also induce neuronal loss when introduced into mice. No evidence was found for the loss of sleep-regulatory hypocretin neurons in Dnmt1 mutant mice. The second project used proximity proteomic methods to identify proteins that regulate human leptin receptor (LEPR) signalling, since leptin signalling is essential for normal body weight regulation. Several candidate proteins were identified that may negatively regulate signalling through LEPR and are of potential therapeutic relevance for obesity.
  • ItemOpen Access
    Genetic and molecular characterisation of metabolic subphenotypes and their contribution to type 2 diabetes aetiology
    Williamson, Alice; Williamson, Alice [0000-0002-7599-9301]
    Systematic assessment of the genetic architecture of type 2 diabetes (T2D) has allowed the identification of hundreds of genetic loci contributing to T2D risk. However, understanding how these loci contribute to the aetiological complexity of T2D has been more challenging. Using an interdisciplinary approach this thesis aimed to investigate the genetic contribution to T2D, and related metabolic subphenotypes, to further the understanding of disease aetiology. This incorporated large-scale population genetic analyses of T2D and fine-scale intermediate traits, with targeted in vitro functional follow up of genes and variants of interest. Focused in vitro and genetic investigation of a single gene, MC3R, of which loss of function was previously thought to result in obesity and metabolic dysfunction, revealed that this was not the case. Combining functional assessment of coding variants in MC3R and association analyses at population-scale revealed a key role of MC3R in relaying nutritional cues to the timing of puberty, accrual of lean mass and height. At the other end of the spectrum, hypothesis-free genome-wide approaches examining traits intermediate to T2D, in combination with in vitro follow up, also have great utility in understanding T2D aetiology. A genetic discovery including > 55,000 individuals was conducted for insulin resistance after a glucose challenge (post-challenge insulin resistance) to approximate insulin resistance specifically in the post-prandial state, a key but understudied contributor to T2D onset. This revealed ten novel genetic loci for post-challenge insulin resistance (P<5x10^-8), including an association at SLC2A4, encoding the key insulin-stimulated glucose transporter GLUT4. Most of these post-challenge insulin resistance loci are shared with T2D, suggesting potential mechanisms behind these disease associations. Functional follow up using cell-based assays identified 9 novel candidate genes involved in insulin-stimulated glucose uptake and GLUT4 trafficking. Finally, integration of information of T2D genetic risk, T2D incidence, and untargeted plasma metabolomics identified distinct metabolomic signatures of specific T2D genetic endophenotypes associated with T2D development, which together with mediation analyses prioritised a subset of potential causal pathways. This work demonstrates the power of integrating population genetic analyses, refined traits intermediate to disease, and tailored in vitro follow up in prioritising key genes and pathways involved in T2D aetiology, and additionally highlights several candidates for further investigation.
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    Identifying key nodes in brain neural circuits controlling glucose homeostasis
    Josipović, Maša
    Understanding the organisation and function of what Santiago Ramón y Cajal referred to as the impenetrable jungle of the brain has been one of the biggest fascinations of ancient and modern science. How does this exquisitely complex structure of close to 100 billion neurons form the basis of who we are, coordinating the processes such as our internal organ function, as well as directing our behaviour, perception and emotion? As a step closer to answering these questions, the work in this thesis aims to unravel the role of the brain in one of the most fundamental processes – maintenance of glucose homeostasis. The brain is constantly informed about the state of our internal environment and the external world; if a disruption occurs, it is able to coordinate physiological and behavioural responses to counterbalance it. This concept holds true for glucose as well. However, interestingly, here the brain not only receives inputs about the level of glucose in the blood, it also has the capability to measure – sense – glucose itself. I hypothesised that blood glucose homeostasis is exerted by glucose-responsive neurocircuitry within the brain, with discrete nodes responding to a rise or fall in glucose. The overarching aim of this thesis was to use mouse models to evaluate the identity and role of these circuits in controlling physiological and behavioural responses to blood glucose changes. I first examined a group of neurons located on the floor of the brain, that we hypothesised might have this exact function: the glucokinase (Gck) -expressing agouti-related protein (AgRP) neurons. The enzyme Gck is well-known for its crucial role within the pancreas, where it enables sensing of glucose levels and leads to glucose-stimulated insulin release. Here, we demonstrate its importance for AgRP neuronal function by genetically removing it from these cells in mice. We show that perturbing the function of this strikingly small population of approximately 3,000 neurons has profound impact on whole-body glucose homeostasis, causing insulin resistance, glucose intolerance, and a reduction in glucose-stimulated insulin secretion. Strikingly, we show that this effect is only seen in female mice, with males exhibiting no disturbances whatsoever. Next, I took a step back and observed the coordinated activity of the neural circuits across the entire brain induced by changes in blood glucose, from a drop (hypoglycaemia) to a rise (hyperglycaemia) in glucose levels. Here, I combined two powerful contemporary techniques, glucose clamps and tissue clearing, to (1) precisely manipulate blood glucose levels causing brain activation, and (2) subsequently render these brains transparent and scan them using light sheet microscopy. We create the first-of-its kind 3D atlas of the whole-brain glucose-responsive activity with a single-cell resolution, and we identify at least a dozen brain regions – potential nodes in the whole-brain glucose-responsive circuitry – showing discrete activity driven by blood glucose changes. Finally, we start to build a foundational framework to investigate the exact function of each of these nodes. Finally, I began to develop and adapt two novel technical approaches to quantify behavioural responses to low blood glucose (hypoglycaemia) in mice. In addition to the more well-known detrimental effects of high blood glucose and diabetes, hypoglycaemia is perhaps even more acutely dangerous as a constant supply of blood glucose is crucial for the brain function and survival. If fully validated, these methods could be used to advance our understanding of the brain circuits engaging protective mechanisms which defend against hypoglycaemia. I hope that this body of work will serve as a strong foundation for future mechanistic studies examining how blood glucose is maintained, how this process goes awry in disease such as diabetes, and answering whether we can specifically target brain neural circuits to find new drugs to treat, or even revert, diabetes.
