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Background Poor dietary habits have been recognized as playing a major role in non-communicable diseases. To improve population dietary intake, governments use food reformulation policies aimed at improving the nutrient profile of foods. This thesis aims at understanding how populations and individuals respond to the reformulation of foods. Methods Two types of data were used to analyse responses to food reformulation in the UK. The National Diet and Nutrition Survey was used to study the changes in dietary intakes following salt and fibre reformulations. Panels of household purchase were used to study households' responses to sugar reformulation. Adjusted time trends were used to study changes over time. A decomposition method was used to understand how changes in the nutrient density of foods consumed or purchased can be explained by changes in consumer demand for, and supply of, foods. Results Food reformulation was linked to improved dietary intake or purchase. These improvements were rather small, but similar across socio-demographic groups. The different decomposition analyses showed that the change in the nutrient density of foods purchased or consumed came mostly from supply-driven effects. Changes in consumer choices had a marginal mean effect, except in the presence of a tax. Further, the extent to which households consumed reformulated products did not influence their change in sugar purchases. Lastly, stronger incentives such as taxes were more effective in promoting reformulation than voluntary initiatives. Conclusions Reformulating foods leads to improved dietary intake. Despite being small, these improvements are not negligible and don't widen existing disparities. Taxing unhealthy products produced a change in consumer food choices, while it did not happen in the absence of such taxes. Given the gap between current and recommended dietary intake, policies to incentivise reformulation should be used alongside other policies aimed at improving food choices at the population level.

Evidence suggests that the ileum is an important player in appetite regulation. The ileum contains a high density of L-cells that secrete appetite suppressing hormones Glucagon Like Peptide-1 and Peptide YY (PYY) in response to luminal nutrients and bacterial fermentation products, short chain fatty acid (SCFAs). Processing of foods may change their structures and reduce the amount of carbohydrates reaching the ileum, which may reduce appetite hormone release. However, access to the human ileum is difficult and little is known about the interplay between food, ileum environment and appetite hormone release. This thesis aimed to understand the impact of food structures on the levels of carbohydrate reaching the ileum and their effect on SCFA production and appetite hormone release. A nasoenteric tube was placed in the distal ileum of healthy participants (n=10) who consumed one of the three intervention diets for four days: High fibre, intact structures (I-HF) High fibre, disrupted structures (D-HF) or Low fibre, disrupted structures (LF). Blood and ileal samples were collected on day 4 in fasted and fed states (hourly for 8h). Disrupting the food structures of a high fibre diet (D-HF) reduced the amount of reducing sugars and bile acids, but not starch, in the ileal space compared to a high fibre intact diet (IHF). The numbers of bacteria and the concentrations of SCFAs in the ileum dramatically dropped from fasted to postprandial states, and this effect was not different between the groups. Postprandial PYY release was higher in D-HF in comparison to LF. Metabolomic analysis of ileal contents identified dynamic changes in several metabolites which were different between groups, including aspartate which was higher in D-HF compared to I-HF and LF. In addition, D-HF had higher serum SCFAs compared to I-HF and LF groups. Therefore a difference in ileal metabolites and/or colonic fermentation may be playing a part in the observed PYY difference. Further studies are needed to better understand the role of food structures and ileal metabolites in appetite hormone release.

Childhood Crohn's disease is associated with growth failure, weight loss and malnutrition. The aetiology of which is multifactorial; chronic inflammation, therapy induced and reduced calorific intake. These factors contribute to alterations in metabolism and body composition. Visceral adipose tissue is the adipose compartment, most strongly associated with chronic inflammation. Intestinal adipose tissue expansion described as 'creeping fat' is well recognized from surgical specimens as a hallmark of Crohn's disease. Expansion of visceral adipose tissue is associated with cardiometabolic risk factors which have been reported in adults and children with inflammatory bowel disease. I performed a systematic review, to understand gaps in the literature in the context of body composition in children with inflammatory bowel disease. 22 studies were included, reporting on body composition in a total of 1477 children with inflammatory bowel disease . Lean mass deficits were more pronounced in children with Crohn's ; 93.6% of Crohn's and 47.7% of ulcerative colitis. Fat related compartment findings were inconsistent, and no clear conclusions could be drawn. Given that children with Crohn's disease had more pronounced deficits in body composition, and intestinal fat expansion is a hallmark of disease, but there is a paucity of data regarding body fat measures; my main aim was to quantify visceral adipose tissue volumes and liver ectopic fat using magnetic resonance imaging (MRI) and Magnetic Resonance Spectrometry (MRS), in this cohort. My secondary aim was to examine relationship of visceral adipose tissue and ectopic liver fat in children with Crohn's disease, disease specific and, metabolic parameters and plasma adipokines. I tested a null hypothesis in that no difference would be observed in abdominal adipose tissue or ectopic liver fat between children with Crohn's disease and healthy children. A prospective cohort of children (7-17 years) with CD and healthy children, were recruited. Visceral adipose tissue , abdominal subcutaneous adipose tissue, abdominal muscle volumes (expressed in litres), and intrahepatocellular lipid (IHCL), was determined using MRS. Blood was analysed for CRP, ESR, albumin, fasting insulin, fasting glucose, lipid profile, adipokines (adiponectin, leptin, visfatin and resistin). Multiple regression analysis was used to identify factors associated with the dependent variable (compartment volume) significance was set at p<0.05. Spearman's rank coefficient for non-parametric data was determined to examine relationship between adipose tissue volumes and disease and blood parameters, R values were reported as per published cut offs. 33 children were recruited (25 CD; 16 males), and 8 controls (5 males), mean age 14.0 ± 2.3 years and 13.4 ± 2.5 years. No participant with CD was receiving concurrent systemic steroids. CD vs. healthy controls; CD was significantly associated with 0.41 litres [0.15 to 0.55] more visceral adipose tissue and 1.71 litres [1.36 to 2.45] more abdominal subcutaneous adipose tissue; after adjusting for sex, weight z score, height z score, and pubertal status (p<0.05). Abdominal muscle volumes were lower in Crohn's disease. There was no significant difference in intrahepatocellular lipid. In children with CD, disease duration, male sex and CRP were significant positive predictors of visceral adipose tissue volume. abdominal subcutaneous adipose tissue had a positive relationship with plasma leptin. Dyslipidaemia was observed, and visceral adipose tissue was associated with plasma triglyceride levels. Visceral adipose tissue was found to positively influence adiposity in CD, namely intrahepatocellular lipid and abdominal subcutaneous adipose tissue. Children with Crohn's were found to have poor quality diets; low calorie, macronutrient imbalance and high percentage non-milk extrinsic sugars . For the first time in paediatric CD patients, we show an association with abdominal adipose tissue obesity (visceral adipose tissue and abdominal subcutaneous adipose tissue) and a trend towards muscle deficit in the context of normal hepatic lipid. Possible drivers of this obesity phenotype in children with Crohn's disease may be the result of dietary inadequacy, energy imbalance, systemic and local chronic inflammation.

Current dietary interventions in the treatment of obesity are struggling to prevent its increasing global prevalence and detrimental side-effects, including non-alcoholic fatty liver disease (NAFLD). Many studies addressing the macronutrient content of the human diet have shown that manipulation of the macronutrient makeup of the diet may have beneficial effects on weight loss, appetite regulation, body composition and metabolic health. However, obesity and nutrition research are flawed by the limitations of current dietary recording methodologies, as there is an inverse relationship between accuracy of reporting of energy intake by all current nutritional methodologies and body weight. This work aimed to determine the effects of specific individual nutrients (a high-protein, hypo-caloric diet, and long chain n3-polyunsaturated fatty acids (n3-PUFA) supplementation with a hypocaloric diet) on weight, adiposity and appetite in overweight participants. In addition to this a pilot study explored the use of a novel lightweight wearable micro-camera in improving the accuracy of dietary intake assessment. Anthropometric measurements were taken using weight, body mass index (BMI), waist circumference (WC), hip circumference (HC), bioelectrical impedance (BIA), and the gold standard for measuring body composition, magnetic resonance imaging (MRI). Energy intake and appetite was measuring using a 3- or 7-day food diary, an ad-libitum meal, visual analogue scale and, for the n3-PUFA study, fMRI was also employed to assess changes in food reward through measuring changes in blood oxygen level dependent (BOLD) signal in response to food images. The results suggest that any changes in body composition, biochemistry and appetite induced by a 12-week hypocaloric diet appear to be independent of protein content (body weight change of -4.06±0.85kg body weight with normal protein vs -4.42±0.85kg with high protein diet), whilst n3 PUFA supplementation during a calorie restricted diet may improve weight loss (-2.53kg±0.59kg, p<0.0001 weight loss seen with n3 PUFA vs -0.05±0.64kg, p=0.419 seen in the placebo group), through improved compliance to the calorie-restricted diet (energy intake compared to baseline was 298 ± 93kcals lower in the placebo group and 524 ± 69kcals lower in the PUFA group). The mechanism behind this improved compliance and enhanced weight loss has not been elucidated, however it seems it is not mediated by changes in appetite or via reward pathways. The microcamera does improve the accuracy of dietary assessment and provides valuable information on macronutrient intake and eating rate. However, there is a need to develop this recording technique to increase its usability and remove user and assessor bias. Our study adds to the research suggesting that n3 PUFA supplementation has a role to play in modifying, particularly reducing, body weight and body composition, and although we found no significant effects with the high protein diet, further work should seek to understand the optimum and minimum effective dose and duration of treatment with n3 PUFA and/or high protein diets.

The association between alcohol consumption and cardiometabolic disease risk has been described as a J or U-shaped curve attributed to a combination of harmful and beneficial effects varying with volume of intake. Diet is an established risk factor for cardiometabolic disease and related traits However, few studies examining this association adequately control for residual confounding by dietary intake. Therefore, the aim of this thesis was to investigate the relationship between alcohol consumption and markers of cardiometabolic health independent of dietary intake. Cross sectional analyses were conducted using data from the Airwave Health Monitoring Study - a British occupational cohort (n = 9,581). Alcohol consumption behaviour was determined from questionnaire and 7-day diet record data. Diet quality was determined by measuring adherence to the Dietary Approaches to Stop Hypertension (DASH) diet using data from the 7-day diet records. Markers of cardiometabolic health included: adiposity (body mass index and waist circumference), blood pressure, cholesterol, HbA1c and C-reactive protein. All analyses were replicated in an independent UK cohort using UK Biobank data (n =146,888). As part of this thesis, genetic analyses were conducted to investigate whether genes implicated in the regulation of HDL-c may facilitate some of the cardioprotective effects attributed to alcohol intake. Heavy alcohol consumption is associated with a deterioration in cardiometabolic health. The risk of cardiometabolic disease as indicated by a cardiometabolic risk score was lowest amongst moderate drinkers and highest amongst never, and heavy drinkers. Increasing alcohol intake was associated with a deterioration in diet quality and had an additive effect of total energy intake. Diet quality did not modify the relationship between moderate alcohol intake and cardiometabolic risk. Alcohol intake did not alter the effect of genes implicated in the regulation of HDL-c. These findings were validated in a sub-sample of the UK Biobank cohort. To conclude, the findings from this thesis show that alcohol consumption plays a key role in determining diet quality and cardiometabolic risk. Specifically, risk of developing obesity.

Introduction: Waist circumference is significantly associated metabolic disease morbidity and mortality, independent of BMI. Characteristics of dietary intakes, such as overall dietary pattern, dietary fibre and fat intake, are recognised as factors associated with waist circumference. Common genetic variants have also been found to explain variation in population susceptibility to having a high waist circumference. However, it is not known whether dietary intakes modify associations between genetic and waist circumference. Therefore, this study's primary aim is to investigate how diet and genetics affect waist circumference across the UK adult's population. This study secondary aim is to investigate the effect of dietary intake on genetic predisposition to having high waist circumference. Method: This cross-sectional study analysed data from the Airwave Health Monitoring Study, a UK-based occupational cohort study that included 45,000 participants (from 2004 to 2018). Diet quality was evaluated based on Dietary Approaches to Stop Hypertension. The genetic risk score associated with waist circumference (GRSwaist) was derived from 91 WC-associated single nucleotide polymorphisms. Multivariable regression models were used to determine the associations between diet, genetics and waist circumference. An interaction term was introduced to multivariable regression models to determine the effect of dietary intake on genetic predisposition to waist circumference. This study was replicated using 442,583 participants from the UK-Biobank Cohort study. Results: Groups with a larger waist circumference (female > 80 cm, males > 94 cm) was positively associated with HbA1c% (OR= 1.08, 95%CI =1.0,1.1, P-value= 0.02). DASH-diet score and fibre intake were negatively associated with waist circumference. A positive association was found between GRSwaist and waist circumference. No interaction was observed between diet scores and genetic predisposition to having high waist circumference. The replicated analysis on the UK-Biobank population was consistent with the Airwave study findings. Conclusion: Waist circumference was positively associated with HbA1c%. Dietary patterns and genetic risk scores are significant factors associated with waist circumference in the UK adults' population. Dietary intake does not modify the influence of genetic on waist circumference.

