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Mycoprotein is a sustainably-produced, fungal-derived dietary protein source and its consumption has been shown to improve acute postprandial glycaemic responses and decrease circulating cholesterol concentrations. However, whether these findings translate to habitual mycoprotein consumption improving physiologically relevant, longer-term changes in insulin sensitivity (IS) and/or glycaemic control has not yet been investigated. Mycoprotein is naturally rich in RNA-derived nucleotides (~10 g per 100 g dry weight), but heat-treated during production to reduce RNA content (to under 2 g per 100 g dry weight), for commercial products to comply with FAO/WHO/UNICEF recommendations limiting the additional dietary nucleic acid load from single-cell protein-rich novel foods to 2 g/day. These recommendations are based on data showing that short term ingestion of high-dose (> 2 g/day) isolated (or yeast-derived) nucleotides results in elevations in circulating uric acid concentrations above clinically acceptable levels (i.e. of > 420 µmol.L-1). Epidemiological and observational studies have reported that serum uric acid concentrations positively correlate with the development of gout, hypertension and metabolic syndrome, and are a predictor of type 2 diabetes, though causal links remain to be established. Despite these concerns regarding dietary nucleotides, serum uric acid concentrations and metabolic health, evidence supports an increased dietary requirement for nucleotides during periods of rapid growth (e.g. infancy) and stress, such as in certain disease states. Muscular exercise is one of the most common states of physiological stress and emerging data imply beneficial effects of dietary nucleotide supplementation on exercise performance and recovery. Dietary nucleotide supplementation has been shown to reduce post-exercise stress hormone response, improve markers of immune health, and result in improvements to muscular strength, force production and time to exhaustion. It has been suggested that dietary nucleotides might positively affect energy production but studies have not yet examined this. Increased production of ATP modulated by dietary nucleotide intake prior to submaximal moderate-intensity exercise might lead to improved exercise performance, potentially by sparing muscle glycogen. Additionally, these potential benefits to energy production might also lead to improved recovery following glycogen depleting exercise. Ribose, a pentose monosaccharide that makes up the structure of nucleotides, has also been proposed as a limiting factor in the production of ATP. As such, dietary ribose (like dietary nucleotide) intake might modulate fuel utilisation and improve energy production and exercise performance in a depleted state, as well as assist skeletal muscle cells in recovery from fatigue; but further research to establish these effects is necessary. The studies described in this thesis, performed in healthy human volunteers, have focused on two main topics. Firstly, the impact of both mycoprotein and dietary nucleotide intake on markers of metabolic health was assessed. Secondly, the potential for dietary nucleotides and ribose to impact muscle ATP and glycogen concentrations and endurance exercise muscle metabolism, performance and recovery. The major findings were that substituting meat/fish for mycoprotein twice daily for 1 week did not modulate whole-body IS or glycaemic control but resulted in changes to plasma lipid composition, the latter primarily consisting of a coordinated reduction in circulating cholesterol-containing lipoproteins. The ingestion of a nucleotide-rich mixed meal was found to increase serum uric acid concentrations for ~12 h but did not influence postprandial blood glucose or serum insulin concentrations. Accordingly, twice-daily consumption of high-nucleotide mycoprotein for one week led to sustained increases in serum uric acid concentrations (above clinically relevant thresholds), but not to any associated deleterious effects in IS, glycaemic control or plasma lipid composition. Finally, the twice-daily ingestion, for 2 weeks, of a nucleotide-rich mycoprotein drink, with or without added ribose, did not influence skeletal muscle ATP content, muscle fuel utilisation during exercise, exercise performance or the metabolic or performance-related recovery from exercise. The present thesis documents the first investigations into the potential impact of mycoprotein on IS and extends on previous observations of the beneficial effects of mycoprotein intake on blood lipid profile by demonstrating how rapidly these benefits ensue. Further novel findings were that high nucleotide intake, even when incorporated into mixed meals, leads to a cumulative increase in serum uric acid concentrations, which after three to five days become clinically significant. Despite this, our results clearly show measures of IS and glycaemic control remained unaffected. Furthermore, it is described in this thesis the first attempt to increase resting skeletal muscle ATP concentrations in humans using dietary nucleotide and ribose supplementation and to assess their impact in muscle fuel utilisation during fatiguing exercise and after acute recovery. The findings of this thesis have theoretical and practical applications regarding the use of sustainable alternative protein sources on the prevention and treatment of cardiometabolic risk factors and add valuable knowledge to our understanding of the role of dietary nucleotides and ribose in muscle fuel utilisation and exercise performance.