  • ItemOpen Access
    Exploring the sex-specific programming of cardiovascular disease by maternal obesity and assessing potential interventions to the mother
    Inzani, Isabella
    There are currently over 1.9 billion adults globally who are overweight or obese. As the prevalence of obesity increases worldwide this includes women of childbearing age. Over half of all women of reproductive age in the UK are currently classed as overweight or obese. Obesity during pregnancy is known to have a negative impact on maternal health, pregnancy outcome, and the long-term cardiometabolic health of her offspring. Maternal interventions in obese pregnancy are needed to prevent transmission of poor cardiometabolic outcomes between mother and child. Maternal exercise in obese pregnancy in both humans and animal models has shown success for the prevention of adverse outcomes. As maternal obesity also results in maternal, placental, and fetal oxidative stress, antioxidant interventions to the mother may be another effective treatment. Human and mouse models of maternal obesity have shown that antioxidant supplementation can prevent an adverse offspring cardiometabolic phenotype. Our lab has previously shown that prenatal exposure to maternal obesity results in long-term consequences for offspring cardiometabolic health in adult male offspring. However, more recent studies have highlighted that there can be sex-specific effects of the maternal environment on offspring health. In the Ozanne lab mouse model of maternal diet-induced obesity, the cardiac phenotype in female offspring is still to be established. There has also been limited exploration of the effects of maternal obesity on either male or female cardiac function in fetal life. Furthermore, there is a lack of literature on the effects of exercise and antioxidant interventions in obese pregnancy on offspring cardiac function. The aims of this thesis are therefore: (1) Chapter 3 - to characterise the sex-specific effects of maternal obesity on cardiac dysfunction in 8-week-old male and female offspring; (2) Chapter 4 - to determine the early cardiac changes in male and female fetuses exposed to maternal obesity; (3) Chapter 5 - A) to identify how the maternal environment is altered in obese pregnancy, and in particular if maternal obesity leads to hypoxic fetal conditions. B) To establish a maternal antioxidant (CoQ10) supplementation intervention during obese pregnancy; (4) Chapter 6 - to establish the effects of maternal exercise intervention during obese pregnancy on male and female fetal cardiac function. In this thesis it was shown in Chapter 3 that young adult mouse offspring of obese mothers had sex-specific mild cardiac systolic, diastolic and electrical dysfunction in vivo, which was more severe in male offspring. This was accompanied by a mild cardiac hypertrophy phenotype, with increased cell size in both male and female offspring of obese dams. The sex-specific differences in the functional and structural phenotypes of the 8-week-old offspring heart in response to maternal obesity may help to explain the different outcomes observed in male and female risk of developing cardiovascular disease. Chapter 4 shows that maternal obesity induced sex-specific changes in the lipidome of the fetal heart, with more changes observed in female fetuses, despite both sexes being exposed to the same maternal milieu. Changes in lipid supply to the fetal heart in obese pregnancies may drive the altered fetal cardiac transcriptome to promote a premature switch from glycolytic to β-oxidative metabolism in both sexes. The changes in lipid composition and metabolism were not accompanied by any alteration or premature maturation of fetal cardiac structure. These effects of maternal obesity on the fetal heart may contribute to the programming of altered cardiac structure and function in later life, though the precise mechanisms for this remain to be determined. In Chapter 5, it is shown that maternal obesity resulted in fetal, but not placental, hypoxia at e13.5 in both sexes. Fetal hypoxia was associated with increased maternal fat mass and hyperinsulinaemia in obese pregnancy. Alterations in either placental size and structure or fetal growth and metabolism did not appear to underlie the development of fetal hypoxia. Obesity induced maternal iron deficiency may contribute to the development of fetal hypoxia. This chapter also established a maternal CoQ10 supplementation intervention protocol in obese pregnancy, confirming supplementation had no effects on diet palatability or maternal appetite, and did not cause any adverse maternal or fetal outcomes. Chapter 6 showed that maternal obesity resulted in fetal growth restriction which was not rescued by maternal exercise intervention. Maternal obesity also reduced fetal insulin levels, which was sex-specifically rescued by maternal exercise in male fetuses only. Maternal obesity affected fetal in vivo cardiac function with altered heart structure and function which was not rescued by maternal exercise. These differences occurred in the absence of any changes in uterine or umbilical blood flow in response to either maternal obesity or exercise intervention. Overall, this thesis showed the effects of maternal obesity on the adult and fetal offspring heart in both males and females. It also determined the effects of maternal exercise intervention on the fetal heart and established an antioxidant intervention protocol. Maternal interventions in obese pregnancy are important for preventing the programming of cardiometabolic disease in offspring. Further studies to fully establish the potential short- and long-term implications of interventions, in both sexes, is important for identifying effective treatment strategies in obese pregnancies to protect the health of future generations.
  • ItemOpen Access
    Production, characterisation and uses of antibodies to the insulin secretory granule membrane
    (1985-11) Grimaldi, Keith Anthony
    The aim of the work described in this thesis was to prepare and characterise a library of monoclonal antibodies that recognise antigens present in the insulin secretory granule membrane. These were to be used in a study of the composition, biosynthesis and turnover of the secretory granule membrane.