Type 2 diabetes (T2D) is a disease with a high prevalence in south Asian countries. Diet is the cornerstone strategy for the management of T2D. South Asians may have increased postprandial blood glucose responses following the same dietary challenge compared to white Europeans. Dietary fibre and protein play a role in regulating blood glucose and appetite via a myriad of mechanisms such as gut microbiota fermentation to short-chain fatty acids. Mycoprotein is a food ingredient high in both fibre and protein which has a positive effect on blood glucose and appetite in humans. Likewise, guar gum is a fibre-rich ingredient that decreases blood glucose and appetite in people with T2D. The aim of this thesis is to determine the effect of mycoprotein on glycaemia and energy intake in humans and the mechanisms underpinning these. In Study 1, the associations between mycoprotein-based food consumers and non-consumers with non-communicable disease markers such as blood glucose, energy intake and diet quality are studied in the UK free-living population using the National Diet and Nutrition Survey. The results showed that there is a positive association between mycoprotein consumers and healthy diet scores, fibre, fasting glucose and glycated haemoglobin. In Study 2, a systematic review of the effects of mycoprotein on glycaemic control and appetite in humans is performed. The results showed that the acute effects of mycoprotein on glycaemia are unclear, there is a consistent decrease in insulinaemia and energy intake in healthy humans. In Study 3, the acute effect of mycoprotein and guar gum in glycaemic control and appetite in people with T2D of South Asian and European ethnicity is investigated via a randomised controlled trial. The results showed that mycoprotein decreases postprandial blood glucose and that enriching chapati with guar gum induces a decreased blood glucose response. South Asians, however, had an increased postprandial glucose response compared to white Europeans. In Study 4, the effects of mycoprotein fermentation by the healthy gut microbiota is investigated using in vitro batch fermentation cultures. The results on breath hydrogen, pH, SCFAs, and bacterial communities, showed that mycoprotein may not be fermented.

Our gut harbours a biosphere of microorganisms constantly responding to the environmental cues we provide for them. From what we eat to how we behave and when we get sick so can our gut microbiome. However, this is not a one-way communication, these microorganisms also work for us in regulating many aspects of our body's biochemical, physiological and immune functions. The gut microbiome lives in different compartments of varying nutrient availabilities, chemical and physiological conditions. The highly fluctuant environment of the small intestine (SI) and the presence bile acids and oxygen render this compartment inhabitable by most species. However, Streptococcus spp. thrive in the SI because they are highly efficient at metabolising simple carbohydrates for energy. Chapter 3 showed certain strains Streptococcus spp. may be in competition with one another for the availability of nutrients in the SI. Inside the colon, these facultative anaerobes were replaced by a diverse community of obligate anaerobes capable for fermenting complex carbohydrates, proteins and even mucin glycans. These microbial communities are resilient and adaptable to short-term Western diet intervention although carbohydrate fermentation activity of the colonic microbiota was decreased as Chapter 3 showed. In Chapter 4, significant alterations in the community structure and metabolic capacity of the colonic microbiota were observed in patients with irritable bowel syndrome (IBS). Lower carbohydrate fermentation and increased protein fermentation were evident in patients with constipation-predominant IBS. Diarrhoea-predominant IBS patients excreted more primary bile acids. Using multi-omics integration, a novel host-microbial co-metabolic pathway involving purine salvage was identified. Chapter 5 showed the passage of short chain fatty acids (SCFA) from the lumen to the mucosa was interrupted by the presence of a mucus layer. A diffusion dynamic for the trans- compartmental passage of SCFA was subsequently hypothesised. This thesis presents a compartmentalised view of the gut microbiome, demonstrating its unique adaptations to dietary change and that disturbances in its community structure and functional capacity underlie the pathophysiology of functional gastrointestinal disorders.

Obesity has become a global health issue, and type-2 diabetes (T2DM) is one of its most common comorbidities. The Roux-en-Y gastric bypass (RYGB) is the most effective surgical procedure for treating obesity, resulting in significant and sustainable weight loss. One proposed mechanism for how RYGB achieves successful weight-loss is based on profound changes in the secretion of gastrointestinal hormones that follow surgery, including increases in Glucagon-Like Peptide-1, Oxyntomodulin and Peptide YY (GOP). Surgically induced changes in physiology are also associated with specific behavioural factors and psychological characteristics, both of which are recognised as important contributors to successful postoperative outcome. This study hypothesised that patients with obesity and T2DM following RYGB have a healthier eating behaviour profile, mediating the long-term sustainable weight loss than Very-Low-Calorie diet (VLCD) or GOP infusion. Eating behaviour, food reward, psychological status, health-related quality of life and food intake were assessed at baseline and following each intervention using a set of validated questionnaires and a 4-day food-diary before and after each intervention. The RYGB showed a reduction in the reward drive to eat at three-month which was maintained up to one-year post-surgery. There were also reductions in external-eating, disinhibition and weight and shape concerns which were not observed following VLCD or GOP, suggesting that calorie-restriction or gut-hormone alone maybe not be responsible for changes in eating behaviour following RYGB. Meanwhile, there were similarities between the RYGB and GOP groups in terms of the increase in the flexible dietary restraint measured by the Dutch Eating Behaviour questionnaire. Moreover, the GOP group did not show any changes in the taste perception as were observed following RYGB. Understanding the behavioural mechanisms that account for such variations could help the development of personalised therapeutic approaches based on the need to improve treatment outcomes and dietary advice offered to patients with obesity.

The beneficial effects of resistant starch on appetite regulation and body weight is well established in the literature; however, the underlying mechanism is not fully understood. Cell walls are the main source of resistant starch in human diets. Changes in dietary patterns over the recent years are dominantly characterised by highly-processed food, which commonly lack their cell-wall structure intact. Industrial processing can cause damage to the native structure of the cell wall, affecting nutrient bioavailability rate and appetitive responses. This thesis aimed to investigate the literature on food structure definition and its effect on energy intake and appetite regulation. Firstly, a systematic review of the effect of food structure on subjective appetite sensations and energy intake was conducted. It has been shown that there is a gap in the current literature on food structure definition. There are only four clinical trials found in the literature that investigated the effect of cellular wall integrity on appetite and energy intake. Secondly, cross-sectional analyses were conducted on two cohort studies to investigate the effect of food structure on energy intake; the National Diet and Nutrition Survey and the Airwave Health Monitoring Study. Lastly, a human clinical study was conducted to investigate the acute effect of different cell-wall structure meals on a subsequent open-buffet meal, and subjective appetite ratings. The work presented in this thesis suggest that there is an inverse association between the intake of minimally-processed (intact cell-wall) food and energy intake on the long-term, however more work is required to investigate the mechanism and acute effect of food structure.

Background: Innovation adoption and diffusion (IAD) in healthcare refers to the essential process of integrating novel practices into systems’ critical functions. IAD is often slow and unsuccessful, leading to patients not receiving optimal care in a timely manner. IAD research has been dominated by descriptive or prescriptive theories with limited benefit to practice applications; these theories are often simplistic and assume IAD to follow a linear and predictable pathway despite emerging evidence portraying healthcare organisations as complex adaptive systems. Therefore, contemporary IAD research is increasingly shifting in favour of complexity theory replacing linear theories. Nonetheless, nursing research in the field is still trailing behind other disciplines on this shift. Aim: This study applied complexity theory to evaluate the process of innovation adoption and diffusion in frontline nursing practice. Objectives: The study explored why some nursing units succeed in innovation adoption and diffusion while others, within the same organisation, fail. Methodology: Drawing on constructivism, this study applied a theoretical framework built on complexity theory as the grand theory, complex adaptive system (CAS) as the middle-range theory, and Atun’s framework at the micro level. The theoretical framework requires a systems thinking approach capable of exploring the system as a whole rather than a collection of isolated, separate individual components. Design: A Comparative case study design was adopted, forming a 2x2 matrix incorporating two cases with four units of investigation. The two cases were two innovations selected as representatives for the phenomenon of IAD in frontline nursing: WHO 5 Moments for Hand Hygiene (5MHH) and Intentional Nursing Rounding (INR). Each innovation (case) was evaluated through its adoption and diffusion into two comparable units of investigation (wards); a ward of successful adoption and diffusion and an unsuccessful comparator ward. The study deployed multiple data collection methods incorporating non-participatory observation for thirty hours in each unit, and semi-structured interviews with nurses across the nursing hierarchy. Results: The study was able to identify several manifestations of complexity in frontline nursing and demonstrate how these impact adoption and diffusion behaviours. Manifestations of complexity were identified in attributing clinical outcomes to staff compliance; in the discrepancy between evidence, policy, performed, practice, and records; and through the disparity in staff values and priorities across the hierarchy. Existing organisational arrangements of clinical governance (policy design, dissemination, monitoring and clinical documentation) did not facilitate IAD at the frontline. Conclusion: Complexity offers an explanation for the variation in frontline innovation behaviours. Future strategies are recommended to improve IAD in frontline nursing through enhancing the adaptability of the innovation, engaging the informal social communication network, recognising frontline priorities, acknowledging staged and gradual improvement in compliance over time, and maintaining patient interest at the core of all frontline practice innovations.

Background: Previous research has demonstrated that acute propionate supplementation in humans can have favourable effects on energy metabolism by raising energy expenditure and lipid oxidation. Moreover, acute propionate supplementation in humans has shown to affect subjective appetite by increasing nausea and triggering the release of the anorectic hormone glucagon-like peptide 1 (GLP-1). Studies investigating the acute effects of propionate administration on glucose homeostasis in humans report conflicting outcomes. However, previous research has generally only investigated the acute impact of propionate supplementation in the overnight fasted state and for relatively short observation periods (<180 min).It is, therefore, presently unknown how raised bioavailability of gut-derived propionate modulates energy metabolism during physical activity and in the postprandial state. Consequently, the aim of the present trial is to investigate the acute effect of sodium propionate supplementation on energy expenditure, substrate oxidation, appetite response and glucose homeostasis in three different energy states (overnight fasted, sub-maximal exercise and post-prandial) and over longer time-periods. Moreover, NMR (nuclear magnetic resonance) spectroscopy was used to investigate changes in serum metabolite phenotype after sodium propionate ingestion. Methodology: The thesis is comprised of three separate randomized controlled double-blind cross-over studies (overnight fasted, submaximal exercise, and postprandial). In each study, following an overnight fast, tablets containing either sodium propionate or sodium chloride (Control) were first administered over 180 min. Overnight Fasted study: 19 volunteers (11 males and 8 females; age: 34.6  4.1 years; BMI (body mass index): 23.1  0.7 kg/m2) completed the two study visits after an overnight fast. The study extended over a total period of 360 min while volunteers remained fasted for the duration of the study. Sub-maximal exercise study: 19 volunteers (14 males and 5 females; age: 42.7  3.5 years; BMI: 24.5  0.7 kg/m2) completed a maximal exercise test visit and two study visits. The study extended over a total period of 240 min. At time-point 180 min, participants exercised at 40% of VO2 max for 60 min. Post-prandial study: 19 volunteers (12 males and 7 females; age: 45.0  3.5 years; BMI: 24.8  0.8 kg/m2) completed two study visits. The study extended over a total period of 300 min. At time-point 180 min, a mixed calorie liquid meal (Ensure Original Vanilla Nutrition Shake: 72.7 g carbohydrate, 13.6 g fat and 20.5 g protein; 500 kcal) was provided to volunteers. Energy expenditure and substrate oxidation were measured throughout these visits using indirect calorimetry. Participants were asked to complete 100mm visual analogue scales (VAS) that assessed subjective appetite (hunger, thirst and nausea) throughout these visits. Insulin resistance and insulin sensitivity were assessed via HOMA-IR and Matsuda Index respectively. The oral disposition index (ODI) was used to assess β-cell function. In the post-prandial trial, GLP-1 release was measured, and 1H-NMR spectroscopy was used to analyse serum metabolite profile associated with propionate supplementation. Results: Propionate supplementation increased energy expenditure in the overnight fasted state, which was mainly observed within the first 180 min of ingestion, and in the post-prandial state. A consistent increase in lipid oxidation was found in the overnight fasted state, however, these effects were not observed during submaximal exercise or in the post-prandial state. A decrease in carbohydrate (CHO) oxidation was also found in the overnight fasted state. Propionate ingestion increased subjective nausea in the overnight fasted and post-prandial states and increased subjective thirst during submaximal exercise. However, no effect on subjective hunger was found was found in the three different energy states. GLP-1 secretion was significantly increased in the overnight fasted state, however, insulin sensitivity and β-cell function were unaffected with propionate ingestion. In the overnight fasted state, low density lipoprotein (LDL)/ very low-density lipoprotein (VLDL), lactate and methanol were upregulated and 3-hydroxybutyrate and lysine were supressed following propionate supplementation. LDL/VLDL, lactate and alanine were upregulated following propionate supplementation in the postprandial state. Conclusion: This thesis is the first to demonstrate that acute oral sodium propionate supplementation in healthy human volunteers can have favourable effects on energy metabolism in different energy states. Should these effects be replicated over longer time periods would suggest that increasing systemic levels of gut-derived propionate appears would be a promising strategy to improve long term energy balance and body weight management.