Obesity and/or diabetes during pregnancy is associated with obesity, metabolic and cardiovascular disease in the offspring. Clinically relevant interventions are required to break the transgenerational cycle of obesity. Metformin is used to treat GDM in the UK and RCTs are investigating metformin as a candidate for implementation in pregnancies complicated by obesity or polycystic ovary syndrome. However, metformin crosses the placenta and long-term offspring follow-up is sparse in both humans and animal models. Data throughout the life-course, investigation of potentially sexually dimorphic effects and cardiovascular outcomes are especially lacking. Using a mouse model, this thesis aimed to determine the short- and long-term effects on adiposity and metabolic health in male and female offspring in early life (neonatal growth trajectory, Chapter 3), young adulthood (8 weeks, Chapter 4) and older age (12 months, Chapter 5). It also aimed to investigate cardiovascular phenotypes in adult offspring at different points across the life-course (3, 6 and 12 months of age, Chapter 6). Exposure to an obesogenic diet from pre-conception and during pregnancy and lactation increased maternal bodyweight, fat mass and food intake. Maternal obesity introduced hypertrophic adiposity in both male and female offspring at 8 weeks of age and this increased adiposity was maintained until 6 months. After 6 months, sex differences emerged: male offspring of obese dams were heavier and showed hypertrophic and hyperplastic adiposity with adipose tissue inflammation and insulin resistance, whereas adiposity in female offspring became less prominent with age. Maternal metformin treatment decreased maternal fat mass and this difference was maintained until late pregnancy. The metformin intervention also increased gestation length and did not prevent the intrauterine growth restriction and catchup growth seen in offspring of obese pregnancy. In offspring, the metformin intervention did not correct the adiposity phenotype programmed by maternal obesity, but instead led to exaggerated adiposity and adipose tissue dysfunction in a sex- and a time-dependent manner. In both sexes, exposure to metformin during obese pregnancy caused adipocyte hypertrophy at 8 weeks of age prior to development of obesity. In males this was associated with adipocyte hyperplasia, inflammation and local insulin resistance. Although male offspring of control-fed and obese dams underwent age-related adipocyte hyperplasia, adipocyte number failed to increase in ageing metformin-exposed male offspring. Combined with more extensive macrophage infiltration and increased liver weight this leads to the hypothesis that adipose tissue expansion capacity is restricted in metformin-exposed males, resulting in adipose tissue dysfunction and ectopic lipid deposition. In female metformin-exposed offspring, adiposity in young adulthood was associated with alternate macrophage activation and an improvement in whole-body insulin sensitivity. However, after 6 months of age female metformin-exposed offspring diverged from other groups in viii body weight and fat mass, showing inflammatory adiposity at 12 months of age associated with both peripheral and systemic insulin resistance as well as hyperinsulinaemia. Maternal obesity also affected cardiovascular health of male and female offspring. In males, offspring of obese dams had diastolic dysfunction leading to pressure overload and hyperdynamic systolic function in absence of changes in blood pressure. In contrast, female offspring of obese dams had increased blood pressure throughout life associated with diastolic dysfunction at 3 months. The metformin intervention corrected some of the changes that were observed with maternal obesity. In male offspring, prenatal metformin halted progression of diastolic dysfunction with age but also introduced other independent cardiovascular changes. In female offspring, prenatal metformin corrected the hypertension seen in young offspring of obese dams, but by 12 months of age female metformin-exposed offspring had developed obesity-induced hypertension. In conclusion, this thesis reports sex- and age-specific effects of maternal obesity and prenatal metformin intervention, illustrating the need for short- and long-term follow-up of both male and female offspring exposed to prenatal interventions.

Non-alcoholic fatty liver disease (NAFLD) is associated with several metabolic comorbidities, and the excessive accumulation of hepatic lipids is strongly related to both hepatic and peripheral insulin resistance. The mechanisms underpinning the link between metabolic dysfunction and NAFLD, however, are not fully understood. Hepatokines such as fibroblast growth factor 21 (FGF21), follistatin, leukocyte cell-derived chemotaxin 2 (LECT2), fetuin-A, fetuin-B and selenoprotein P (SeP) have been identified as liver-secreted factors which may influence metabolic homeostasis in distal tissues. Energy balance plays a key role in the regulation of these hepatokines and further research is required in humans to determine the responsiveness of these proteins to acute exercise and dietary interventions. Hepatic lipid composition, which can now be assessed using proton magnetic resonance spectroscopy (1H- MRS), has also been highlighted as a potential factor impacting metabolic health in NAFLD. A hepatic lipid profile consisting of more saturated and less polyunsaturated lipids is thought to be more metabolically harmful, and the relationship between hepatic lipid composition and metabolic dysfunction in NAFLD requires further characterisation. Chapter 3 of this thesis demonstrates that a single bout of continuous, aerobic exercise transiently increases circulating FGF21 and follistatin concentrations in healthy men; with the FGF21 response being intensity-dependent. Chapters 4 and 5 show that circulating concentrations of FGF21, LECT2 and fetuin-A are elevated following short-term (1-7 days), high-fat overfeeding in healthy men; however, the time-course varies considerably between hepatokines. Specifically, the FGF21 response is rapid and transient; whilst the LECT2 response is more gradual and sustained over seven days. Initial increases in circulating fetuin- A may also begin to occur after seven days. Chapter 6 demonstrates that 1H-MRS at 3.0 tesla is a valid tool to non-invasively assess hepatic lipid composition in NAFLD; however, the technique may not be suitable for fat fractions below 5.56%. Using this methodology, Chapter 7 found that in men with NAFLD, the hepatic lipid composition of individuals with impaired glucose regulation consists of a greater proportion of saturated and a lower proportion of unsaturated/polyunsaturated lipids compared to individuals with normal glucose regulation. Furthermore, this hepatic lipid profile is associated with a lower cardiorespiratory fitness and lower levels of daily physical activity. Collectively, the studies in this thesis demonstrate that hepatokines such as FGF21, follistatin, LECT2 and fetuin-A are sensitive to acute perturbations in energy balance induced through exercise and/or overnutrition. Furthermore, H-MRS is a valid tool to assess hepatic lipid composition in NAFLD, which appears to be more saturated and less polyunsaturated in those with impaired glucose regulation compared to those with normal glucose regulation. These findings may support a potential a role of these two factors (i.e. hepatokines and hepatic lipid composition) in the link between NAFLD and impaired metabolic health; however, further experimental evidence in humans is required. Future research should investigate the impact of more chronic lifestyle interventions on these factors to explore their potential as therapeutic targets for improving metabolic health in NAFLD.