  • ItemOpen Access
    The Role of Thada in Energy Homeostasis
    Lin, Yu-Hung
    The high prevalence of obesity worldwide has fuelled research to discover new treatments that tackle obesity safely and effectively. Over the last decades, we and others have considered that developing strategies focused on increasing organismal energy expenditure may help fight obesity and its associated comorbidities. For this purpose, our research focused on a gene called Thada. THADA encodes a 220 kDa protein that has been recently reported in Drosophila melanogaster to uncouple the heat dissipation of SERCA from its Ca2+ pumping action. We anticipated that this mechanism could contribute significantly to the energy metabolism in endotherms, such as mammals. We envisaged THADA could contribute to the muscle non-shivering thermogenesis—in addition to sarcolipin, another uncoupler of SERCA. Our working hypothesis was reinforced by the fact that mutations in THADA have been positively selected during the last 45-30K years in the Arctic populations, which the authors of the research and we interpret as a potential adaption to the extreme cold. In this thesis, I have performed in vitro and in vivo approaches and confirmed that the ablation of THADA caused increased ATP hydrolysis and Ca2+ transport efficiency of SERCA and affected cellular bioenergetics in primary skeletal muscle cells. Interestingly, phenotypic characterisation of the Thada KO mice has revealed that the genetic ablation of Thada causes an impairment in the maintenance of core body temperature in response to cold exposure. This effect can only be revealed under specific environmental conditions when the brown adipose tissue (BAT) is cancelled/neutralised. Our physiological data are supported by a robust transcriptomic signature where skeletal muscle, BAT and inguinal subcutaneous white adipose tissue of the Thada KO mice presented primary and compensatory fingerprints associated with the environmental temperatures tested. The findings of this thesis provide evidence of THADA’s contribution to energy homeostasis in mammals and open the possibility of using THADA as a viable therapeutic target to increase basal metabolic rate and hence energy expenditure to tackle obesity.
  • ItemOpen Access
    Oligodendrocytes of the adult median eminence
    Buller, Sophie
    Oligodendrocytes (OLs) are the myelin forming cells of the CNS. Once thought to be generated exclusively during development, recent advances have shown that new OLs are generated in the adult brain from oligodendrocyte progenitor cells (OPCs) in response to physiologically relevant stimuli such as motor skill learning. Emerging evidence suggests that OL genesis is influenced by nutritional stimuli in the healthy and diseased brain, with recent studies highlighting that OPCs in the adult median eminence (ME) are highly proliferative and rapidly differentiate in response to nutritional signals. As such, these data raise questions about new OL and myelin generation in the ME at baseline, the fate of pre-existing OLs here, and the functional significance of OLs generated in the healthy adult ME given its diverse roles in the regulation of energy balance, glucose homeostasis, and neuroendocrine function. Data presented in this thesis demonstrate that in contrast to the corpus callosum (a well- characterised white matter tract), new myelin forming OLs are generated at a high rate and rapidly turnover in the healthy adult ME, which results in the appearance of relatively stable population of OLs in the ME over time in adult mice. Examining the impact of metabolic state on these processes revealed nutritional regulation of the ME OL lineage. Diet induced obesity, for example, blunts OL generation and turnover and increases ME myelin amounts, whereas caloric restriction reduces ME OPC differentiation and myelination. Intriguingly, blocking new OL generation in the adult brain using Pdgfrα-Cre/ERT2;Rosa26-YFP;Myrffl/fl mice mimics key metabolic and neuroendocrine adaptations to energy deficit, and is associated with cellular and structural remodelling of the ME, resulting in increased ME-arcuate nucleus (ARC) barrier permeability. Exploring potential mechanisms regulating OL lineage plasticity revealed that myelin debris generated during myelin turnover recruits immune cells to the ME, which are required for ongoing OL plasticity and, together with newly formed OLs, may contribute towards local perineuronal net remodelling. Collectively these observations indicate that 1) OL lineage cells are highly plastic and sense, adapt, and respond to nutritional stimuli, 2) ME OL plasticity plays a physiological role in the adaptative responses to states of negative energy balance, 3) ME OL plasticity is required for physiological hypothalamic functions via regulating the access of circulating factors to key hypothalamic feeding centres and 4) microglia contribute towards ME OL lineage plasticity and function, implicating a novel role for microglia in health.