Cardiovascular diseases (CVD) continue to be the leading cause of death worldwide. Hypercholesterolemia (HC) is a modifiable risk factor in the development of CVD. Diet-based therapies, such as dietary fibres (DF) are first-line treatment options to be used solely or in conjunction with pharmacological options for cholesterol management. DF have been shown to reduce HC, but underlying mechanisms are unknown. Existing literature suggest that the hypocholesterolemic effect may be as a result of an interplay of diet, host metabolism, and gut microbiota (GM) which leads to the increased faecal excretion of bile acids (BA). Faecal BA excretion (FBAE) leads to a reduction of circulating cholesterol via a cascade of reactions, therefore increasing FBAE may be a cardioprotective mechanism. A novel mechanism was thus hypothesised. DF may modulate GM to promote the polymerisation of secondary BA deoxycholic acid (DCA) to polydeoxycholic acid (PDC). PDC cannot be reabsorbed; it is subsequently excreted, triggering compensatory reactions that lead to reduced plasma cholesterol and its associated CVD risk. This PhD project explored the mechanisms involved in and the effects of diet on PDC production in humans using a combination of cutting edge metabolomic and metagenomic tools. An ultra-performance liquid chromatography-based method was developed for high throughput and sensitive detection and quantification of PDC. PDC was reliably detected in human faeces (up to 90% of total faecal DCA) and was found to correlate with anti-inflammatory butyrate producing GM and established beneficial GM like Bifidobacteria, and also with a high fibre high protein diet. More investigations are warranted to further elucidate the role of PDC in host physiology. Current indications suggest a beneficial role for PDC. Abbreviations: CVD cardiovascular diseases; HC Hypercholesterolemia; DF dietary fibres; BA bile acids; GM gut microbiota, FBAE faecal bile acid excretion, DCA deoxycholic acid; PDC polydeoxycholic acid.

Asthma is a chronic inflammatory disease characterized by decline in lung function, goblet cell metaplasia and tissue remodeling. Recent years have demonstrated the interplay between the airways and the gut microbiota, showcasing the dampening of airway inflammation by gut-microbial metabolites short-chain fatty acids (SCFAs) in rodent models. No studies have investigated the presence and effect of SCFAs in the airways of asthmatics and limited studies have investigated the metabolic profile of asthmatics and the effect of fibre supplementation. We demonstrate the presence of SCFAs (mainly acetate and propionate) in the airways of healthy controls and asthmatics, with an increase in acetate in asthmatics. The levels of SCFAs detected in the airways exceeded what has previously been described in peripheral blood. SCFA concentrations in asthmatic airway were correlated with specific airway microbial genera, suggesting a possible production of SCFAs by the airway microbiota. This was further confirmed by in vitro batch culture, where airway bacterial species produced higher amount of SCFAs in the presence of mucin, indicating proteolytic activity by the airway microbiota. In order to determine the direct effect, which local SCFA level could have on the airway epithelium, the effect of acetate and propionate were investigated on primary airway epithelium. Both SCFAs promoted a modulation of cytokine gene response upon, stimulation on both apical and basolateral side, which was further enhanced by IL-13 treatment. These results suggest that the microbial-airway-derived SCFAs promote a local change in the inflammatory response in vitro, which is different to the response seen with gut-microbial-derived SCFAs in preclinical data. Furthermore, the metabolic profile of asthmatics was investigate using 1H NMR spectroscopy and UPLC-MS, in order to identify potential urinary biomarkers that would aid in the diagnosis of asthma and sub-populations of asthma. Several urinary biomarkers were found to be either elevated or decreased in asthmatics belonging to purine, polyamine and fatty acid metabolism. Interestingly, a panel of carnitines were able to distinguish between sub-population of severe asthmatics. Lastly, investigating the effect of prebiotic supplementation to individuals with exercise-induced asthma, did not generate any statistically significant effect on SCFAs concentrations nor lung function, possibly due to the low levels of prebiotic supplementation.

Severe anaemia (Hb<5.0g/dl) affects almost 1 in 20 children in sub-Saharan Africa, and up to 1 in 4 child hospital admissions in this region (Muhe et al., 2000; Kiggundu et al., 2013). Micronutrient deficiency, malaria, infections and genetic factors contribute to its development. Severe childhood anaemia is associated with a high risk of mortality (19% within 6 months) (Phiri et al., 2008) and survivors experience long term health effects. Bacterial co-infection from gut-derived pathogenic bacteria is a frequent cause of death (Church and Maitland, 2014). Diet, altered metabolism, and the gut microbiota are thus implicated in severe anaemia, however interactions between these factors are poorly understood. The Transfusion and treatment of severe anaemia in African children study (TRACT) which assessed optimum transfusion dose, micronutrient supplementation, and prophylactic antibiotic treatment provided an opportunity to address these factors. The aim of this study was to characterise the diet of children in Eastern Uganda, and assess the impact on recovery from anaemia. It also aimed to assess whether interactions between diet and host metabolism or the gut microbiota were significantly altered in severely anaemic children, and whether changes in these were associated with recovery. A pilot study (n=19) was conducted to validate a 24-hour dietary recall method, which showed that the method had low inter-assessor variability, and high validity when compared to weighed food records. A subset (n=339) of children from the TRACT study in eastern Uganda were recruited to undergo dietary assessment and provide urine, plasma and faecal samples at admission, day 28 and day 180. A majority of children had diets which lacked diversity, reliant on a single carbohydrate-rich but micronutrient-poor food source. Food intake on admission was restricted in energy and micronutrients and was inadequate for a majority of children, but quantity and diversity increased as children recovered. Urine and plasma metabolic profiles reflected acute inadequate energy intake on admission (ketosis) and metabolic stress associated with critical illness (glycosuria and lipoprotein dysregulation), in addition to condition-specific effects including malaria-associated hyperphenylalaninaemia. These metabolic derangements resolved with treatment as children recovered. The gut microbiota of children in this study on admission lacked diversity and was dominated by potentially pathogenic Enterobacteriaceae and Enterococcus faecium. High levels of antibiotic use, in addition to low dietary diversity and dietary fibre intake contributed to reduced diversity and richness in species observed. Alterations in diet, metabolism and the gut microbiota were identified in this study in severely anaemic children, reflecting the causes and severity of this condition. Potential routes to reduce the impacts of these derangements were identified for future research.

Glucose is a major molecule for energy supply. Thus, the movement of glucose from food into tissues is necessary for survival. However, since high levels of glucose are toxic to the body, glucose homeostasis plays a key role in preventing a number of diseases, including type 2 diabetes, obesity, coronary heart disease and many types of cancer. Empirical evidence has shown that postprandial glucose and insulin sensitivity are not only governed by the content of food but also by other factors that may play a role in the digestive process, including food structure, rheological properties and food breakdown pattern. The combination of different levels of structure—from the molecular to the micro and macro levels—develops the rheological behavior of food that impacts the availability and digestibility of nutrients. In this work, I brought together nutritional physiology and rheology to explore the impact of peas and chickpeas on postprandial glycaemia and to develop a better understanding of how their physical properties change the physiological response. This in turn will give insight into the effect of food structure and its rheological properties on postprandial metabolic responses. The main finding of this work highlighted the importance of an intact cell wall in lowering postprandial glycaemia and subsequent metabolic responses when compared to a ruptured structure. Also, this work showed that starch structure played a role in postprandial glycaemia and insulinemia when the cell wall is absent. Further investigation was performed on the rheological properties of foods with different structures, relating these results with metabolic responses. Results from studies assessing the rheological and mechanical properties of food strongly suggested that they indeed affect the food breakdown pattern during digestion. It was shown that an intact structure’s rheological properties slowed the breakdown of food during digestion. Also, different intact structures had different rheological properties that suggested an impact on the food’s behaviour during digestion and hence on postprandial metabolic responses. Throughout this thesis, an indirect relation between food structure, its rheological properties, food breakdown during digestion in relation to postprandial glycaemia and related metabolic responses was realised.

The structural elements within a food, known as food structure, are thought to play a critical role in appetite regulation and energy homeostasis. Food structure can refer to the spatial arrangement of cellular structural elements, such as starch granules, or the physical form of a food. Manipulating the starch structure within a food system, such as in peas, can limit starch digestibility and thus improve the quality of the starch eaten. Bypassing upper gastrointestinal (GI) tract digestion can increase microbiota fermentation activity, which is responsible for the beneficial effects associated with resistant starch consumption. Short-chain fatty acids, fermentation by-products, have previously been reported to regulate glucose homeostasis, reduce energy intake and increase peripheral anorexigenic gut hormone concentrations in humans. A large part of this thesis aimed to investigate the acute effects of manipulating the starch structure within peas by inactivating the starch-branching enzyme of the pea seeds (SBE-deficient peas) on appetite regulation and energy homeostasis in humans. The first study presented in this thesis investigated the effect of food structure within peas on gastric emptying rates using a 13C breath test. The data suggest that manipulating the structure of the peas improves glycaemic response without affecting the gastric emptying rate. The second study presented in this thesis investigated the effect of upper GI digestion on different pea structures. SBE-deficient pea seeds were less available at the small intestinal level to be broken down into glucose. The third study presented in this thesis investigated the effects of lower gut fermentation on different pea structures. SBE-deficient pea seeds produced more short chain fatty acids (SCFAs) as a result of fermentation. However, these pea studies did not cause a change in subjective appetite or in appetite-regulating hormones. Propionate, one of the main SCFAs produced from microbial fermentation, is thought to play a crucial role in appetite regulation and energy homeostasis. Supplementation with inulin-propionate ester (IPE), which selectively enriches colonic propionate, has been reported to increase peripheral anorexigenic gut hormone concentrations and reduce energy intake acutely in humans. Long-term IPE supplementation prevented weight gain and reduced intra-hepatocellular lipid content. Another part of this thesis aimed to investigate the effects of colonic 4 propionate on appetite regulation and energy homeostasis in humans following a lower energy diet through IPE supplementation over a 12-week period. IPE reduced subjective appetite and led to significant weight loss compared to baseline. However, these effects were independent of changes in anorexigenic gut hormones. The majority of the work presented in this thesis supports a role of fermentation in energy and appetite homeostasis. However, the exact underlying mechanisms remain unclear.