Intermittent fasting may be an effective strategy for managing obesity and the associated dysfunction. It involves punctuating typical patterns of nutritional intake with scheduled periods of abstinence from all energy-providing nutrients. The few prior studies of this approach have yielded promising insights; yet there remains a dearth of knowledge regarding how intermittent fasting affects energy metabolism and health in humans, which this research sought to address. Initially, diurnal variations in subjective appetite ratings were established as a robust foundation upon which to design temporal nutritional interventions (Chapter 4). Specifically, appetite increased throughout the day to a peak in the evening, despite the apparent inversion of the accompanying rhythm in key regulatory peptides. This led to the development of a novel intermittent fasting intervention in which a complete fast was applied in alternating 24-hour periods for 20 days, with transitions from fasting to feeding occurring at 15:00 each day. In separate lean and overweight/obese cohorts, the impact of this diet, both eucaloric (i.e. complete refeeding in fed periods) and with a 25% calorie restriction (i.e. 50% refeeding during fed periods), was contrasted against a standard 25% calorie restriction. The experiments featured measures of postprandial metabolic responses and free-living physical activity (combined heart rate/accelerometry). In lean adults (Chapter 5), combining intermittent fasting with calorie restriction decreased physical activity thermogenesis relative to intermittent fasting or calorie restriction in isolation, largely due to reduced spontaneous activity during fasting. However, there were no improvements in metabolic health, whilst intermittent fasting also resulted in smaller declines in fat mass than daily calorie restriction. A similar pattern was seen for body composition in overweight/obese individuals (Chapter 6) but none of the interventions caused adaptive changes in energy expenditure. Instead, combining intermittent fasting with calorie restriction reduced postprandial insulinaemia and improved fasted and postprandial plasma lipid concentrations. Collectively, this suggests that the amount and timing of energy intake exert interactive effects on metabolism and health, with baseline adiposity being an important determinant of responses.

Obesity is the most extended metabolic alteration worldwide increasing the risk for the development of cardiometabolic alterations such as type 2 diabetes, hypertension, and dyslipidemia. Body mass index (BMI) remains the most frequently used tool for classifying patients with obesity, but it does not accurately reflect body adiposity. In this document we review classical and new classification systems for phenotyping the obesities. Greater accuracy of and accessibility to body composition techniques at the same time as increased knowledge and use of cardiometabolic risk factors is leading to a more refined phenotyping of patients with obesity. It is time to incorporate these advances into routine clinical practice to better diagnose overweight and obesity, and to optimize the treatment of patients living with obesity.

Over forty percent of eastern Kentucky residents are classified as obese. From a biomedical perspective, obesity is linked to a range of chronic diseases, including heart disease, diabetes, and high blood pressure and is caused by particular lifestyle behaviors that lead to an increase in calorie consumption and decrease in calorie expenditure. However, these links – individual behavior leads to obesity which leads to chronic disease - do not take into account a wide range of personal, social, environmental, political, and economic conditions. In addition to the assumptions of what it means to become and be obese, Kentucky is regularly ranked among the unhealthiest states in the country. In other words, assumptions are made about how people live based on their bodies as well as their geographical location. This project explores some basic questions that form the bedrock of these assumptions. I discuss three food aid programs that developed in response to the Covid-19 pandemic in the spring and summer of 2020. Participants received boxes of (mostly processed) foods and expressed both gratitude and frustration. Standardized, shelf stable food, after all, is not the most nutrient dense and often contains one or more allergens. Boxes provided food during a time of fear and uncertainty but undercut the agency of families to determine their own food needs. Next, I dive into the metabolic and anthropometric data collected over three years (2017-2019) from twenty-five participants in a fruit and vegetable walking program. Positive patterns emerged from the data, specifically concerning measures related to diabetes and heart disease. Biology is influenced by cultural products, such as access to medical care and a living wage. To contextualize the walking program and its suggestive but limited findings, I present key concerns from walking program participants and other members of the community. Then I present another factor that contributes to health and disease – disordered eating. Disordered eating is a non-standardized term and phenomenon that affectively moves between bodies. Through a combination of a validated survey (the Eating Attitudes Test) and in-depth interviews, I explore what disordered eating is, how it affects individuals and those around them, and from the various cultural and relational places it emerges. Finally, this project contests the narrative of absence that often overshadows nutritional studies of eastern Kentucky, specifically when it comes to food. Through interviews and farm visits, I paint an alternative portrait of eastern Kentucky as overflowing with gardens and fresh produce in order to encourage a turn towards food sovereignty in program development and implementation.

Polycystic ovary syndrome (PCOS) is a common and complex endocrinopathy with significant metabolic and reproductive manifestations, carrying a major health and economic burden. Exercise has consistently been found to improve clinical outcomes, but shortfalls with exercise prescription are evident, with little known about the impact of exercise intensity for improving health outcomes. Research suggests that high-intensity interval training (HIIT) is feasible, well tolerated and enjoyable for people with, or at risk of, chronic disease and can address many of the shortfalls and barriers to exercise participation. Despite this, there is limited research exploring the efficacy of HIIT in comparison to standard moderate-intensity exercise recommendations for improving the health of women with PCOS. Therefore, the optimal exercise intensity for improving health outcomes remains unknown. The overall aim of this thesis was to determine the effectiveness of exercise, and more specifically exercise intensity, on the metabolic, reproductive and mental health and health-related quality of life of women with PCOS. The first two studies of my thesis were systematic reviews (Chapters 2 and 3), one that included a meta- analysis, to summarise and evaluate the current exercise intervention literature on key clinical metabolic, reproductive and anthropometric outcomes and on mental health and health-related quality of life (HRQoL) in women with PCOS. Briefly, results from these reviews suggested that exercise is beneficial for improving both the physical and mental health of women with PCOS. Furthermore, exercise of vigorous intensities provided additional benefits for increasing cardiorespiratory fitness, body composition and insulin resistance. There was insufficient evidence to be able to analyse the impact of exercise intensity on mental health and HRQoL, however, the available evidence indicated that exercise is effective for improving health-related quality of life and PCOS symptom distress. Exercise also shows some efficacy for improving symptoms and/or prevalence of depression and anxiety in women with PCOS. Following this, a protocol for a clinical trial was designed (Chapter 4), and conducted to investigate whether HIIT could confer greater benefits than standard moderate intensity continuous training (MICT) on cardio-metabolic outcomes (Chapter 5), reproductive outcomes (Chapter 6) and mental health and HRQoL (Chapter 7) in overweight women with PCOS. To do this, we employed a two-arm, randomised clinical trial where twenty-four overweight women, aged 18-45 years with diagnosed PCOS were randomised to either MICT (60-75% peak HR [HRpeak]) or HIIT (>90% HRpeak). We had 13 women complete the HIIT intervention and 11 women complete the MICT intervention. In regards to cardio-metabolic outcomes, both HIIT and MICT improved VO2peak (HIIT; 23.4 ± 10.1%, P