  • ItemOpen Access
    The molecular, hormonal and symptomatic responses to hypoglycaemia in mice
    Staricoff, Emily
    I hypothesised that mouse models could be used to investigate the molecular, hormonal and symptomatic responses to acute and recurrent hypoglycaemia. To test this hypothesis, I used hyperinsulinaemic clamps to create carefully controlled experimental manipulations of blood glucose. It is well established that the hypothalamus plays a fundamental role in glucose sensing and coordination of the concerted effort to maintain blood glucose within a tight homeostatic range. However, little is known about the molecular changes that take place in the hypothalamus under conditions of hypoglycaemia or hyperglycaemia. Therefore, I aimed to use single-nucleus RNA sequencing (snRNAseq) to reveal the hypothalamic molecular signatures and gene expression profiles of different blood glucose levels. Hyperinsulinaemic clamps were used to create carefully controlled hypoglycaemic, euglycaemic and hyperglycaemic experimental interventions. Upon sequencing of the hypothalami, two thirds of nuclei were identified as neuronal. Across the dataset, there was relatively low expression of immediate early genes (IEGs), which could evidence support for the successful minimisation of experimentally induced stress during the hyperinsulinaemic clamp technique. Numerous genes that were differentially expressed under hypoglycaemic or hyperglycaemic conditions were identified. Increased understanding of the glucose responsive transcriptome in health will provide the basis for subsequent investigations into changes that take place in pathophysiological states, such as impaired awareness of hypoglycaemia (IAH). IAH resulting from recurrent exposure to hypoglycaemia (RH) remains a major obstacle when intensive insulin therapy is used to treat diabetes. RH can cause attenuation of the usual protective physiological (hormonal and behavioural) responses, termed hypoglycaemia associated autonomic failure (HAAF). HAAF has been reproduced experimentally in both humans and rats, however a mouse model of HAAF has not yet been comprehensively validated. In this thesis I found that four consecutive days of hypoglycaemia in mice was sufficient to significantly reduce hormonal responses to a subsequent hypoglycaemic episode, and therefore create a murine model of HAAF. Using this 4-day HAAF model I examined glucoprivic feeding to assess hunger, as an important symptom of hypoglycaemia awareness, and found a non-significant trend towards attenuation of the feeding response following HAAF induction. I also began to probe the neurocircuitry of IAH through designing and piloting an experimental paradigm to investigate restoration of HAAF. Validation of a murine HAAF model is essential to ensure future basic research is reproducible and accurately reflects the clinical condition. This will allow application of powerful modern molecular techniques to investigate neurocircuitry implicated in HAAF development. Improved mechanistic understanding of HAAF will facilitate the identification of potential therapeutic targets to either prevent the loss of, or restore, awareness to hypoglycaemia in the clinic.
  • ItemOpen Access
    Characterisation of human enteroendocrine cells in organoid models
    Miedzybrodzka, Emily
    Gut hormones secreted by enteroendocrine cells (EECs) coordinate the postprandial response to a meal by regulating digestion, absorption, circulating nutrient availability and satiety. EECs are specialised endocrine cells scattered throughout the gastrointestinal epithelium that release hormones in response to a wide range of luminal and systemic signals. This thesis focused on characterising the secretory mechanisms of three important gut hormones: glucagon-like peptide 1 (GLP-1, which has potent insulinotropic and anorexigenic effects), motilin (a key regulator of gut motility which is not expressed in rodent models) and serotonin (5-HT, a monoamine neurotransmitter with wide-ranging effects within and outside the GI tract). Several in vitro models have been developed to study the mechanisms of EEC regulation; however, at the outset of this project, it was not possible to identify human EECs without prior fixation and immunostaining which precluded functional studies. We therefore used CRISPR-Cas9-mediated knock-in to generate human intestinal organoids with fluorescently labelled EECs. Organoids are three dimensional self-renewing cultures grown from the adult stem cells of intestinal crypts which express mature epithelial cell types, including EECs. The properties of labelled human organoid EECs were investigated using a combination of fluorescence-activated cell sorting (FACS), bulk RNA sequencing, gut hormone secretion assays, single cell Ca²⁺ imaging, immunohistochemistry and peptidomics. GLU-Venus ileal organoids were first used to explore the mechanisms regulating GLP-1 release from human L-cells. We optimised protocols for the study and differentiation of EECs in human organoid culture, and used these to determine that human L-cells are highly phenotypically similar to their murine counterparts. GLP-1 secretion was stimulated by glucose, angiotensin II, arginine vasopressin and synthetic agonists of receptors for bile acids (GPBAR1), long chain fatty acids (FFA1) and monoacylglycerols (GPR119). Bulk RNA sequencing performed on human L-cells will guide future investigations. We next generated MLN-Venus and MLN-GCaMP7s duodenal organoids, and used these to carry out the first in-depth transcriptomic and functional characterisation of human motilin-expressing M-cells. Several receptors important for the postprandial and interdigestive regulation of motilin release were identified, including GPBAR1, FFA1 and GPR119. Acidification also stimulated acute M-cell activation, which was dependent on acid-sensing ion channels (ASICs), and ASIC-independent motilin secretion. To study 5-HT secretion from intestinal enterochromaffin cells (ECs), organoids with labelled tryptophan hydroxylase 1 expressing cells (TPH1-Venus) were established from human duodenum, ileum and rectum. Bulk RNA sequencing was performed on duodenal ECs, and considered in parallel with single cell RNA sequencing data of small intestinal EECs (isolated based on chromogranin A expression). Several highly enriched receptors were identified and initial Ca²⁺ imaging data suggested a possible stimulatory role for the short chain fatty acid acetate and the aromatic amino acid tryptophan in some duodenal ECs. Finally, we developed new methods and tools which are expected to prove useful for future studies of human EEC function. Organoids expressing an L-cell localised cyclic AMP sensor will enable intracellular responses to a wider range of stimuli to be monitored. We also established robust protocols to knockout genes of interest in human organoid cultures, which will allow for the detailed investigation of factors regulating EEC activation and differentiation. In summary, this thesis has demonstrated the utility of CRISPR-Cas9-modified intestinal organoid models for the study of stimulus-secretion coupling in human EECs, and opened several new avenues for further investigation. We hope that the insights obtained will improve our understanding of enteroendocrine physiology and the role of gut hormone therapies in the treatment of type 2 diabetes, obesity and motility disorders.