Obesity is a major global health issue. As high-protein and high-fibre diets are associated with increased satiety and weight loss, uncovering the mechanisms by which dietary protein- and fibre-derived metabolites are sensed in the gut and regulate appetite may reveal targets for anti-obesity therapies. The work presented in this thesis sought to further this endeavour with a two-pronged approach, in vitro and in vivo. The in vitro intestinal organoid model recapitulates the major features of the in vivo intestinal epithelium. I cultured intestinal organoids from mouse duodenum, jejunum and ileum, and demonstrated that basal GLP-1 content increased distally along the small intestine, in line with the in vivo L-cell density pattern. Research within our group had identified that the amino acid L-Phenylalanine has anorectic properties and highlighted the calcium-sensing receptor as a potential target through which L-Phenylalanine mediates GLP-1 release. My work built on this by showing that L-Phenylalanine significantly stimulates GLP-1 release in mouse ileal organoids, an effect significantly attenuated by a calcium-sensing receptor antagonist. Investigation of the underlying signalling mechanisms indicated that L-Phenylalanine-induced GLP-1 secretion may not be mediated via a Gi/o-coupled pathway, and that Ca2+ influx through L-type calcium channels may be required. The in vivo dietary intervention study I concurrently ran in healthy humans led to observations that a high-protein, high-fibre test meal stimulated GLP-1 and PYY release that remained above fasting levels for the 6-hour sampling period. Furthermore, acute repeated exposure of the gut to a protein- and fibre-enriched diet may have beneficial effects in terms of sustaining post-prandial plasma PYY responses and modulating glucose homeostasis. Gastrointestinal contents from the terminal ileum and proximal colon were obtained from participants via a specialised nasoenteric sampling tube, and microbial profiling of these together with stool samples revealed region-specific differences within the distal gut, indicating that stool may not be an accurate proxy for either microbial diversity or composition of the terminal ileum or proximal colon. Additionally, acute temporal shifts in microbial composition, perhaps due to dietary intervention, were identified in the gut contents samples. The work in this thesis has contributed to our knowledge of how ingested protein and fibre affect anorectic gut hormone release, glucose homeostasis and appetite, and has shed light on how differences in microbial diversity and composition can exist in different regions of the distal gut. It has also enhanced our understanding of the mechanisms that sense L-Phenylalanine in the gut. Further investigations are required to determine the therapeutic potential of protein- and fibre-derived metabolites as appetite-suppressing agents, with an end-goal of developing novel strategies to prevent and treat obesity.

Obesity is a major global health concern. Current anti-obesity therapies are either ineffective, or highly invasive and difficult to provide on a global scale. Therefore, there is a growing need for new treatments. High protein diets have been shown to be effective at promoting weight loss, improving glucose tolerance and supressing appetite, but compliance is low. Understanding the mechanisms underlying the beneficial effects of high protein diets could aid the development of new anti-obesity therapies. Acute studies have shown protein loads to stimulate the secretion of anorectic hormones which can promote satiety and decrease food intake. However, the long-term effects of high protein intake on the regulation of gut hormones is less clear. This thesis presents a series of studies investigating the mechanisms which may mediate the effects of protein on energy and glucose homeostasis. The long-term effects of high protein diets on the synthesis and secretion of gut hormones in mice was investigated, and a trend was observed for increased PYY expression in the jejunum, the site of protein digesta absorption. Amino acids are products of protein digestion and have been suggested to regulate satiety and appetite in response to protein intake. Findings from our group have identified various appetite supressing amino acids including L-Cysteine, L-Arginine and L-Phenylalanine. The mechanism underlying L-Phenylalanine's anorectic effects were further investigated. L-Phenylalanine decreased food intake, without apparent behavioural side effects, increased neuronal activation in the dorsal vagal complex (DVC), and stimulated the secretion of anorectic gut hormones and pancreatic hormones. The secretion of GLP-1 and PYY were blunted by a calcium sensing receptor (CaSR) antagonist in vitro, suggesting the CaSR may mediate the anorectic effects of L-Phenylalanine. Vagal afferents transmit peripheral sensory information from the gut to the central nervous system via nuclei in the DVC. The activation of the DVC in response L-Phenylalanine suggested the vagus nerve might be involved in mediating these responses. The CaSR is one of the few nutrient sensors expressed in the vagus. Knockdown of the CaSR in the vagal afferents of mice inhibited protein induced glucagon secretion. This suggests the CaSR may play an important role in an entero-pancreatic vagal circuit that requires further investigation. The effect of CaSR knockdown on L-Phenylalanine's anorectic effects were inconclusive. These studies examined the effects of L-Phenylalanine and high protein diets on appetite regulating hormones. They contribute to our understanding of the mechanism underlying the satiating effects of protein and identify vagally expressed CaSR as a potential nutrient sensor important in the regulation of pancreatic hormone secretion in response to protein intake. Further studies are required to determine the physiological importance of CaSR signalling in the vagus and the therapeutic potential of the CaSR system in the management of obesity.

Undernutrition is a predictor of morbidity and mortality in older adults and those living with conditions of accelerated aging such as COPD. Nutritional screening is recommended to identify at-risk individuals but there are concerns that this initial step in a pathway of nutritional care is not always completed or communicated. In addition, there is little research in community-dwelling older populations regarding how best to organise care at the hospital-community interface. Exploring existing pathways of nutritional care experienced by community dwelling older adults will help us to understand potential determinants of undernutrition that may influence their trajectory of recovery and highlight any gaps in care. The aim of this thesis was to examine nutritional risk, physical function and quality of life in a cohort of community-dwelling older adults. An observational prospective cohort study of community-dwelling, older adults demonstrated that individuals at medium and high risk of undernutrition were at higher risk of mortality and suffered a worse trajectory of recovery following hospital admission compared to their counterparts at low nutritional risk. However, cross-sectional analyses using data from a large cohort of COPD patients attending pulmonary rehabilitation between August 2009 and January 2015 showed that whilst medium/high nutritional risk was associated with worse strength and function prior to a period of respiratory rehabilitation, it did not influence performance, with similar benefits observed in low and medium/high nutrition risk groups. This thesis suggests that irrespective of nutritional status, older adults recently discharged from hospital or attending outpatient rehabilitation programmes exhibited reduced ability to perform activities of daily living, however, that individuals at nutritional risk experience a reduced rate of recovery, suggesting that worse nutritional status may compound adverse effects on physical function and quality of life following hospital admission. However, nutritional support may have the potential to reduce further deterioration in at risk participants.

Obesity is a fast-growing epidemic that poses a major challenge to the public health. There is a current lack of safe and effective anti-obesity treatment options, therefore an improved treatment option is critical. Short chain fatty acids (SCFAs), produced in the colon via fermentation of non-digestible carbohydrates by gut microbiota, activates FFA2 and FFA3, G-protein coupled receptors (GPCRs) that stimulate the release of anorectic gut hormones GLP-1 and PYY. However, the underlying molecular mechanisms stimulating their release are poorly understood. A fundamental mechanism controlling the signalling capacity of GPCRs is via receptor trafficking to diverse cellular compartments such as early endosomes (EE), or very early endosomes (VEE). A subpopulation of VEEs contains the adaptor protein APPL1, essential for driving receptor recycling from the VEE and regulating endosomal G protein signalling. I therefore characterised the signalling pathways exerted by SCFAs in intestinal enteroendocrine cells and colonic organoids and elucidated the trafficking properties of FFA2 that regulate anorectic gut hormone release. In enteroendocrine cells, SCFAs are unable to elicit Gαq/11-signalling but robustly activates Gαi/o signalling which is important for propionate induced GLP-1 secretion. FFA2 undergoes both constitutive and ligand induced internalisation. Following ligand-induced internalisation, FFA2 traffics to the VEE to activate Gαi endosomal signalling that is regulated by APPL1. In addition, by employing high resolution single vesicle imaging, I unveiled propionate-induced FFA2 recycling is APPL1 dependent. I also examined the dependence of receptor internalisation and found that receptor internalisation is critical only for propionate induced- Gαi/o signalling, p38 activation and GLP-1 release, while Gαq/11 signalling occurs from the plasma membrane. Together these findings suggest an important spatial requirement for propionate-mediated activity and uncovers novel mechanisms regulating the release of anorectic gut hormone, GLP-1.

Evidence suggests that dietary resistant starch (RS) has a positive impact on controlling blood glucose levels possibly via increased colonic fermentation and production of short chain fatty acids (SCFA), and hence reducing susceptibility to T2D. Naturally occurring mutations of plants lacking the starch branching enzyme (SBE) result in lower digestible carbohydrate content and higher amounts of RS delivery to the colon (Bhattacharyya et al., 1990). Drawing on insights from basic plant science and genetics, this thesis aimed to improve our understanding of how the consumption of differently processed plant-based foods leads to physiological effects associated with health benefits. Wild type (RR) peas were used as the control and mutant (rr) peas as the intervention group. The effect of further processing and associated health outcomes was studied using and comparing pea flour and pea derived products from both genotypes (RR and rr). Study 1 investigated the effect of peas and pea flour on gastric emptying (GE) and acute glycaemia. GE analysis showed no differences between RR and rr peas or peas flour. However, analysis indicated different glucose kinetics. The rr genotype exhibited significantly lower glucose concentrations as opposed to the RR genotype. Study 2 investigated the effect of peas and pea flour on gastric and small intestinal digestion. Metabolomic profiling indicated different digestion patterns between the two genotypes: different glucose related biomarkers outcomes were observed with the rr genotype exhibiting lower concentrations. Study 3 investigated the effect of peas and pea flour consumption on colonic fermentation. The rr genotype significantly increased the production of SCFAs propionate and butyrate. Study 4 investigated the effect of 28 days of RR and rr pea derived products consumption on glucose homeostasis via a gut dependent mechanism. Glucose homeostasis parameters were not improved despite changes in gut microbiota composition between or within groups. The physico-chemical characteristics of the rr genotype coupled with limited starch digestibility (high RS content) had the greatest impact on lowering postprandial blood glucose in the acute studies. Changes in gut microbiota are a consequence of lower carbohydrate digestion in the small intestine and increased delivery of RS to the colon and did not alter glucose homeostasis biomarkers over 4 weeks of consumption.

Type 2 diabetes mellitus (T2D) and obesity are two of the key health challenges of modern society. With an ageing population and increasing life expectancy, advanced T2D, including those treated with insulin, is now frequent within clinical practice. Effective treatment of glycaemic control in those treated with insulin relies at present on insulin intensification, that although effective at managing blood glucose, is associated with weight gain and an increased risk of cardiovascular disease (CVD) mortality. Bariatric surgery has been shown to be one of the most effective treatments for obesity and T2D. One key mechanism by which bariatric surgery aids diabetes control is through energy restriction. The use of formula low energy diet (LED) to replicate this and attendant significant weight loss has been observed to bring about T2D remission in those with recently diagnosed T2D patients. However, evidence for the use of these formula diets is lacking in those T2D patients concurrently treated with insulin. The work presented in this thesis aimed to investigate the role of formula low energy diets in obese type 2 diabetes patients concurrently treated with insulin. Investigation 1 studied the effect of a formula LED, over a 24-week period, on weight loss insulin usage, and glycaemic control in obese T2D patients treated with insulin compared with gold standard clinical care (GSC). The presented data demonstrates that those on an LED had greater weight loss, improvement in HbA1c, and reduction in insulin usage compared to GSC. Investigation 2 studied the effect of weight loss induced by an LED on surrogate markers of CVD. Compared with GSC, there were key reductions in triglycerides, diastolic blood pressure and high sensitivity C-reactive protein. Based on CVD risk algorithms, there may be a CVD benefit of weight loss through LED, at least in the short term. The work presented in the thesis helps to support the use of formula LED as a therapeutic treatment for complex T2D patients. The exact mechanisms by which the LED brings about the observed improvements in glycaemic control and reduction in insulin usage remains be determined.

There is a high prevalence of T2DM and CHD in South Asians who have migrated to developed countries including UK, compared to their Caucasian counterparts. Studies investigating genetics, obesity, and physical activity have not been able to explain the prevalence of metabolic disorders in South Asians. Dietary intake might play a role in the development of metabolic diseases suggested by published work showing contradictory result. We hypothesized that detailed dietary assessment will lead to understanding the high prevalence of metabolic disorders in South Asians. The data of a sub-cohort from LOLIPOP was used to re-examine the dietary intake and metabolic disorders in groups without and with CHD in South Asians versus Caucasians. Initially the estimated dietary records were analysed which revealed higher carbohydrate but lower SFA and alcohol intakes in both South Asian groups, a dietary profile which is not associated with metabolic disorders. It is possible that food components which are not shown in traditional dietary assessment play a role in metabolic diseases. Thus, I undertook metabolomics profiling method for serum and duplicate diet samples with the intention to get further insight in the relationship between dietary intake and metabolic disorders. 1HNMR technique was applied for both samples. The analysis for serum and food samples showed no difference between South Asians and Caucasians in the UK. But the comparison between South Asians in the UK versus India revealed higher levels of valine, citrate, and glucose but, lower dimethylglycine and phenylalanine. Food analysis revealed higher levels of citrate, glucose, alanine, and glycerol whereas, fumarate and phenylalanine were lower in South Asians in UK. Most of the identified compounds play a role in energy metabolism which raises the possibility of the effect of diet composition on metabolic disorders. Further investigation is required applying a targeted metabolomics study to identify specific pathways.