Abstract: Fermented dairy products such as kefir have long been associated with a host of health benefits; however, the role that microbial composition has in these benefits has not been properly examined. This research aimed to examine how kefir impacts host metabolic health, as well as how alterations to the microbial populations of the kefir impacts these health benefits and the fermentation dynamics. In order to examine how kefir impacts metabolic health, four traditional and one commercial kefir were administered to mice using a high fat diet-induced obesity model. Two traditional kefirs decreased weight gain and plasma cholesterol levels in mice and one also lowered liver triglycerides. Conversely, commercial kefir had no beneficial effect. Improvements to liver triglycerides corresponded with decreases in the expression of fatty acid synthase, a gene involved in liver lipogenesis. This study shows that traditional kefir has the potential for improving metabolic dysfunction associated with obesity and indicates that differences in kefir microbial populations may influence the ability of traditional kefir to positively impact host metabolic health. As the metabolic health benefits associated with kefir were only associated with traditional examples, we set out to create a kefir product that utilized microbes from traditional kefir while maintaining a production method that is viable on an industrial scale. This was accomplished through the isolation of a large collection of organisms from a variety of traditional kefirs representing the most abundant species. Characterization of the isolates was carried out, and individual isolates were selected for use in the pitched kefir based on the characteristics of both the isolate and the kefir from which it was isolated. This proof of concept study showed that it is possible for traditional kefir organisms to ferment milk outside of a kefir grain, and that the resulting product resembles kefir in microbial density and pH. Following the development of the novel pitched kefir, we investigated the ability of this pitched culture kefir to recapitulate health benefits observed with a specific traditional kefir. This was accomplished using a mouse model of diet induced obesity. Additionally, we examined how differences in the microbial composition of kefir impacted these benefits. Both the traditional kefir and its pitched culture equivalent decreased plasma cholesterol and liver triglyceride levels by similar amounts when compared to commercial kefir. Furthermore, a pitched kefir produced without yeast and pitched kefir produced without lactobacilli did not show cholesterol-lowering effects. The traditional and pitched kefir with the full complement of microbes were able to impart corresponding decreases in the expression of multiple cholesterol and lipid metabolism genes in the liver. These results show that traditional kefir organisms can be utilized to create a more health promoting commercial kefir, while also highlighting the importance of microbial interactions during fermentation in the ability of fermented functional foods to beneficially impact host health. To better understand how alterations to the microbial composition of kefir fermentations impact the dynamics of the fermentation, we used two-dimensional gas chromatography and time-of-flight mass spectrometry (GCxGC-TOFMS) to examine the metabolite profiles of traditional and pitched culture kefir, including pitched culture kefirs that had the yeast or lactobacilli removed. Interestingly, despite similarities in their ability to improve host metabolic health, the Pitch and traditional kefir differed significantly in the profile of metabolites present, especially in yeast associated metabolites such as ethanol and esters. In addition, the removal of lactobacilli from the Pitch fermentation resulted in lower levels of organic acid formation during fermentation, while the removal of yeast appeared to result in little alteration in the metabolite profile. This thesis provides insight into how microbial composition impacts kefir fermentation, and presents a new strategy for the development of fermented functional foods.

Americans are exposed to multiple per- and polyfluoroalkyl substances (PFAS), chemicals used in consumer products including stain-resistant treatment and food packaging. Strong evidence of health effects from high exposures to these chemicals has recently emerged, but there is less information about the health effects of typical exposure levels. In Chapter 1, we tested the extent to which early pregnancy PFAS concentrations were associated with gestational weight gain and postpartum weight changes, using 1614 women in the Project Viva pre-birth cohort. Women gained more weight during pregnancy per doubling of 2-(N-ethyl-perfluorooctane sulfonamide) acetic acid (EtFOSAA), retained more weight at one year postpartum per doubling of perfluorooctanoic acid (PFOA), and gained more weight at three years postpartum per doubling in PFOA. We concluded that concentrations of certain PFAS in pregnancy were associated with greater gestational weight gain, weight retention, and weight gain after pregnancy. In Chapter 2, we examined the extent to which PFAS plasma concentrations during pregnancy were associated with postpartum anthropometry and biomarkers, again using participants in Project Viva. Higher pregnancy concentrations of EtFOSAA, PFOA, and perfluorooctanesulfonic acid (PFOS) were associated with greater adiposity, and higher PFOS was associated with higher systolic blood pressure at three years postpartum. We also reported that EtFOSAA concentrations were associated with biomarkers of inflammation and worse cardiovascular health at three years postpartum. We concluded that higher concentrations of PFOS, PFOA, and EtFOSAA were associated with a shift towards a higher-risk postpartum profile across measures of anthropometry, blood pressure, and biomarkers of cardiometabolic health. In Chapter 3, we tested whether PFAS concentrations were associated with metabolites previously shown to predict incident type 2 diabetes, using participants in the Diabetes Prevention Program. Higher concentrations of Sb-PFOA (branched isomer of PFOA) were associated with higher leucine and lower glycine, while higher total PFOA and n-PFOA (linear isomer of PFOA) were associated with lower glycine. Several PFAS were positively associated with multiple triacylglycerols, diacylglycerols, and glycerophospholipids. Our results suggest that PFAS may increase risk of type 2 diabetes through alterations in amino acid, glycerolipid and glycerophospholipid metabolism. Further research is needed to elucidate mechanisms.