  • ItemOpen Access
    Investigating the impact of non-alcoholic fatty liver disease on the pathogenesis and progression of heart failure with preserved ejection fraction
    Leggat, Jennifer
    Heart failure with preserved ejection fraction (HFpEF) is a growing public health concern due to its increasing incidence alongside a lack of effective therapeutics. Understanding how this condition develops is therefore of the utmost importance in order to develop preventative strategies. As such, investigation of the cardiac functional effects of HFpEF risk factors is crucial; one such risk factor is non-alcoholic fatty liver disease (NAFLD). NAFLD has been shown to both predispose individuals to HFpEF and to increase HFpEF severity, independently of cardiovascular risk factors. However, whether the coexistence of these conditions is mediated independently by comorbid metabolic dysregulation, or whether the steatotic liver itself can exert direct cardiotoxic effects, is as yet unknown. Accordingly, the aims of this thesis are to establish whether hepatic steatosis in itself is sufficient to detrimentally impact myocardial diastolic function, and to investigate mechanisms by which this might occur. In Chapter 3, the association between NAFLD and HFpEF is established in a murine model, demonstrating that murine models are suitable for use in this research. In Chapter 4, appropriate models of specific hepatic steatosis and diastolic dysfunction are developed for use in Chapter 5, in which we demonstrate that hepatic steatosis is sufficient to exacerbate both surgically- and chemically-induced diastolic dysfunction. Potential mechanisms underlying this association are investigated in Chapter 6, in which we demonstrate that factors secreted by fatty hepatocytes are capable of altering cardiomyocyte physiology. We further identify that diastolic dysfunction in NAFLD is associated with increased myocardial lipogenic lipid content, and demonstrate that altering myocardial lipid composition is sufficient to alter cardiomyocyte morphology and function. In Chapter 7, we demonstrate that reducing hepatic de novo lipogenesis attenuates diastolic dysfunction, in association with reduced serum lipids and reduced myocardial lipogenic lipid content, suggesting that hepatic steatosis may drive dyslipidaemia which may alter myocardial lipid handling, resulting in diastolic dysfunction. Together, these data demonstrate that hepatic steatosis can directly contribute to the pathogenesis of cardiac dysfunction, and allude to dyslipidaemia and cardiac lipid mishandling as potential mediators of liver-heart cross-talk in the context of NAFLD and HFpEF. Following further study to fully characterise this mechanism, this work could therefore facilitate the future development of targeted therapies against dyslipidaemia to prevent the pathogenesis of HFpEF in NAFLD patients.
  • ItemOpen Access
    The effect of maternal obesity/GDM and metformin intervention on maternal, placental and fetal health
    Hufnagel, Antonia Sophie
    In many populations worldwide over 50% of women have a body mass index above 25. Therefore, an increasing number of women enter pregnancy overweight or obese. This increases the risk for pregnancy complications such as Gestational Diabetes Mellitus (GDM). Placental function is often affected in obese and GDM pregnancies, which affects fetal growth and development. Recent years have implicated placental extracellular vesicles (EV) in fetal-maternal communication, with their microRNA (miRNA) content affecting maternal metabolism and potentially the fetus. Overall, it is well-established that obese and glucose-intolerant pregnancies have short- and long-term health consequences for mother and child. Women with GDM are therefore treated to control maternal glycaemia and thereby prevent effects on the fetus caused by excessive glucose exposure. In many countries metformin (an oral glucose-lowering agent) is now the first line pharmacological treatment for GDM as it can control maternal glycaemia and reduce gestational weight gain. However, metformin can cross the placenta and thereby directly affect the fetus. A few studies in humans and in animal models have reported increased adiposity in offspring exposed to metformin during pregnancy. However, information regarding the immediate actions of metformin on the placenta and fetus is limited. This thesis therefore aimed to assess the impact of metformin treatment of an obese and glucose-intolerant pregnancy in a mouse model on (i) the mother (described in chapter 3), (ii) fetal growth and placental structure (described in chapter 4) and (iii) on the placental lipidome and transcriptome (described in chapter 5). A last aim of this thesis was (iv) the establishment of isolation of placental EVs and characterisation of their miRNA content in an obese and glucose-intolerant pregnancy (described in chapter 6). In this thesis it is shown (chapter 3) that feeding mice a diet high in sugar and fat prior to and throughout pregnancy increased maternal fat mass and impaired glucose tolerance. Additionally, a preeclampsia-like phenotype with impaired uterine artery compliance and increased serum sFlt levels was induced. All these parameters were improved by treatment of the dams with metformin at clinically relevant doses prior to mating and throughout pregnancy. These findings are consistent with metformin being beneficial for maternal metabolic health, as observed in human studies, including recent data highlighting the potential of metformin to prevent preeclampsia. Chapter 4 highlights that placentas from obese dams had calcium depositions and a reduced labyrinthine zone. Calcium deposits have been observed previously in pregnancies complicated by GDM and obesity, showing that our model resembles the human situation. The obesity-induced placental pathologies together with the reduced uterine artery supply likely led to the fetal growth restriction observed. Metformin treatment did not rescue any of these obesity-induced changes in the fetus and the placenta. Metformin crossed the placenta and entered the fetal circulation at levels equivalent to maternal concentrations and was also taken up into fetal tissues. Metformin can therefore exert direct effects on the placenta and/or the fetus that may explain why fetal growth restriction and placental impairments are not rescued by metformin despite the improvement in uterine artery compliance. Reduced body weight in babies exposed to metformin in utero has previously been shown in human studies. Metformin did not affect placental AMPK and mTOR signalling as previously reported, but increased apoptosis markers in the male placenta. As the placenta accumulated substantial levels of metformin and is the key interface between mother and fetus we investigated in chapter 5 effects of metformin on the lipidome and transcriptome by comparing obese untreated and obese metformin-treated placentas. Metformin reduced triglycerides with low carbon numbers and free carnitine. A reduction of carnitine has previously been linked to preterm birth. Additionally, phosphatidylserines (PS) and sphingosine (18:0) were increased in the male placenta and lyso-phosphatidylcholine was reduced in the female placenta upon metformin treatment. A few human studies reported previously increased rates of preterm births in pregnancies exposed to metformin, thereby the reduced carnitine levels together with increased PS levels that could be linked to apoptosis in the male placenta warrant further investigation. Placental transcriptome analysis identified no major changes in mRNA expression upon metformin exposure but highlighted overall sex differences within the placenta that were consistent with increased in utero vulnerability of male fetuses to maternal obesity. Lastly, chapter 6 of this thesis shows the feasibility of extracting placental EVs released from placental explants. In a pilot study EVs and their miRNA cargo from male control placentas were compared to those from male obese placentas. miRNAs previously identified as playing a role in IGF2 signalling, mitochondrial dysfunction and preeclampsia were altered in obese placental EVs. Therefore, further follow-up could identify the role of placental EVs in matching maternal supply and fetal demand via modulating IGF2 signalling and could add evidence for their use as biomarkers to identify impaired placental function and preeclampsia. Overall, this thesis shows beneficial effects of metformin on maternal metabolic health and adds evidence to the current discussion of metformin as a preeclampsia treatment. However, it also highlights that metformin can have direct effects on the fetus and the placenta, with some evidence that at least some of these effects are sexually dimorphic. The use of metformin in pregnancy is complex and might be good for some women where beneficial effects of metformin in pregnancy might outweigh potential short-and long-term effects on the offspring. However, this might not be the case in other women. Therefore identifying treatment strategies/metformin formulations that retain maternal benefits whilst reducing fetal exposure should be the important goal of future studies.