Fermentable carbohydrates have been shown to exert beneficial effects on dysregulated metabolic conditions such as obesity and diabetes by improving insulin sensitivity and glucose homeostasis, independent of weight loss. Short-chain fatty acids produced by the fermentation of these carbohydrates are considered the primary mediators of these beneficial effects. This thesis aims to evaluate the effect of fermentable carbohydrates on metabolic profiles of pre-diabetic individuals at high risk of developing diabetes and the effect of colonic propionate on glucose and lipid homeostasis in obese individuals. The work presented in this thesis comprises of two axes. The first one is centered on the development and validation of an analytical approach of quantifying SCFAs in human plasma, serum and urine. The applicability of the assay in nutritional studies is also assessed in the second aim. The second part explores the metabolic profiles of pre-diabetic and obese individuals with the employment of 1H nuclear magnetic spectroscopy and mass spectrometry analysis. Concentrations of SCFAs and alteration of the lipid profile were also assessed. The responses to dietary interventions were compared using multivariate statistics. The fermentable carbohydrate inulin increased the production of postprandial SCFAs in pre-diabetic individuals but did not exhibit a significant effect on the overall metabolic profile. Colonic propionate improved the postprandial lipaemia in obese individuals and exerted higher microbial derived metabolites suggesting a role for propionate in the lipid homeostasis in obesity. Propionate levels in the circulation were not affected but systemic acetate significantly increased during the meal test. Finally, the controlled intervention with the non-fermentable carbohydrate cellulose improved lipid profiles in pre-diabetic and obese individuals.

Long hours and shift work have been associated with cardiometabolic disease risk. Despite diet being an established modifiable risk factor, few studies have examined how dietary behaviours vary in relation to working hours. The aim of this thesis was to investigate the relationship between working hours and diet quality with markers of cardiometabolic health. Cross-sectional analyses were conducted using data from The Airwave Health Monitoring Study - a British police occupational cohort (n=5,849). Number of weekly working hours was determined from questionnaire data. The Dietary Approaches to Stop Hypertension score was calculated using 7-day diet data to measure diet quality. Markers of cardiometabolic health included: adiposity (body mass index, waist circumference and body fat), blood pressure, cholesterol, HbA1c and C-reactive protein. Sub-group analyses were conducted in participants with available shift work data (based on police radio records) (n=2,323). As part of this thesis a revised food diary and shift work questionnaire were developed and piloted for use in future studies. Male employees (n=3,332) working >49hrs per week (vs. 35-40hrs) were more likely to have a dietary pattern associated with elevated cardiometabolic risk. There was a positive dose-response relationship across working hours (!35-40, 41-48, 49-54, !55hrs per week) with markers of adiposity in male employees. Diet quality did not modify this association. Based on limited shift work data: night workers (vs. day) were found to consume a higher quantity of sugar-sweetened beverages. However shift work was not associated with increased cardiometabolic risk. This thesis suggests a sex specific positive association between weekly working hours and adiposity that is independent of established risk factors. Temporal eating pattern and previous shift work data collected using the revised food diary and shift questionnaire will be important to future studies exploring the relationship between diet, work hours and health.

Propionate is a short chain fatty acid produced in the colon via fermentation of undigested foods. Elegant in vitro and in vivo studies have shown that increased production of propionate in the gut can reduce body weight and energy intake, and improve glucose homeostasis. These positive effects have been attributed to increased levels of the gut hormones, PYY and GLP-1, increased energy expenditure and improved beta-cell function. Furthermore, bariatric surgery increases levels of colonic propionate and this likely contributes to the huge weight loss seen with this treatment. Additionally, other short chain fatty acids and the consumption of fermentable carbohydrates to increase levels of short chain fatty acids can delay gastric emptying and affect the central regulation of appetite. Taken together, these findings suggest propionate could have several beneficial health effects in humans. Inulin-propionate ester was developed to deliver propionate to the colon. Effects of acute delivery of propionate to the colon with a single dose of inulin-propionate ester was investigated in healthy human volunteers on energy intake, subjective ratings of appetite and satiety and plasma glucose and insulin levels. Potential mechanisms for any effects were explored by assessing plasma levels of PYY and GLP-1, gastric emptying using 13C-octanoic acid, energy expenditure using indirect calorimetry, and central effects using fMRI. In addition, encapsulated sodium propionate was used to assess delivery to the small intestine on appetite and glucose and insulin levels. Acutely increasing colonic levels of propionate significantly reduced energy intake, increased satiety, increased levels of PYY and GLP-1, and reduced anticipatory food behaviour in humans. No effects were observed on gastric emptying, plasma levels of glucose and insulin and energy expenditure. In addition, delivery of propionate to the small intestine did not affect appetite or plasma levels of glucose and insulin. Colonic propionate significantly reduced energy intake and reduced appetite likely through increasing levels of GLP-1 and PYY and reducing anticipatory food behaviour. In addition, propionate does not affect appetite or glucose and insulin levels via the small intestine. As obesity is a growing epidemic, it is likely that interventions that increase colonic propionate levels could prevent weight gain or drive weight loss by regulating energy homeostasis.

Propionate, a product of microbial fermentation, is thought to play a key role in appetite regulation and energy homeostasis. Supplementation with inulin-propionate ester (IPE), which selectively increases colonic propionate production, has previously been reported to reduce energy intake and increase peripheral anorexigenic gut hormone concentrations acutely in humans. Furthermore, long-term IPE-supplementation prevented weight gain and reduced intrahepatocellular lipid content. This thesis aimed to further investigate the effect of colonic propionate on appetite regulation and energy homeostasis in humans. Study 1 investigated the effect of L-rhamnose, a propiogenic non-digestible carbohydrate, on gut transit rates using a dual 13C breath test combined with breath H2. The presented data suggest that L-rhamnose delays gastric emptying rates, increases circulating propionate concentrations and is fermented in the small intestine despite it previously being considered to reach the colon intact. Study 2 investigated the effect of IPE on brain responses to food picture stimuli during an fMRI food picture evaluation task. IPE reduced brain responses to food picture stimuli in the striatum, a brain region involved in reward processing, and reduced their subjective appeal, and reduced energy intake. Study 3 investigated the effect of IPE following a 7-day supplementation period with food products containing IPE (IPE-FP). IPE-FP reduced energy intake. IPE-FP also significantly increased fasting and post-prandial energy expenditure. However, this effect was lost after correcting for lean mass. There was also a reduction in fasting non-esterified fatty acid concentrations following IPE-FP. Finally, study 4 investigated the effect of propionate on liver fat accumulation in two human hepatocyte cell lines. Propionate did not reduce liver fat accumulation in vitro, which suggests the effects observed in vivo may be indirect. The majority of work presented in this thesis supports a role for propionate in energy homeostasis. However, the exact underlying mechanisms remain to be elucidated.

Metabolic flexibility is defined as the ability of an organism to adapt from fuel oxidation to fuel availability. It is well established that obesity results in loss of metabolic flexibility, however, the exact mechanism remains unclear. Therefore, it is critical to understand the mechanism underlying metabolic flexibility to tackle the epidemic of obesity. An energy-restricted diet will lead to weight loss. However, little is known regarding the mechanisms behind the improvement in metabolic flexibility with weight loss following an energy-restricted diet. In investigation 1, I compared the effects of an energy-restricted diet and a control diet over 12 weeks on overweight and obese volunteers to elucidate the underlying mechanism of metabolic flexibility. The results demonstrated that weight loss is associated with significant reductions in body fat composition, waist circumference, hip circumference, visceral fats, blood pressure, and an improvement in postprandial insulin sensitivity. This translates into an improvement in metabolic health and metabolic flexibility. However, the underlying mechanism remained unclear. Furthermore, dietary modification using short chain fatty acids provides an attractive avenue to improve metabolic flexibility. However, the evidence for the role of short chain fatty acids in improving human metabolic flexibility is lacking. In order to investigate the effects of short chain fatty acids on metabolic flexibility, I used encapsulated sodium propionate, which is a novel method to explore its effects on glucose homeostasis. Investigation 2 consists of two parts. The first part of the study was to demonstrate the pharmacokinetic profile of encapsulated sodium propionate. The results demonstrated no significant changes in serum propionate concentrations following ingestion of encapsulated sodium propionate. The second part of the study was designed to establish the most effective dose of sodium propionate in improving glucose homeostasis. Results from this part of the study demonstrated no significant change in beta cell function.

Background: Cardiovascular disease (CVD) is one of the leading causes of death worldwide, driven by various metabolic risk factors and diabetes. Diet is a key modifiable component in the development of these risk factors. This study aimed to investigate cardio-metabolic risk factors associated with diet. Secondly, dietary interaction with genetic risk of diabetes. Methods: A cross-sectional analysis was conducted on 5,864 participants’ baseline data from the Airwave Health Monitoring Study. Participants completed a 7-day food diary and a health screening including blood sample, blood pressure and anthropometrics. Diet quality was assessed according to UK Dietary Reference Values (DRV) using the novel DRV index and the established UK nutrient profile model. The nutritional genomic association with diabetes and HbA1c% was studied using a polygenetic risk score (GRS). Results: A higher DRV score was associated with a lower waist circumference ( -0.56 p < 0.0001), total cholesterol ( -0.06, p < 0.0001) and glycated haemoglobin A1c ( -0.02, p=0.003). Individuals with a higher DRV score (top quartile vs bottom quartile) were less likely to have diabetes (OR 0.75, p=0.04), elevated blood sugar (OR 0.80, p=0.01) and abdominal obesity (OR 0.64, p < 0.0001). GRS was associated with a higher HbA1c ( 0.03, p < 0.0001) and higher risk of diabetes (OR 1.13, 95%CI 1.04-1.24). Interactions between GRS and higher diet quality modified the genetic effect on HbA1c (-0.017, pinteraction=0.02) in high-risk individuals. High alcohol intake (>16g/day) modified the genetic effect on HbA1c ( 1.22, p=0.002). Obesity (BMI >30kg/m2) increased the effect on HbA1c by =1.90 pinteraction =0.0006. Conclusion: Individuals consuming a high quality diet (high in complex carbohydrates, fibre, fruit and vegetables and wholegrain) aligned with the UK guidelines were associated with fewer metabolic risk factors for CVD and diabetes. A high quality diet also reduced the genetic effect on HbA1c and diabetes in high-risk individuals. These findings support the importance of encouraging a healthy diet, along with moderate alcohol consumption, as part of public health advice for a lower risk of CVD and diabetes, especially in high genetic risk groups.

The current treatment for cancer is focused on chemotherapeutic treatments which can lead to many side effects including nausea, vomiting and chemoresistance. Combating chemoresistance is a key target in cancer therapy, along with enhancing the effects of current chemotherapy treatments. One progressive novel approach being investigated is pre-treating (“priming”) cells prior to chemotherapeutic treatment. Evidence has shown that “priming” of cancer cells can sensitizes the mitochondria to respond better to chemotherapy and hence reduce severity of treatment and side effects. In this thesis, I aim to define the mechanism by which “priming” enhances cell death through the mitochondria. Firstly, cannabidiol (CBD), a phytocannabinoid that has shown to have beneficial properties in inflammation, pain relief and in reducing cancer cell progression was examined for a potential “priming” agent. Secondly, the effect of the chemotherapy agent, cisplatin, was fully defined for its role in mitochondrial function along with a combination therapy of CBD and cisplatin. And finally, “priming” with CBD was assessed for any potential benefit to cancer treatment. I have demonstrated here that “priming” with CBD can enhance the killing effect of cisplatin in cancer cells and not in control cells. More specifically, the underlying mechanism for “priming” by CBD was shown to be through the modulation of mitochondrial function by means of the high stress two-phase system p53/Nrf2 together with voltage dependant anion channel 1 (VDAC1) receptor activation. From this body of work, “priming” has been shown to have a potential in enhancing current chemotherapeutic treatment.