High-throughput transcriptomic profiling has become a ubiquitous tool to assay an organism transcriptome and to characterize gene expression patterns in different cellular states or disease conditions, as well as in response to molecular and pharmacologic perturbations. Refinements to data preparation techniques have enabled integration of transcriptomic profiling into large-scale biomedical studies, generally devised to elucidate phenotypic factors contributing to transcriptional differences across a cohort of interest. Understanding these factors and the mechanisms through which they contribute to disease is a principal objective of numerous projects, such as The Cancer Genome Atlas and the Cancer Cell Line Encyclopedia. Additionally, transcriptomic profiling has been applied in toxicogenomic screening studies, which profile molecular responses of chemical perturbations in order to identify environmental toxicants and characterize their mechanisms-of-action. Further adoption of high-throughput transcriptomic profiling requires continued effort to improve and lower the costs of implementation. Accordingly, my dissertation work encompasses both the development and assessment of cost-effective RNA sequencing platforms, and of novel machine learning techniques applicable to the analyses of large-scale transcriptomic data sets. The utility of these techniques is evaluated through their application to a toxicogenomic screen in which our lab profiled exposures of adipocytes to metabolic disrupting chemicals. Such exposures have been implicated in metabolic dyshomeostasis, the predominant cause of obesity pathogenesis. Considering that an estimated 10% of the global population is obese, understanding the role these exposures play in disrupting metabolic balance has the potential to help combating this pervasive health threat. This dissertation consists of three sections. In the first section, I assess data generated by a highly-multiplexed RNA sequencing platform developed by our section, and report on its significantly better quality relative to similar platforms, and on its comparable quality to more expensive platforms. Next, I present the analysis of a toxicogenomic screen of metabolic disrupting compounds. This analysis crucially relied on novel supervised and unsupervised machine learning techniques which I specifically developed to take advantage of the experimental design we adopted for data generation. Lastly, I describe the further development, evaluation, and optimization of one of these methods, K2Taxonomer, into a computational tool for unsupervised molecular subgrouping of bulk and single-cell gene expression data, and for the comprehensive in-silico annotation of the discovered subgroups.

Development of type 2 diabetes (T2D) is preceded by prediabetes, which is a metabolically "atypical" state associated with chronic low-grade inflammation, overweight and obesity, lack of exercise, and detrimental changes to the gut microbiome. Dietary intake and exercise are modifiable lifestyle factors for reducing T2D risk; however, several questions remain unanswered related to the efficacy and role of prebiotics and exercise, and their respective influences on gut microbiome composition, intestinal permeability, insulin sensitivity and metabolic flexibility. Sedentary to recreationally active overweight and obese adults 40-75 years old at-risk for T2D were recruited (n=22) and randomized to either supplementation with inulin, a prebiotic dietary fiber, (10g/d) or maltodextrin while consuming a controlled diet for six weeks. At baseline and week 6, participants completed a stool collection, a 4-sugar probe test, an intravenous glucose tolerance test (IVGTT), and high-fat meal challenge with skeletal muscle biopsies to evaluate changes in the gut microbiome composition, intestinal permeability, insulin sensitivity and metabolic flexibility, respectively. There were no baseline group differences (all p>0.05). Following the intervention, Bifidobacteria operational taxonomic units increased in the intervention group ([placebo: Δ 9.5 ± 27.2 vs inulin: 96.3 ± 35.5][p=0.03]). There were no other group differences over time in any other outcome variables with the exception of changes in metabolic flexibility. Secondarily, a systematic review of literature was conducted to determine the influence of exercise engagement on gut microbiome composition. Overall, exercise interventions appeared to diversify taxa within the Firmicutes phylum, and specifically in several taxa associated with butyrate production and gut barrier function. Due to unclear risk of bias in all studies and low quality of evidence, additional research is needed using well- designed trials. In summary, the respective influences of prebiotics and exercise on human gut microbiome composition and their subsequent effects on metabolic function and disease risk are not well understood.

RELASHIONSHIP BETWEEN F2 – ISOPROSTANES AND ADULT METABOLICALLY HEALTHY OBESITY By Tatiana Victoria Piccoli 2017 Introduction: Obesity-related morbidity continues to increase worldwide. Obesity is one of the primary conditions for the development of metabolic syndrome which is characterized by a combination of different components, including metabolic, physiological, and biochemical factors that influence the development of cardiovascular disease, diabetes mellitus type two and contribute to all-cause mortality. The development of metabolic abnormalities is related to oxidative stress which increases among obese individuals. The obese population is not homogenous and represented by individuals with altered and with a normal metabolic profile, those with the normal metabolic profile are recognized as Metabolically Healthy Obese (MHO). The lipid peroxidation is the main hallmark of oxidative stress and this process can be evaluated via the measurement of the end products such as F2 -Isoprostanes. Aim: The goal of this study was to investigate the relationship between metabolically healthy and unhealthy obesity with markers of oxidative stress (urinary F2 -Isoprostanes) and assess whether free radical-induced oxidative stress influences transition from metabolically healthy to the metabolically unhealthy group. Metabolic health was defined as blood pressure below 130/85 mmHg, fasting blood glucose level as below 126 mg/dl and HDL-cholesterol above 40 mg/dl for males and above 50 mg/dl for females. Method: The cohort of 857 participants included Hispanic, non-Hispanic black, and non-Hispanic white from 40 to 69 years of age and had an obesity prevalence of 29% (n=244) at baseline. Among this group, 11.07% (n=27) were metabolically healthy and 88.93% (n=217) were metabolically unhealthy based on criteria for MHO. Among the MHO group, after 5-year follow-up, 37% remained metabolically stable and 63% developed metabolic abnormalities and among the MUO group, 5.6% became metabolically healthy and 94.4% remained metabolically unhealthy. The association between different types of metabolic health and F2 -Isoprostanes species was measured using Wilcoxon rank-sum test. Result: The MUO status had a direct association with greater weight, Hispanic ethnicity, impaired glucose tolerance, decreased insulin sensitivity, and decreased fasting insulin level. No association was found between metabolic health status and levels of F2 -Isoprostanes at baseline and follow-up. Conclusion: The result suggests that elevated levels of F2 -Isoprostanes do not promote the transformation of metabolically healthy obesity into metabolically unhealthy obesity. Keywords: Obesity, metabolic health, metabolically healthy obesity, oxidative stress, F2 -Isoprostanes