  • ItemOpen Access
    A systems biology approach to the pathogenesis and progression of Non-Alcoholic Fatty Liver Disease
    Kamzolas, Ioannis
    Non-alcoholic fatty liver disease (NAFLD) refers to a spectrum of diseases ranging from simple steatosis (isolated hepatic fat accumulation) to steatohepatitis (NASH; hepatic fat accumulation with lipotoxicity, hepatic cell damage and inflammation), eventually progressing to fibrosis, cirrhosis and potentially hepatocellular carcinoma. NAFLD is a common disease highly associated with the Metabolic Syndrome. Hence, it has been rising in prevalence in parallel with the increasing incidence of diabetes and obesity. The transition between the different stages of the disease could be a discriminant for a benign prognosis or a higher mortality risk due to cardiovascular or chronic liver disease. The diagnosis of NAFLD can be based on imaging studies (e.g., ultrasound technology or MRI spectroscopy). However, the specific stage of the disease, the presence of hepatocellular damage, inflammation, and amount of fibrosis in the liver can be detected only by biopsies. Such invasive methods cannot be applied outside specialist practice, present a significant risk of complications and are subject to sampling error; thus, they constitute an imperfect gold standard. To date, there is a lack of 1) tractable non-invasive biomarkers that could aid the diagnosis, risk stratification and monitoring of patients; 2) approved therapies; 3) reliable pre-clinical models of the disease to test drug and biomarker effectiveness/accuracy. A better understanding of NAFLD/NASH pathophysiology in humans will help achieve identifying new targets, improving the management of NAFLD patients. During my PhD, I have integrated and analysed transcriptomics data from rodent models and human NAFLD patients of different stages of the disease to identify specific mechanisms that explain the progression between the different stages of the disease. I have also compared these data to preclinical disease models to rank them against human pathophysiology and point to the most desirable ones. The thesis is organised into six chapters. In addition to the “Introduction” and the “Final Discussion”, the four “Results” chapters address the following topics: 1. Molecular characterisation of rodent models to determine their suitability for preclinical studies using human data as reference. Using an unbiased approach that allows ranking the murine models based on metabolic phenotyping, histology, and transcriptomics (compared to human data), I have ranked multiple preclinical models of NAFLD based on their “proximity to human disease”. My results suggest that rodents fed with diets enriched in refined carbohydrates, saturated lipids, and cholesterol (Western Diets), with/without sugar water (American Lifestyle approach) are the closest models to human NASH and, therefore, the most representative of human pathophysiology. Additionally, some genetic models of obesity (ob/ob, MC4r) augment liver damage induced by these diets, making them valuable tools to achieve more advanced disease stages and/or faster models. 2. Identification of the molecular landscape of NAFLD progression. All previous work describing NAFLD progression has been based on the division of the datasets in artificially defined, discrete disease stages. Here, I implemented a pseudotemporal ordering method that successfully captures the disease trajectory in a continuum. Based on the pathway enrichment and upstream regulator analysis results, I show that my analysis matches the results of the discrete approach supervised by histology, providing additional granularity and better defining the transition among disease stages. An expression-module-based analysis also defined relevant processes representing the molecular signature of the disease during its progression. Using Random Forest models, I identified a list of genes predictive of the disease progression; intriguingly, this list of hits features multiple drivers of NASH well characterised mechanistically plus novel targets, worth future investigation. I expect this detailed molecular profile of NAFLD progression to help understand the mechanisms underpinning NASH progression and improve the stratification of patients and the ranking of pre-clinical models. 3. Comparison of NAFLD progression for patients with and without Type 2 diabetes. Using transcriptomics data of NAFLD patients with and without diabetes, the aim is to answer whether there are differences between the two populations in NAFLD progression. Utilising the pseudotemporal ordering approach mentioned above, I identified specific T2DM-associated biological processes that due to the complex nature of the disease and the limitations of relatively small human datasets, could not be identified with standard bioinformatics approaches. 4. Contribution of MBOAT7 and INSIG1 in NAFLD progression in mice and humans. This chapter provides a proof of concept of how transcriptomic studies can be reverse-translated into mice to study the contribution of the targets I identified in mechanisms of NASH progression. Here I show that the partial genetic ablation of MBOAT7 alters the hepatic transcriptome and the dependence of these effects on the dietary challenge. Additionally, I show that an INSIG1 ablation induces an increased lipid remodelling and cholesterol biosynthesis, acting as a protective mechanism that prevents NASH progression. Overall, my work paves the way for a better understanding of the NAFLD disease progression and defines new approaches to study NASH with system biology and translational approaches, exploiting novel methods that had never been used in NAFLD research.