Intestinal microbiota play an important role in determining susceptibility to obesity. Their ability to ferment non-digestible carbohydrates, to produce short-chain fatty acids (SCFAs), is thought to be important in influencing host metabolism. However, the underlying mechanisms are poorly understood. Using mice that lack the short-chain fatty acid receptor; free fatty acid receptor 2 (FFAR2), it was found that this receptor is essential for transducing the effects of fermentable carbohydrate to reduce food intake and prevent body weight gain. These beneficial metabolic effects were associated with an increase in the anorectic gut hormone peptide YY (PYY) due to an FFAR2-dependent increase in the density of PYY-expressing cells within the proximal colon. Inulin supplementation also increased GLP-1 cell density in the proximal colon, resulting in increased circulating GLP-1 concentrations. However, this was not dependent on expression of FFAR2. Further investigation revealed that the increase in PYY cell density was associated with increased expression of the paired-box transcription factor Pax4 known to function in terminal differentiation of PYY cells. It is, therefore, apparent that SCFAs, produced by microbial fermentation of inulin, act via FFAR2 to increase Pax4 expression and thereby enhance PYY cell proliferation. The ability of SCFAs to influence enteroendocrine cell fate and potentiate anorectic gut-brain signalling represents a paradigm shift in the understanding of nutrient sensing and highlights the potential for cellular engineering approaches for the treatment obesity.

Background: Epidemiologic evidence is sparse on the role of dietary patterns that may be important drivers of high blood pressure (BP) and body mass index (BMI) levels. Additionally, dietary fibre intake in association with BP and BMI yielded inconsistent results. Objective: Investigate the relationships of eating frequency, dietary energy density, diet quality, evening energy intake, GI, GL, and dietary fibre to BP, BMI using cross-sectional data from the INTERnational study on MAcro/micronutrients and blood Pressure (INTERMAP) of 4680 men and women aged 40-59 y from Japan, China, the United Kingdom, and the United States of America. Methods: During 4 visits, eight BP, four weight and height measures, four 24-hour dietary recalls, and two 24-hour urine samples were collected. Consumption of all solid foods was aggregated into eating occasions. Nutrient density is expressed using the Nutrient Rich Food index. Multivariable adjusted linear regression models were used to estimate BP and BMI differences per 2SD higher intakes of eating occasions, dietary energy density, Nutrient Rich Food index, evening energy intake, GI, GL, and dietary fibre. Results: Compared to participants with <4 eating occasions/24-hours, those with ≥6 eating occasions/24-hours had lower average: systolic BP: 116.4 vs. 121.4 mm Hg; BMI: 27.3 vs. 29.0 kg/m2; total energy: 2127 vs. 2521 kcal/24-hours; dietary energy density: 1.5 vs. 2.2 kcal/g; and higher Nutrient Rich Food index score: 35.1 vs. 26.8. Additionally, insoluble fibre higher by 4 g/1000 was inversely associated with systolic BP (p<0.05), while soluble fibre and GI, GL showed no associations with BP and BMI. Conclusions: Results suggest that higher meal frequency may be associated with improved diet quality and lower BP and BMI. Higher intakes of insoluble fibre may contribute to lower BP and BMI. This may have implications for behavioural approaches to controlling high BP levels and the obesity epidemic.

The link between early life and development of metabolic disorders and obesity in later life has been the focus of many studies over the past decades. Fetal life and early infancy are two of the most critical periods of physiological and metabolic development and plasticity, and thus, are periods whereby a stimulus can cause long term consequences on the health of an individual (developmental programming). Accelerated weight gain during early life per se or in combination with extreme birth weights (low/high birth weight) have been postulated as factors that can affect the development of individuals with later consequences in health. However, there is not a clear understanding of the specific contribution at different ages to impaired health neither of the mechanisms involved in these alterations. In this thesis, I used an isogenic murine model with natural birth weight variation within a normal range to investigate the effects of extreme birth weights on body composition and appetite regulation at different stages of life. I compared low and high birth weights phenotypes during lactation, at the time of weaning, and a young adulthood as well as early matured age. Mice were challenged to a moderate high fat diet for 12 weeks after weaning in order to assess the effects of both birth weight and a hypercaloric feed on body composition and hypothalamic neural activity. At weaning, adiposity was positively related to birth weight and weight gain but negatively related to growth rate. Low birth weight male mice (LBWm) had a lower plasma glucose concentration but similar levels of insulin to High birth weight male mice (HBWm), indicating a degree of hyperinsulinemia. Low birth weight females (LBWf) were hyperinsulinemic and hyperglycemic compared to High birth weight females (HBWf). There was an upregulation in the expression of genes related to insulin signaling, adipogenesis/lipid metabolism and thermoregulation in Subcutaneous Adipose Tissue (SAT) of LBWm mice compared to HBWm mice but the contrary was seen in LBWf mice in respect to their counterpart. LBWm mice caught up in weight with HBWm mice at younger age than the equivalent catch up in female mice. Birth weight and diet impacted body fat patterning and appetite regulation differently in both young males and females. However, at week 51 of age (early matured age), diet seemed to override the effects of birth weight on total body fat. LBWm mice tended to have smaller adipocytes than young and matured HBWm mice, especially when fed a HF diet, and this pattern was independent of fat mass. In conclusion, the current study suggests that extreme birth weights in an isogenic mouse model (within natural birth weight variation), as well as postnatal nutrition influenced growth, glucose / lipid metabolism, body fat patterning and appetite regulation in an age-gender dependent manner.

Subjects with prediabetes are at much higher risk of developing diabetes than healthy subjects. Weight loss helps reduce the risk of developing diabetes in these subjects, but long-term weight loss is difficult to achieve due to increases in appetite. Consumption of fermentable carbohydrate has been shown to reduce food intake and body weight, and also improve insulin sensitivity independent of weight loss. Therefore, fermentable carbohydrates may help prevent the development of diabetes in subjects with prediabetes via a two-pronged effect. This thesis comprises three investigations which examine the effects of inulin compared to cellulose control on 1) appetite and food intake, 2) measures of glucose homeostasis and 3) a long-term randomised control trial examining the effect of inulin on weight loss maintenance. In each investigation, subjects take 30g/inulin a day following a 4-week dose-escalation period. In investigations 1 and 2 all subjects take both inulin and cellulose supplements for a 6-week period each, separated by a 4-week wash-out phase. Investigation 3 is a randomised control trial comprising a 9-week weight loss phase, during which participants aim to reach a 5% weight loss at 9 weeks, and a 9-week weight maintenance phase during which subjects are asked to maintain the weight they have lost. In this body of work I demonstrate that inulin reduces appetite, food intake and weight in subjects with prediabetes. I also demonstrate that inulin was significantly associated with an increase in early insulin secretion and GLP-1, potentially due to an improvement in the incretin effect. Inulin also appears to improve insulin sensitivity in subjects with a specific subtype of prediabetes only. Finally, I demonstrate that inulin supplementation results in significantly greater weight loss maintenance, alongside changes in body composition likely to be beneficial long-term.

Accumulation of ectopic fat in liver and pancreas are seen as key indicators of the metabolic syndrome affecting over a third of the population in the western world. Deposition of these ectopic fat depots is linked to insulin resistance, a major risk factor in the development of type 2 diabetes. Quantifying ectopic fat deposition, without the use of biopsy, has always been a challenge. Magnetic resonance (MR) techniques have emerged as the gold-standard for estimating overall fat deposition. However, little is known on the regional variation of fat deposition in liver and pancreas in human subjects. The aim of this project was to determine, using an in vivo Multi-Echo MR imaging technique, regional variations in ectopic fat deposition in liver and pancreas and assess the potential impact of environmental factors, including BMI, age, and gender. The overall ranges in liver and pancreas fat ranged from 0.69- 32.27 % and 0.40 - 24.18% respectively. There was a strong correlation between liver fat, body weight, waist-to-hip ratio and waist circumferences. A significant correlation between pancreatic fat and waist-to-hip ratio, particularly in men, was also observed. The relationship between liver and pancreatic fat was surprisingly weak, suggesting different mechanism of accumulation. Regional variations in liver and pancreatic fat were assessed by using surgical segmentations as anatomical markers, resulting in 8 anatomical segments for the liver and 3 for the pancreas. Intra-abdominal adipose tissue and age had the largest effect on liver and pancreatic fat deposition. Relative physical activity influenced the overall levels of liver ectopic fat content but did not significantly affect regional distribution in the pancreas. No gender differences in fat distribution in the liver were detected but a strong correlation with pancreas and men was detected. Life-style interventions, such as weight loss programs, showed significant reduction in overall liver and pancreatic fat content. As for bariatric interventions, more significant reduction of overall ectopic fat in liver was seen with an increase in total and regional pancreas fat content. In conclusion, I have shown, utilising an anatomical compartmentalisation of the liver and pancreas, that there are significant heterogeneity in fat deposition in the pancreas, and that different regions respond variably to lifestyle interventions. There is a more homogeneous fat deposition in the liver, which is more consistently modulated in response to lifestyle changes.

In the present context of worldwide-prevalent obesity, optimisation of existing weight loss strategies and development of novel population-wide interventions is critical. Roux-en-Y gastric bypass (RYGB) is considered the gold-standard treatment for obesity. Weight loss is recommended prior to surgery to reduce liver size and risks of complications. However, little is known regarding the optimal pre-operative diet type and duration. In a first investigation, I examined the effect of three pre-operative diets on liver fat content (IHCL), as a proxy of liver volume, in obese patients undergoing RYGB. I compared the effects of a 2-week and 6-week liquid formula 800kcal/day low-calorie diet (LCD) with a low-carbohydrate, high-protein 1000kcal/day conventional food diet (control) prescribed routinely in bariatric clinics. The 2-week and 6-week LCD induced a 40% and 67% reduction in IHCL respectively compared with the control diet, which did not induce any significant loss in IHCL. In the overweight population, dietary changes remain the first-line treatment strategy to induce weight loss. Certain food components promote satiety by altering gastro-intestinal (GI) hormone release, and may therefore play a role in the prevention of obesity and type-2 diabetes mellitus (T2DM). Mycoprotein, a novel fungal food containing protein and fibre, reduces energy intake acutely and improves glucose homeostasis in lean individuals. In a second investigation, I explored the effect of mycoprotein on energy intake, glucose homeostasis, and GI hormones release in overweight volunteers. Mycoprotein significantly reduced acute energy intake by 10% compared with chicken in overweight individuals, without significantly altering GI hormones, and gastric emptying. Furthermore, mycoprotein reduced post-prandial insulin and improved insulin sensitivity compared with chicken, showing its potential health benefit in overweight individuals. These studies suggest that a 2-week pre-operative LCD may be optimal in the preparation for RYGB and that mycoprotein might be of benefit in the dietetic prevention of obesity and T2DM.

The current obesity epidemic poses a major challenge to public health. Apart from bariatric surgery, a safe and effective long-term treatment for obesity has yet to be identified. Therefore, an improved understanding of the physiology of energy homeostasis is now more critical than ever. Epidemiologically, there is an inverse association between consumption of fermentable fibre and weight gain. This is supported by experimental studies, which demonstrate that increasing the consumption of fermentable fibre can reduce appetite and body weight. It is thought that these effects may be attributable, at least in part, to the bacterial fermentation of fibre in the colon yielding short chain fatty acids (SCFAs). SCFAs have been identified as ligands for the G-protein coupled receptors GPR41 and GPR43. There are fundamental gaps in our current understanding of the physiological roles of SCFAs and their receptors in energy homeostasis, and of their specific effects in different endocrine tissues. This thesis investigates the effects of colonic SCFAs on anorectic gut hormone release from enteroendocrine L cells, and the role of GPR43 in mediating these effects. I established mouse and human primary L cell models in our laboratory and used them to demonstrate that SCFAs, and particularly propionate, stimulate the release of anorectic gut hormones peptide YY (PYY) and glucagon-like peptide 1 (GLP-1). I confirmed that colonic administration of propionate increases plasma gut hormone concentrations in vivo, in rats and mice. I then explored the mechanisms underlying propionate-induced gut hormone release in vitro. I demonstrated that Gpr43-/- L cells exhibit significantly attenuated PYY and GLP-1 secretion in response to propionate; a result which was also confirmed for the first time in vivo using Gpr43-/- mice. These findings suggest that colonic SCFA signalling via GPR43 may play an important physiological role. Further investigation is now required to characterise the intracellular signalling mechanisms underlying GPR43-activated gut hormone secretion and to determine the wider role of GPR43 in energy homeostasis.