The early postpartum period is a challenging time for lactating mothers as it is a transitional period when healthy routines are often profoundly disrupted and may lead to low quality diet and thus adverse lipid profiles. Diets of high quality as assessed by DASH score have been shown to be strongly predictive of a reduced risk of future development of all-cause mortality, cardiovascular disease, cancer, type 2 diabetes, and neurodegenerative diseases. Our study objectives were to examine in a Midwestern setting: 1) metabolic health differences in postpartum lactating women with higher versus lower diet quality, and 2) overall diet quality and micronutrient adequacy in early postpartum lactating women, specifically focusing on those micronutrients critical to milk quality. A sample of 18 early postpartum lactating women were recruited from the Greater Cincinnati area. Dietary intake was assessed from a 24-hour dietary recall, physical activity was assessed via the Pregnancy Physical Activity Questionnaire, and metabolic health was assessed via bloodwork and anthropometric data. Metabolic Syndrome Severity Score (MetS score), a metabolic health z-score, was used to quantify overall metabolic risk among study participants. DASH score results were divided at the median to form lower versus higher diet quality groups, then Student’s t-tests were run to compare mean values between these DASH score groups. Median total DASH score was 39 points out of a maximum of 80 points, and ranged from 22 to 52. The DASH score groups differed significantly in total DASH score; and number of servings of dairy, protein foods, sodium, saturated fats and added sugars. Although not a part of the DASH Score, the DASH score groups also differed significantly in total calories and empty calories. The lower DASH score group displayed significantly greater mean body weight, BMI, waist circumference, plasma triglycerides and MetS score. There was no significant difference in metabolic health parameters between physical activity groups. The lower diet quality DASH group consumed significantly greater amounts of choline and selenium but fewer than 50% of all participants met the recommended intake for choline, Vitamin A and Vitamin D. Our pilot study results support the concept that mothers with better diet quality will have better metabolic health, and in addition diet quality of early postpartum lactating mothers in the Midwest region is poor. Further studies should consider a greater number of days of dietary recalls, more detailed dietary information like supplementation and food avoidances, and exercise intervention as opposed to a self-reported method of assessment.

Despite tremendous progress in biological understanding of metabolic diseases and development of novel imaging techniques over the past several decades, clinical assessments and epidemiological studies for metabolic disease are largely limited to simple surrogate measurements such as body mass index. Recent advances in dual-energy X-ray absorptiometry (DXA) and 3D optical surface scanning have enabled new opportunities for integrative and clinically-accessible metabolic health assessment.The focus of this dissertation was to develop novel 3D and DXA imaging methods for detailed metabolic risk assessment that are fast, safe, and accessible. This work centered on the hypothesis that detailed descriptors of body shape and compositional distribution of tissues throughout the body better reflect health risk than currently-used metrics.I present derivation of high-resolution quantitative fat, lean, and bone images from DXA, and describe statistical appearance models of these images that can be used to accurately predict metabolic syndrome and diabetes status across ethnicities. I demonstrate clinically-viable body composition estimation from commercial 3D optical body scans using traditional anthropometric measurements, then improved accuracy and precision using more detailed 3D statistical shape models that efficiently capture 95% of body shape variance. Finally, I describe integrative methods including a combination DXA and bioelectrical impedance technique to provide rapid, accurate, and precise four-component (4C) body composition. 4C assessment is useful for monitoring of many metabolic conditions including over/de-hydration, malnutrition, obesity, and sarcopenia, and this technique enables practical implementation in the clinic.These works collectively provide new tools to researchers, clinicians, and even individuals around the world to assess metabolic status and track, visualize, and predict personalized body changes towards improved health.

Diet-quality scores (DQS), which are developed across the globe, are used to define adherence to specific eating patterns and have been associated with risk of coronary heart disease and type-II diabetes. We explored the association between five diet-quality scores (Healthy Eating Index, HEI; Alternate Healthy Eating Index, AHEI; MedDietScore, MDS; PREDIMED Mediterranean Diet Score, P-MDS; Dutch Healthy Diet-Index, DHDI) and markers of metabolic health (anthropometry, objective physical activity levels (PAL), and dried blood spot total cholesterol (TC), total carotenoids, and omega-3 index) in the Food4Me cohort, using regression analysis. Dietary intake was assessed using a validated Food Frequency Questionnaire. Participants (n = 1480) were adults recruited from seven European Union (EU) countries. Overall, women had higher HEI and AHEI than men (p < 0.05), and scores varied significantly between countries. For all DQS, higher scores were associated with lower body mass index, lower waist-to-height ratio and waist circumference, and higher total carotenoids and omega-3-index (p trends < 0.05). Higher HEI, AHEI, DHDI, and P-MDS scores were associated with increased daily PAL, moderate and vigorous activity, and reduced sedentary behaviour (p trend < 0.05). We observed no association between DQS and TC. To conclude, higher DQS, which reflect better dietary patterns, were associated with markers of better nutritional status and metabolic health.