  • ItemOpen Access
    The role of the mitofusins in adipose tissue
    Mann, Jake; Mann, Jake [0000-0002-4711-9215]
    Monogenic mitochondrial disease is known to cause tissue-selective manifestations, typically affecting the nervous system, liver, and muscle. However, until recently, there was relatively little human genetic evidence for a role of mitochondria in disorders of energy balance (i.e. weight) or insulin action. Several groups have identified that biallelic p.Arg707Trp mutations in MFN2 (MFN2R707W) cause a striking leptin-deficient partial lipodystrophy syndrome. It is not known why this mutation affects adipose tissue when other mutations in MFN2 cause a peripheral neuropathy. More generally, it is not clear how mitochondrial dynamics affect adipogenesis and adipokine secretion. Therefore, we aimed to broaden and deepen our understanding of the roles of the mitofusins (MFN1 and MFN2) in adipose tissue by: (i) assessing the role of the mitofusins on murine in vitro models of adipogenesis, and (ii) examining the phenotype of a Mfn2R707W knock-in mouse model. Wild-type (WT), Mfn1-/-, and Mfn2-/-, mouse embryonic fibroblasts (MEFs) were differentiated into mature adipocytes. Despite the disruption of their mitochondrial morphology, Mfn1-/- MEFs manifested a marked increase in adipogenesis relative to WT or Mfn2-/- MEFs, as reflected by increased lipid accumulation, expression of mature adipocyte markers (Plin1, Glut4, Adipoq), and insulin-stimulated glucose uptake. In contrast, though Mfn2-/- MEFs accumulated similar amounts of lipid to wild-type cells, they showed reduced PPARG and GLUT4 expression with minimal insulin-stimulated glucose uptake. RNA sequencing revealed a pro-adipogenic transcriptional profile in Mfn1-/- MEFs including upregulation of PPARG. These findings were replicated using siRNA knock-down in 3T3-L1s, where knock-down of Mfn1 was associated with a marked increase in lipid accumulation and knock-down of Mfn2 caused a reduction in expression of Pparg, Glut4, and Plin1. Homozygous Mfn2 p.Arg707Trp knock-in (KI) mice were generated through CRISPR-Cas9 mutagenesis. Male mice were fed either a chow diet (CD) or a 45% high-fat diet (HFD) for up to 50 weeks. Mfn2 was expressed at similar levels in wild-type (WT) and KI animals. Mitochondria were assessed morphologically using transition electron microscopy (TEM), which showed that mitochondria from brown adipose tissue (BAT) and white adipose tissue (WAT) were more circular with no morphological change in non-adipose tissues. There was no difference in body weight, fat mass, or glucose tolerance in KI but there was evidence of activation of the integrated stress response in WAT and BAT only. KI had lower leptin and adiponectin in their serum and at mRNA level, which was replicated in adipose explants. In conclusion, loss of Mfn1 but not Mfn2, results in enhanced adipogenesis due to a combination of increased Pparg expression and a pro-adipogenic transcriptional profile. Mice homozygous for Mfn2 p.Arg707Trp show an adipose-specific mitochondrial stress response with low leptin, which is partially transcriptionally mediated. These findings underscore the importance of mitochondrial dynamics in adipose tissue function.
  • ItemOpen Access
    Optimisation of molecular imaging using 11C-methionine PET in the detection of pituitary adenomas
    Bashari, Waiel; Bashari, Waiel [0000-0002-2204-9169]
    Decision-making in pituitary disease is critically dependent on high-quality imaging of the sella and parasellar region. Magnetic resonance imaging (MRI) is the investigation of choice, and for the majority of patients combined T1 and T2 weighted sequences provide the information required to allow surgery, radiotherapy (RT) and/or medical therapy to be planned and long-term outcomes to be monitored. However, in some cases standard MRI sequences are indeterminate and additional information is needed to help inform the choice of therapy for a pituitary adenoma (PA). Pituitary molecular imaging using 11C-methionine positron emission tomography (11C-met-PET) is well described. This thesis adds to the existing literature reporting potential roles for molecular imaging in patients with different subtypes of PA. Specifically, it describes a series of novel approaches to pituitary imaging using 11C-met-PET with particular focus on optimisation to facilitate detection of occult PAs, and thereby allow definitive treatment to be recommended for a subset of patients who would otherwise be deemed unsuitable for surgery and/or RT. The first chapter provides an overview of pituitary adenomas and the modalities used in pituitary imaging, with emphasis on molecular imaging approaches described to date. In the second chapter, methods commonly applied during the conduct of the different research studies are described and, when required, these are further considered in each subsequent chapter. Chapter three reports findings from two retrospective cohort studies examining the potential roles of of 11C-met-PET in patients harbouring prolactin-secreting PAs, and in patients with residual acromegaly due to parasellar disease extension. In chapters four and five, two prospective studies describe novel approaches to image optimisation using biochemical suppression of 11C-met-PET signal in normal, and in adenomatous pituitary tissue, respectively. In the chapter six, results from a prospective study involving 15 patients with different types of PAs undergoing hybrid 11C-met-PET/MR are reported. The final chapter (seven) draws the various study findings together and considers how these can inform the design of future research projects and adoption by the NHS.