Dietary supplementation with fibre has been shown to ameliorate features of the metabolic syndrome and inhibit malignant growth in certain types of cancer. These effects have been linked to short-chain fatty acids (SCFA), mostly acetate. However, the ubiquitous role of SCFAs in metabolism, combined with a short tissue half-life and the non-targeted nature of oral and peripheral administrations make achieving phenotypically relevant levels of SCFA by standard delivery techniques challenging and limit their therapeutic potential. Liposomal encapsulation of a therapeutic agent overcomes these issues by protecting against degradation, increasing circulation time and passively targeting both the liver and tumour tissue. In this research project, I have designed a bifunctional liposome formulation to transport SCFA, monitored their distribution and uptake utilising visualisation by MRI, PET/CT and fluorescence microscopy. These bifunctional liposomes were useful for effectively encapsulating small molecules within their aqueous core, which in this case was acetate, and capable of acetate delivery into cells while also being amenable to cellular imaging. I have shown that preferential delivery of liposome encapsulated acetate (LITA) nanoparticles to key sites of metabolic control provide beneficial therapeutic effects in animal models of both obesity and cancer. Chronic administration of LITA nanoparticles in an obeseogenic model led to a significant reduction in adiposity, intrahepatocellular lipid, inflammatory tone and genetic indication of a decrease fatty acid synthesis in the liver. Application of LITA in a murine xenograft model caused an inhibition of tumour growth in three colorectal cancer cell lines: HT-29, HCT116 p53+/+ and HCT116 p53-/-. The mechanisms for these two outcomes are not fully defined; however cellular energy homeostasis of both scenarios was restored. These results indicate that LITA nanoparticles can be used to improve multiple metabolic pathways, in vivo.

Loss of appetite is frequently observed during ageing; termed the ‘anorexia of ageing’. Some evidence suggests that with advancing age there is an increase in satiety hormones, such as CCK and PYY, and a decrease in the hunger hormone ghrelin which may contribute to the anorexia of ageing. However, not all studies agree, emphasising the need for more in depth research to clarify age related changes. My first study developed a rat model to replicate the hormonal responses observed in humans. However, the results of this study did not replicate human studies; there were no alterations gastrointestinal hormones. I then designed a human study to examine more comprehensively, the effect of ageing on gastrointestinal peptide hormone release. Healthy human volunteers aged 20 to 92 years were studied in fasting and post-prandial conditions. The results demonstrated an increase in PYY concentrations post-prandially in older volunteers. In order to understand the mechanism behind this, I followed this study with an investigation looking at the changes in gastrointestinal hormones in ageing mice colons. The data produced results conflicting with the human study, since no increase in PYY levels was observed. Dietary manipulation provides a good opportunity to alter gastrointestinal appetite hormone release, since release differs depending on the macronutrient content of the food consumed. My final studies focused on high protein diets. Results demonstrated that older volunteers were insensitive to the satiating effect of protein, where younger volunteers ate less following a high protein test meal but the older volunteers food intake did not change. Gastrointestinal appetite hormones concentrations did not change significantly between the high and low protein meals in the older and younger adults. These results suggest that age-associated reductions in appetite could be due to a resultant increase in the satiety hormone PYY. More studies need to be conducted to understand the role of protein on appetite and food intake in older adults.

AIMS The purpose of this thesis was to fully quantify the dysregulation between body mass index (BMI) and body adiposity in 3 phenotypically different groups of adults for whom BMI may be particularly unreliable. The 3 groups were: 1. The ‘Thin Fat’ , 2. The ‘Fat Fit’ 3. South Asians. A secondary purpose of the thesis was to evaluate the accuracy of 2-C body composition devices and proxy measurements to accurately assess regional and whole body adiposity. METHODS In order to establish a cohort baseline, whole body and regional adiposity were quantified using MRI and MRS. Cohort = 500 healthy adults. Participant’s adiposity data obtained were: TAT, SAT, ASAT, NASAT, IAAT, IHCL, S-IMCL, S-IMCL. Anthropometric data included: height, weight, waist circumference, hip circumference (and skinfolds in some sub-groups). Study 1: (A). In 21 healthy non-obese, males; 4 different 2-C body composition techniques (UWW, BIA, SKF, ADP) were compared to MRI adiposity data. Study 1: (B). In 74 adult Caucasian (40 females and 34 males) abdominal adiposity was measured using an abdominal BIA device (Viscan) and compared to MRI adiposity. Study 2: In 477 participants (343 male & 234 female) an in-depth comparison of BMI was conducted to identify TOFI individuals by developing a clinical index from the abdominal internal fat: subcutaneous abdominal fat (IAAT/ASAT) ratio for a normal range. Study 3: 50 males, fitness tested using VO2 max and then categorized by their fitness (fit vs unfit), and fatness (fat vs slim) according to MRI adiposity data. Study 4: 260 participants (68 Asian & 192 Caucasian) – age and BMI matched. Proxy measures WHR, WC etc compared. Apply study 3, TOFI cut-off to Asians adiposity data. RESULTS From the baseline adiposity data I confirmed that there is a wide range of regional body fat distributions (internal abdominal adipose tissue, IAAT; and abdominal subcutaneous adipose tissue, ASAT) by BMI, and that individuals with similar BMI values can show great variation in IAAT and ASAT. Study 1. (A). When whole cohort data were compared to MRI adiposity data there was no significant difference between the measures derived. However when the cohort was divided by ethnicity (Asian vs Caucasian) differences were more apparent. Caucasian adiposity was overestimated by up to 3% and Asian adiposity was underestimated by up to 11%. BodPod would be best suited to measuring Asian adiposity and BIA devices would be best suited to measuring Caucasian adiposity. Study 1. (B). The abdominal adiposity device (Viscan) using BIA method was not able to accurately measure IAAT in obese males and females. It appeared better at measuring subcutaneous adiposity (ASAT). It also appeared to be influenced by organ volumes in some cases – particularly the liver. Study 2. The ‘Thin on the Outside – Fat on the Inside’ (TOFI) phenotype can be defined using the ratio of IAAT and ASAT (IAAT/ASAT). The resulting TOFI index provides a quantitative means of comparing intra-abdominal fat deposition and thereby identifying “at risk” individuals. In Caucasians, cut-off values of >1.0 in males and >0.45 in females are proposed for TOFI definition. Additionally, anthropometric measurements such as waist circumference (WC) and waist to height ratio (WHtR) are not appropriate for classifying the TOFI phenotype. This is because these surrogates generally correlated more with total and subcutaneous adipose tissue stores than internal or ectopic depots. Study 3. IAAT and liver fat are lower in men who are fat, fit and active than in men who are fat, unfit and inactive. These ‘metabolically healthy’ individuals have the capacity to store excess fat in insulin-sensitive abdominal subcutaneous adipose tissue (ASAT) and this may help explain why the risk of chronic disease is lower in the ‘fat-fit’ than the ‘fat-unfit’. As a consequence, aerobic activity and the pursuit of physical fitness may be more appropriate goals in the battle against chronic disease than weight loss. Study 4. Asian Indian males were found to be significantly ‘fatter’ with significantly higher subcutaneous fat depots compared to similar Caucasian males. Given the increased metabolic risks seen in the Asian population increased IAAT measures were not found to be significantly higher. Additionally, the TOFI classification was not useful in identifying ‘at risk’ individuals in the Asian group. Also, waist circumference measurements did not identify Asian males that had significantly elevated ASAT. However, elevated liver fat stores were seen in Asian males and females compared to Caucasians. Liver fat may therefore be a potential ‘at risk’ identifier in this ethnic group. CONCLUSION The results of this thesis confirm BMI may be an inexpensive, non-invasive measure of obesity for predicting the risk of related complications, but its accuracy is limited by its dysregulation with adiposity. While obesity means excess body fat, the current definition of obesity using BMI is based on body weight regardless of its composition. The studies in this thesis have highlighted that fact that there are several different sub-populations of individuals for whom BMI does not tell the whole story. The Fat-Fit, the TOFI and the Asian Indian are specific phenotypic examples of these sub-populations. This is evidence of the fact that BMI should not be considered as the only measure of obesity. The results of this thesis also confirm that some techniques to measure adiposity are suboptimal for measuring percent body fat. For this reason MRI and other high quality (and high cost) imaging methods are still the best method for health risk based research.

This thesis investigates the ability of fermentable carbohydrates to stimulate anorectic gut hormones, which in turn reduce appetite and body weight. Fermentable carbohydrates have been shown to suppress food intake and body weight in rodents via the release of satiety hormones, glucagon-like peptide 1 (GLP-1) and peptide-YY (PYY). However, evidence of the effect of fermentable carbohydrates on modulating body weight in humans is contradictory. In this thesis, supplementing oligofructose over eight weeks has been shown to significantly reduce hunger and increase PYY secretion in overweight volunteers. However, there was no suppression on energy intake or body weight when compared with cellulose supplementation. Studies in rodents also suggested that reductions in food intake and body weight following intake of fermentable carbohydrates are associated with activation in the central nervous system. In contrast, oligofructose supplementation in this study had no significant effect on reducing activation in pre-selected brain reward regions in response to visual food cues, as measured by functional MRI. Surprisingly, the activation in these brain regions was reduced by cellulose intake. The negative findings from the oligofructose study may be due to insufficient concentrations of short chain fatty acids (SCFAs) in the colon. SCFAs, the fermentation end-products of ingested fermentable carbohydrates are thought to play a significant role in modifying energy homeostasis by stimulating PYY and GLP-1 from enteroendocrine L-cells in the colon. By applying a novel method of delivering propionate to the colon using a propionate carrier molecule (PCM), it has been shown that supplementation with PCM in healthy lean volunteers reduced hunger and energy intake in pilot studies via elevated PYY secretion. PCM also has a dose-dependent appetite inhibiting effect. The preliminary results in this thesis open up an interesting possibility of the development of the PCM as a dietary supplement to aid weight loss.

Muscle mass loss in the critically ill is substantial, between 1-2% per day. Muscle wasting and weakness delays patient recovery and rehabilitation. Understanding the relationship between nutritional and inflammatory markers will help to identify a notional “nutritional tipping point”, where anabolism exceeds catabolism, which will allow us to target nutritional therapy more successfully. Muscle depth on ultrasound, inflammatory markers, cytokines, urinary markers of protein breakdown and nitrogen balance data were collected in 78 critically ill patients. Patients lost a significant amount of muscle depth during their intensive care unit (ICU) stay. Results indicate that a nutritional tipping point is unlikely to exist during an ICU admission. Data suggests that nitrogen balance remains negative over 20 days, indicating that whilst patients should continue to receive standard nutrition support during their ICU stay, any additional nutritional strategy should be targeted outside of the ICU environment. Multilevel modelling indicated that each additional day spent on ICU was significantly associated with decreased C-reactive protein, muscle depth and interleukin-10 levels. With every year increase in a patient’s age muscle depth and 3-methylhistidine levels were significantly reduced and interleukin-10 levels were significantly increased. Sample size estimates from current data provide some guidance for future studies, however estimates should be viewed with caution. The feasibility of providing a leucine-enriched essential amino acid supplement to trauma ICU patients was furthermore assessed. Inflammatory and nutritional makers were assessed along with protein turnover. Although it was feasible to use the supplement in the trauma population, studying this patient group was more complicated than anticipated. Whole body protein turnover was enhanced in trauma patients with breakdown far exceeding synthesis. These studies suggest that although it may be feasible to provide amino acid supplementation to critically ill patients, this type of intervention should be targeted outside of the ICU environment.

Dysphagia can be problematic for many patients after laryngectomy surgery. While laryngectomy patients have a low risk of aspiration, they often experience other symptoms of dysphagia. There is no standard protocol for swallow assessment and no consensus on the best evaluation tool to use post laryngectomy. The purpose of the first part of this study was to establish Fiberoptic Endoscopic Evaluation of Swallowing (FEES) as an alternative tool to Videofluroscopy for the evaluation of dysphagia after laryngectomy and to develop a rating scale suitable for use with both evaluation tools. The purpose of the second part of the study was to examine whether voice prostheses have an effect on swallowing and whether an optimum voice prosthesis exists for swallowing and voice. A prospective observational study was carried out on 30 laryngectomy subjects who had simultaneous videofluroscopy and FEES swallow exams. These were rated by 3 expert examiners to determine whether endoscopy could be established as swallow evaluation tool suitable for laryngectomy patients. This was followed by a second prospective observational study on 41 subjects which involved each subject undergoing both swallowing and voice evaluations on 4-6 different voice prostheses. Results of the first part of this study establish FEES as an alternative tool to videofluroscopy. A rating scale was successfully developed for use with both videofluroscopy and FEES post laryngectomy swallow evaluations. Results of the second part of this study indicate that both swallow and voicing performance can change for some laryngectomy patients with the use of different voice prostheses. The hypothesis that quality of life is altered by swallowing impairment post laryngectomy is confirmed. This work improves the options available to patients and clinicians for the evaluation of swallowing post laryngectomy. In addition, it has highlighted that choice of voice prosthesis may influence swallowing ability in some laryngectomy patients.