The rapid rise in obesity prevalence appears to be a reflection of the changes in dietary and behavioural patterns, with eating habits shifting to higher consumption of energy-dense foods which are rich in fats and sugars, while at the same time, levels of physical activity are decreasing. These differences in energy intake and expenditure, often referred to as energy balance, have direct implications for weight regulation, with even small deviations in daily energy balance resulting in large body weight changes over the long term. Diet and exercise interventions aiming to shift the energy balance towards negative by either decreasing caloric intake and/or increasing physical activity have shown to be effective for weight loss. Many iterations of such dietary and physical activity interventions have been proposed, but intermittent fasting (IF) and high intensity interval training (HIIT) have recently been purported as effective strategies. Despite their effectiveness, the molecular mechanisms by which these lifestyle interventions induce their effects are unclear. Therefore, the purpose of this thesis was to examine the effects of IF and HIIT, alone and in combination, on anthropometric and metabolic health parameters in a model of diet-induced obese mice. To elucidate possible mechanisms of action, we investigated the impact of both lifestyle interventions on mRNA-miRNA regulatory networks, but more importantly, how such changes may translate into exercise- induced and diet-induced improvements in body composition and metabolic health. The findings from the thesis demonstrate that intermittent fasting with or without high intensity interval training resulted in significantly less weight gain in male mice despite concurrently consuming a high fat and sugar diet. The reduced weight gain was predominantly in the form of lower fat mass accumulation, with no significant loss in lean mass. These observations were supported by enhanced expression of adipose tissue genes relating to fragmentation of unilocular lipid droplets, lipolysis, fatty acid oxidation and efflux. Moreover, lower expression levels of leptin, pro-inflammatory markers and markers of hypoxia were also observed. These changes were also reflected by changes in miRNA-24, -222, -145 and -143. Within the skeletal muscle, the combination of diet and exercise demonstrated minimal impact on mRNA and miRNA expression markers relating to energy metabolism, however IF alone displayed significantly lower expression of all markers compared to control mice or other intervention groups. Interestingly, the changes in body composition, glycaemic control, lipid panels and expression of mRNA and miRNA seem to be gender specific with different responses, independent of intervention, demonstrated in male mice compared to the female mice. In conclusion, the novel results from this thesis have demonstrated superior effects on body composition and lipid profiles following combined IF and HIIT compared to either diet or exercise intervention alone while concurrently consuming a high fat and sugar diet. These observations are likely due to the physiological and biochemical changes that occur within the adipose and skeletal muscle tissue when creating a negative energy balance shift. The gender specific responses to the same diet and/or exercise intervention could indicate potential hormonal differences influencing metabolic control/adaptation in mice. Identification of important regulatory miRNAs through this thesis could provide potential therapeutic targets for obesity treatment and management.

Obesity is a worldwide problem, and as such a large body of research has focused on potential treatments to decrease excess body weight and the associated loss of adequate metabolic health. Obesity risk is influenced by more sedentary lifestyles, higher energy intakes and greater consumption of convenience and processed foods. Worldwide evidence indicates that these factors occur as part of nutrition transitions, typified by an increase in energy density and plant derived fats, refined carbohydrates and a reduction in the variety of food consumed. It is believed that this increase in availability of particular plant derived fats can influence health, with the monounsaturated fatty acid (MUFA) oleic acid (OA) appearing to preserve metabolic health, and the polyunsaturated fatty acid (PUFA), linoleic acid (LA), having potential negative impacts, though this has not been fully explored. The first study of this thesis aimed to investigate changes in the Australian diet from historically available population level food disappearance data and how the availability of common dietary fats, specifically OA and LA changed, and what foods have influenced this. This study found that over the time period 1961-2009 total available energy (TAE) from lipids increased 16.67 %, with a slight reduction in TAE from carbohydrates. Cumulative change in TAE from LA was +120.48 %, found to result from total plant oils having a + 627.19 % cumulative change in LA availability. As the effect of these fatty acids (FA) on weight gain and metabolic health is yet to be fully elucidated, a rodent study was performed, with 60 Sprague Dawley rats being fed a high fat ‘Western’ style diet to induce obesity for 9 weeks. The animals were then switched to diets with varying FA compositions, namely a standard ‘Western’ style diet, a high OA ‘Mediterranean’ style diet and a high LA diet for 6 weeks. The impact of these diets on body weight and composition, energy intake, glucose handling and adipose tissue and skeletal muscle metabolism was also investigated. As adiponectin is an insulin sensitising adipokine the ability of this to interact with FA and influence gene expression relating to FA metabolism in skeletal muscle was also investigated through incubation of excised muscles in physiological levels of globular adiponectin. 6 weeks of specific high fat diet feeding in male Sprague Dawley rats resulted in no significant differences between body weights, food intake, blood pressure, percentage body fat, or adipose depot specific change to FA metabolism genes for the 3 high fat fed groups. Despite this, this study does show that there is a potential for the type of dietary fat to modulate food intake and energy efficiency (week 6 high OA vs. high LA, 0.020 ± 0.005 and 0.037 ± 0.003, g gain/mJ, p=0.033), with reductions in both of these occurring for the high OA consuming animals towards the end of the study. Additionally, investigation of changes to gene expression in the muscles from these animals found the ability of adiponectin incubation to increase FAT/CD36, β-HAD and CPT1 mRNA expression in both the EDL and soleus from high OA fed animals (all p≤ 0.05 compared to tissue incubated without adiponectin), a response which was not seen in any of the other diet groups. This may indicate that high OA feeding preserves adiponectin sensitivity in both of these muscle types and may confer metabolic benefits not seen in the other high fat diets tested here. Finally, the ability of different FA to modulate acute appetite and hormonal responses following consumption of isoenergetic breakfasts containing elevated contents of carbohydrate, OA and LA respectively, was investigated in eight overweight and obese individuals (age 45.8 ± 3.6 years, BMI 32.0 ± 1.3 kg/m2 ) in a 3-way crossover single-blinded study. Consumption of all meals resulted in a significant increase in fullness and a reduction in desire to eat, with the control and high OA meals decreasing prospective food intake (p= 0.006 and p= 0.049 respectively), though this did not occur following ingestion of the high LA meal (p= 0.183). Additionally, following the consumption of the LA meal there was a significant increase in ghrelin production (compared to the control meal, p= 0.018). This was coupled with a spike in resistin production at the one hour time point, indicating potential impairment of insulin signalling. Taken together this indicates that high LA meals may promote excess energy intake and impair glucose handling, though further investigation is required. The results of this thesis indicate that LA availability in Australia has increased over the preceding 5 decades as a result of increased plant oil availabilities, as has total fat, possibly contributing to the increasing rates of obesity and obesity associated co-morbidities. Results from the animal and human studies indicate that a higher OA intake as opposed to LA may help to preserve skeletal muscle FA metabolism and potentially limit food intake, indicated by the reduction in food intake and energy efficiency seen in the high OA diet consuming rats in the rodent study and the increase in the appetite and food intake stimulating hormone ghrelin following consumption of the high LA meal in the human study.