  • ItemOpen Access
    Novel functions of N-acetyltransferase 10 (NAT10) in DNA repair and replication with potential implications for premature ageing syndromes
    Lam, Jonathan
    Hutchinson-Gilford Progeria Syndrome (HGPS) is an invariably fatal disease with a range of diverse symptoms that are normally associated with those of advanced age. In recent decades, clinicians and scientists together have made great progress in deriving the mechanisms of disease initiation and progression. Nonetheless, the discovery of a cure remains elusive. Inhibition of the acetyltransferase activity of the N-acetyltransferase 10 (NAT10) protein has been shown to have profoundly positive effects on both cellular and mouse models of premature ageing; however, the pathways it is involved in remained largely unexplored. This project set out to identify, characterise and derive mechanistic detail surrounding the functions of NAT10 to better determine its cellular function and potential candidacy as a drug target in HGPS. To this aim, I set out to investigate both the interactome and acetylome of NAT10. While the latter approach was unsuccessful, my interactome studies were able to identify several hits of potential interest that had not been previously identified in the literature. This project focussed particularly on a putative interaction of NAT10 with the complex of three proteins: MRE11, RAD50 and NBS1/NBN (MRN complex). This complex plays an important role in the sensing and repair of DNA double-strand breaks (DSBs), highly genotoxic lesions that can result in unrestrained cellular lethality or genomic instability. During this project, I showed that NAT10 also plays a role in the repair of DSBs. Specifically, I observed that NAT10 localises to DSBs, dependent on the activity of the protein PARP1, and promotes repair by one of the main DSB repair pathways, homologous recombination, most probably by allowing the initial MRE11-mediated resection step to occur. Supporting this repair-promoting role, I also showed that depleting NAT10 leads to increased cellular sensitivity to a range of genotoxic agents. In searching for direct NAT10 acetyltransferase substrates, I showed that NAT10 does not acetylate MRN components. Instead, I observed that NAT10 acetylates PARP1, corroborating recently published findings from others. Among other roles, PARP1 is known to modify the chromatin state around DSBs. As I showed that NAT10 depletion prevents chromatin decompaction after DNA damage induction, my hypothesis is that NAT10-mediated acetylation of PARP1 is involved in chromatin decompaction to allow DNA repair. This is currently under investigation in the lab. While investigating homologous recombination, I observed that NAT10 depletion leads to an accumulation of cells in S-phase of the cell cycle. This was likely due to increased replication stress in these cells, as I observed global replication fork slowing and increased chromosomal abnormalities after NAT10 depletion. I also showed that NAT10 interacts with the replication fork component PCNA. Finally, I attempted to connect these novel pathways to HGPS, but it is still unclear how or whether inhibiting these specific functions of NAT10 might be beneficial for HGPS.
  • ItemOpen Access
    Functional validation of human rare missense variants associated with body fat distribution and cardiometabolic risk
    Dong, Liang
    Body fat distribution is a predictor of metabolic and cardiovascular disease independent of body mass index (BMI) and is heritable. To understand the genetic determinants of the differences observed in fat distribution in the general population, genome-wide association studies (GWAS) have emerged as an extremely useful approach and have convincingly associated many single nucleotide polymorphisms (SNPs) with body fat distribution. Unfortunately, it has proven very difficult to link specific genes or genetic variants with these findings, reducing their translational impact. However, massive increases in the size of human genetic studies have increased the power of studies to confidently link rare missense (coding) variants with a range of phenotypes. In collaboration with colleagues in the MRC Epidemiology Unit in Cambridge, we recently identified rare missense variants in ALK7 (p.I195T and p.N150H), CALCRL (p.L87P), PLIN1 (p.L90P) and PDE3B (p.R783X) which were associated with a favourable body fat distribution and with protection from cardiometabolic disease, while a variant in gene PNPLA2 (p.N252K) was associated with an adverse phenotype (1). To understand the roles of these genes, initial loss-of-function studies were carried out using siRNA-mediated knockdown in 3T3-L1 (pre)adipocytes, as all the genes are expressed in adipose tissue and have been at least loosely implicated in the regulation of intracellular lipolysis. In this cell model, Plin1 depletion resulted in an increase in basal lipolysis while isoproterenol stimulated lipolysis was suppressed. Pnpla2 depletion, on the other hand, impaired lipolysis under both basal and stimulated conditions. Alk7 or Calcrl knockdown resulted in a consistent decrease in lipolysis while the impact on adipogenesis was variable. Depletion of Pde3b did not have any impact on adipogenesis or lipolysis. Subsequent studies focussed on functional analysis of a subset of the specific missense variants. mRNA expression of the PNPLA2 p.N252K variant was lower in the heterozygous carriers of the mutant allele than in homozygous wild-type allele carriers, although notably the variant retained its ability to target lipid droplets and also its catalytic activity. Functional analysis of the ALK7 variants indicated that the p.I195T variant failed to initiate signal transduction upon stimulation with known ligand Nodal, while the impact of the p.N150H mutant was far more subtle. Meanwhile, the PLIN1 p.L90P variant seemed to manifest a stronger interaction with HSL compared to wild type in the basal state. However, further investigation suggested that this variant suppressed stimulated lipolysis while the ability to promote lipid accumulation was unaffected. Collectively, these findings provide some early insights into the potential impact of variants convincingly linked with altered body fat distribution and metabolic disease risk, but further work is required to more clearly understand exactly how they affect adipogenesis and/or lipolysis and thereby modify fat distribution and disease risk in vivo.