Finding effective life style interventions is paramount in the control of obesity and its comorbidities such as NAFLD. In this thesis I investigate the effects of a short chain fatty acid (SCFA) on obesity and liver fat, using firstly two different carbohydrates, inulin (an indigestible but fermentable carbohydrate) and isomaltulose (a digestible carbohydrate). I go on to investigate the effects of acetate, through the development and implementation of a novel nanoparticle carrier to study the effects of this SCFA on hepatic metabolism. To study the effects of isomaltulose and inulin on adiposity, magnetic resonance imaging (MRI) and spectroscopy (MRS) techniques were used on mice fed on a high fat diet supplemented with these two carbohydrates. Manganese enhanced MRI (MEMRI), was used to monitor hypothalamic brain activation. A significant decrease in hypothalamic activation was detected in both inulin and isomaltulose supplemented animals in the absence of detectable phenotypic changes, including body adiposity and liver fat. As some of the effects of fermentable carbohydrates are thought to occur through the increased production of SCFAs, the potential physiological effects of one of the SCFA, acetate, were further explored. PET imaging was employed to study whole body 11C-acetate biodistribution on a murine model. The highest uptake of 11C-acetate was observed in the heart followed by liver, colon, brown adipose tissue (BAT), brain, fat and muscle (20, 10, 4, 3.6, 3, 2.9 and 2 peak %ID/g respectively). Colonic administration caused significant difference in uptake pattern of heart, liver, brain and BAT (p=0.001), muscle (p=0.0001) and colon (p=0.004) compared with i.v. No difference was observed in fed vs fasted animals. Pre-administration of “cold” acetate prior to systemic administration of 11C-acetate increased uptake of the latter in the liver, heart, brain and BAT suggesting that priming with CA saturates either the GPR43/41 receptors or the transport system of acetate In order to assess the potential beneficial effects of acetate, it was necessary to administer this SCFA in a chronic and consistent manner. Administration of large concentrations of pure SCFA to mice or human is known to have significant detrimental effects, so an alternative nanoparticle based strategy was developed for this purpose. Acetate was encapsulated in liposomal nanoparticles, capable of carrying millimolar concentrations of SCFA. In a murine model fed on normal fat diet, liposomal-encapsulated acetate significantly decreased liver adiposity, but not total body fat, while serum markers of obesity were reduced although they did not reach significance. In a murine model fed on high fat diet, liposomal-encapsulated acetate decreased whole body adiposity, liver fat content and serum free fatty acid (FFA) concentrations and serum markers of liver disease were significantly reduced whereas ketone concentrations in serum were significantly increased. This thesis shows that alterations in dietary carbohydrate composition can lead to significant effects on appetite, probably through the increase production of SCFAs. Furthermore, the use of liposomal-nanoparticles for direct SCFA delivery appears a potentially fast and effective way to treat some of the physiological and metabolic abnormalities associated with obesity.

Dietary fatty acids are thought to play a causal role in weight gain and obesity. However, data suggests that fatty acids are not all similarly obesogenic. Long chain n-3 polyunsaturated fats have been suggested to favour negative energy balance and a reduction in visceral adiposity. Their use as a functional dietary component to prevent or treat obesity is unclear in rodents and relatively unexplored in humans. I therefore aimed to investigate the effects of long chain n-3 fatty acid dietary enrichment in rats and humans on energy homeostasis and adiposity. I first investigated the effects of long chain n-3 fatty acids on energy homeostasis in rats by comparing a diet rich in fish oils, a naturally rich source of long chain n-3 fatty acids, to an isoenergetic diet based on anhydrous milk fat, a fat rich in saturated and monounsaturated fats and used in the manufacture of processed foods. Male Wistar rats were fed an ad libitum diet based on either 20% or 40% energy from fish oils or anhydrous milk fat for 12 weeks. At high fat levels (40%), fish oil feeding was associated with reduced energy intake compared to the isoenergetic anhydrous milk fat diet. However, there was no significant difference in body weight or adiposity between groups. This suggests that these two different dietary fat types may induce changes in metabolic efficiency or energy expenditure. At moderate fat levels the fish oil fed rats consumed more energy and gained more weight than anhydrous milk fat fed rats, but did not have significantly greater adiposity. I found no change in several markers of energy expenditure (uncoupling protein-1 mRNA expression, circulating thyroid hormone concentration or active behaviour), but further work is required to elucidate the mechanisms through which different dietary fats elicit the observed differences in energy intake and body weight. The high levels of long chain n-3 fatty acid supplementation delivered in rodent feeding studies are not practicable in humans. Therefore, I subsequently investigated the effects of more modest long chain n-3 fatty acid supplementation (2.25g per day of n-3 fatty acids) on energy homeostasis and adiposity in overweight and obese humans in a randomised, double-blind, placebo-controlled pilot study. The effect of long chain n-3 fatty acid supplements on adiposity, metabolic parameters and appetite were measured following 12 weeks of supplementation and following 12 weeks of supplementation in combination with a hypocaloric diet. After 12 weeks of supplementation alone, the long chain n-3 fatty acid supplemented group had non-significant reductions in intrahepatocellular lipid and plasma concentrations of the adipokine leptin. However, there was no significant change in total or visceral adipose tissue. When the supplements were taken in combination with a hypocaloric diet, there was no significant change in either total or visceral adiposity in the long chain n-3 supplemented group. Ad libitum energy intake at a test meal, energy intake calculated from a 72 hour food diary and visual analogue scales also suggested there was no effect of long chain n-3 fatty acid supplementation, on appetite or energy intake, whether alone or in combination with a hypocaloric diet. However, long chain n-3 fatty acid supplementation was associated with an increase in reported food appeal scores. In line with a possible effect of long chain n-3 fatty acid supplementation on the non-homeostatic control of food intake, I specifically examined the effects of long chain n-3 fatty acid supplementation on brain reward centre activation using blood oxygen level dependent functional magnetic resonance imaging (BOLD fMRI) in response to food picture presentation. The effect of 24 weeks of n-3 fatty acid supplementation and a 12 week energy restriction on brain reward centre revealed no significant differences in BOLD activation in any single region of interest. However, when regions of activation were grouped into a reward pathway neuronal network, activation tended to increase in those participants subjected to a hypocaloric diet and placebo supplementation, whereas those given the long chain n-3 fatty acid supplements tended to have a decrease in BOLD activation. These data suggest that long chain n-3 fatty acids may modulate non-homeostatic reward centre signalling. The studies described in this thesis suggest that long chain n-3 fatty acids may have specific beneficial effects on for example, intrahepatocellular lipid and brain reward centre activation. However, further work is required to conclusively determine whether long chain n-3 fatty acids are useful in the prevention and treatment of obesity and obesity-associated disease.

Background: To understand the pathophysiology of obesity, the role of hedonic eating needs to be considered. Neuroimaging research has suggested that activity in brain reward systems is modulated by the rewarding properties of food, nutritional state, and individual differences in eating behaviour. The interaction between these factors and the influence of gut hormones, such as the stomach-derived orexigenic hormone ghrelin, on these systems remains unclear. Methods: In the first study, 20 non-obese subjects were either fasted or ate a filling breakfast of their choice (fed). In the second study, 18 non-obese subjects were either fasted or given a fixed breakfast and received a subcutaneous saline or ghrelin injection. Subjects underwent functional MRI scanning while viewing and rating the appeal of food and object pictures and completed appetite and psychological questionnaires. Results: The first fMRI study showed that fasting increased activation to pictures of highcalorie over low-calorie foods in the ventral striatum, amygdala, anterior insula, medial and lateral orbitofrontal cortex (OFC). Fasting also biased the appeal towards high-calorie foods. Dietary restraint positively correlated with activation in executive systems when viewing high-calorie compared to low-calorie foods, but negatively correlated with activation in affective systems. Reward drive positively correlated with activation in the ventral striatum to high-calorie foods. In the second fMRI study activation to high-calorie foods in the insula and/or OFC when fasted and fed was shown to correlate with hunger and predict subsequent food intake. Administration of ghrelin was shown to mimic fasting to increase the appeal of high-calorie, especially sweet foods, and OFC activation to high-calorie foods. Conclusion: The results suggest an interaction between homeostatic and hedonic aspects of eating behaviour, with fasting and ghrelin biasing brain reward systems towards highcalorie foods.

Peptide tyrosine tyrosine (PYY) and glucagon like peptide-1 (GLP-1) are endogenous, anorectic gut hormones released from entero-endocrine L cells. The aims of this thesis were to: investigate the breakdown of PYY using peptide analogues in vitro and in vivo, determine the stimuli for release of PYY and GLP-1 from L cells in vitro and finally to attempt to stimulate the endogenous secretion of PYY and GLP-1 and so suppress appetite in man. The breakdown of PYY was studied using specially designed PYY analogues with changes to known enzyme cleavage sites. Degradation of the analogues was studied by incubation with proteolytic enzymes. Receptor binding assays were carried out to confirm that changes to the PYY structure had not altered binding to the endogenous PYY receptors. In vivo studies in rodents previously confirmed an extended pharmacological profile of these analogues. In order to study the various stimulants for PYY and GLP-1 release, a primary L cell model was developed. This was used to study the effect of various nutrients: glucose, amino acids and short chain fatty acids (SCFA) on PYY and GLP-1 release. In mouse and human primary L cell cultures the SCFA propionate increased PYY release significantly compared to basal levels, indicating that propionate is a potent stimulus of PYY release. To further investigate the results of the in vitro work, a randomised, double blind, crossover study was carried out in human volunteers to evaluate the effect of propionate on appetite. Administration of propionate ester over six days reduced energy intake at a buffet meal by 18.8% compared to control (P < 0.05, n = 20). However, there was no significant change in plasma GLP-1 or PYY levels between the groups, possibly suggesting an alternative explanation for the reduction in appetite seen. This may provide an interesting avenue for future studies. These studies of the physiological mechanisms underlying release and degradation of PYY and GLP-1 may contribute towards the development of an anti-obesity therapy based around L cell stimulation.

Patients with chronic kidney disease (CKD) are at increased risk of mortality and cardiovascular (CV) disease and those on maintenance haemodialysis (MHD) are at particularly high risk. Insulin resistance (IR) is known as a contributing factor to many of the conditions classified as "classic CV risk factors" and understanding changes in IR is thought to be of key importance in targeting potential medication. A prospective observational study of individuals with CKD was designed to better establish the relationship between CV risk factors and events, before and after the onset of MHD. In addition, a subgroup study was set up to examine if changes in IR affect CV outcome and mortality. 519 patients were recruited for this study, 210 were on MHD at entry and 309 had CKD stages 3, 4 and 5. Subjects were followed prospectively for 24 months and the relationship between body mass index (BMI), blood pressure (BP), total cholesterol (TC), triglycerides (TG), diabetes status and glycosylated haemoglobin (HbA1c) and CV events analysed in both MHD and non-MHD groups using logistic regression. A small study was also carried out to determine the reliability of HbA1c in MHD subjects. 106 of these subjects were recruited into the subgroup study, where baseline assessment of their IR (using the HOMA-IR model) was carried out as well as other relevant tests. 103 subjects reached an endpoint, and analysis of the whole cohort identified high SBP and diabetes as significant CV risk factors. TC was recognized as a protective factor as lower TC was associated with higher CV risk. The mean HOMA-IR increased through the CKD stages 3-5 before significantly falling in the MHD group. HbA1c showed poor association with glycaemia as measured by use of 48-hour continuous glucose monitors (CGM). These results suggest that CV risk factors may vary at different stages of CKD. The “reverse” TC result only in the non-dialysis group differs from current theories, where low cholesterol is considered a CV risk factor putatively as a surrogate marker of malnutrition. This study shows that IR increases as renal function deteriorates but is significantly decreased with MHD. In this cohort changes in IR were not predictive of CVE or mortality.