Non-Hispanic Black (NHB) and Hispanic children are at an increased risk of obesity and metabolic disease in the United States. Increasingly obesity is viewed as a multifaceted phenotype, beyond simply excess body weight. Research indicates that homeostatic mechanisms as well as hedonic systems are altered in obese individuals. The objective of this study was to examine how sugar sweetened beverage (SSB) intake, a highly modifiable dietary behavior, impacts both metabolic health and hedonic perceptions. This study is made up of three cross sectional analyses: the first two are secondary analyses of data from the University of Southern California, and the third was preformed at the University of Texas at Austin. The first examined the relationship between SSB intake, perceived hunger and satiety, and endocrine biomarkers in overweight and obese NHB and Hispanic adolescents (14-17 y). SSB intake of two servings or more per day was associated with decreased satiety and suppressed ghrelin compared to subjects that consumed one or less servings of SSB. The second examined the association between SSB intake, visceral fat accumulation (VAT), and cortisol awakening response (CAR) in a similar sample of 60 overweight and obese NHB and Hispanic adolescents (14-17y). SSB intake of two servings or more was associated with increased VAT and increased CAR compared to subjects that consumed one or less servings of SSB. The third examined the effect of SSB intake on reward pathways in the brain using functional magnetic resonance imaging (fMRI), as well as how free-living dietary and dietary intake at an ad libitum meal impacts hunger and metabolic biomarkers in 41 overweight and obese Hispanic children (7-10 y). Although analysis of the fMRI data was uninterruptable, increased hunger and decreased satiety at an ad libitum meal was related to added sugar intake greater than 10% of the subject’s daily calories. In adolescents, SSB intake is associated with decreased feelings of fullness and an unfavorable metabolic profile. In young children, added sugar intake is associated with appetite independent of homeostatic factors.

Insulin and FGF21 can change the course of metabolic disease. Chronic insulin signalling aggravates metabolic disease, while FGF21 reverses it. To partially address how this occurs, I investigated whether insulin compromises cellular protein homeostasis, and I characterised the FGF21 signalling network. Insulin potently regulates protein metabolism. As a result hyper-activation of the insulin signalling pathway, as occurs during metabolic diseases and aging, may lead to dysregulated protein homeostasis (proteostasis). To investigate this hypothesis I generated a time-resolved map of insulin-regulated protein turnover in adipocytes using metabolic pulse chase labelling and high-resolution mass spectrometry. Insulin did not affect protein degradation, despite suppressing autophagy and it increased the synthesis of 46% of detected proteins. Elevated synthesis did not cause proteostatic stress, such as carbonylation or protein aggregation, but rather enhanced protein stability and folding. I attributed this unexpected increase in protein stability to induction of proteostasis machinery, including ribosomal, proteasomal, chaperone and ER/mitochondrial UPR proteins. Thus I conclude that hyper-stimulation of the insulin signalling pathway in adipocytes improves rather than impairs cellular proteostasis due to upregulation of the machinery for protein folding and clearance. Hence, assuming similar mechanisms occur in vivo it is unlikely that impaired proteostasis accounts for insulin’s effects on metabolic disease and aging. FGF21 improves the metabolic profile of obese animals through its actions on adipocytes. To elucidate the signalling network(s) responsible for FGF21’s effects, I quantified the dynamic changes in the phosphoproteome of adipocytes following acute exposure to FGF21. FGF21 regulated a network of 821 phosphosites on 542 proteins. A major FGF21-regulated signalling node was mTORC1/S6K. In contrast to insulin, FGF21 activated mTORC1 via MAPK signalling rather than through the canonical PI3K/Akt pathway. The activation of the mTORC1/S6K signalling axis by FGF21 was surprising as this branch of signalling is thought to contribute to deleterious metabolic effects such as obesity and insulin resistance. Rather, mTORC1 mediated many of the beneficial actions of FGF21 in adipocytes, including UCP1 and FGF21 induction, increased adiponectin secretion, and enhanced glucose uptake. This study provides a systems view of FGF21 signalling and reveals a new role for mTORC1 in maintaining nutrient homeostasis.

To clarify the protective benefits of physical activity (PA), epidemiologists and public health researchers continue to seek improved methods of assessing PA. In particular, accelerometers have gained acceptance with researchers as they provide reliable estimates of PA and can record both the amount and intensity of ambulatory movement. However, there is concern that accelerometer data reduction techniques may not provide quantitatively accurate measurements of time spent in various PA intensity categories. One way to circumvent these inaccuracies is to use the accelerometer-derived total activity counts (TAC), which is a more direct expression of what the monitor records. In order to explore the efficacy of TAC as a measure of PA, this dissertation used data from the 2003 - 2006 National Health and Nutrition Examination Survey to: 1) investigate whether TAC was more strongly associated with cardiometabolic biomarkers than minutes of moderate-to-vigorous PA (MVPA), 2) determine population-referenced TAC percentiles for the U.S. population, and 3) determine which accelerometer-derived measure(s) of PA intensity and volume provided the best fit for assessing the association with the metabolic syndrome. The first study demonstrated that TAC had stronger associations with cardiometabolic biomarkers than time spent in MVPA bouts of ≥ 10 minutes, suggesting TAC is a more robust measure of PA (Part IV). In the second study, age- and gender- specific population-referenced percentiles for TAC, MVPA, and light PA (LPA) were developed (Part V). This is a different approach to accelerometer data reduction that complements the current method of looking at time spent in intensity sub-categories. The third study used structural equation modeling to examine whether TAC, MVPA, or MVPA plus LPA provided the best fit for assessing the relationship with the metabolic syndrome (Part VI). This study also assessed the relative contribution of LPA, MPA, VPA, and TAC to the reduction in the prevalence of the metabolic syndrome. Results indicated a model with TAC provided the best fit for assessing the relationship between PA and the metabolic syndrome. These findings suggest TAC, may be a better measure of PA when examining the reduction in the metabolic syndrome prevalence.