The objective of this thesis was to further the understanding of the effect of diet, insulin and exercise on the regulation of carbohydrate metabolism in health and type 1 diabetes. Three studies were undertaken in both non-diabetic healthy volunteers and patients with type 1 diabetes. The first study determined the influence of high fat diet on biochemical and molecular regulators of whole body and muscle metabolism in healthy volunteers. The second study examined the influence of insulin on whole body and muscle metabolism in patients with type 1 diabetes during moderate exercise. The final study compared the influence of insulin and a high carbohydrate diet on liver glycogen concentrations and substrate oxidation during exercise between patients with type 1 diabetes and healthy volunteers. The main results were, a) 6 days of high fat/low carbohydrate dietary intake did not induce whole-body insulin resistance but caused a shift in intramuscular glucose metabolism from oxidation to glycogen storage when compared to a normal balanced diet. Insulin-stimulated carbohydrate oxidation and muscle PDCa activity were blunted after the high fat diet and this was associated with an up regulation of muscle PDK4 mRNA and protein expression, b) Exercise under hyperinsulinaemic conditions in patients with type 1 diabetes did not spare muscle glycogen utilisation at a time of high exogenous glucose utilization and oxidation, and finally c) Changes in liver glycogen concentration and substrate oxidation during exercise occurred at comparable rates in patients with type 1 diabetes and in healthy controls despite the presence of relative hyperinsulinaemia in the former compared to the latter group. The key conclusions are, 1) in healthy humans short-term high fat feeding does not induce whole body insulin resistance but impairs basal and insulin-stimulated carbohydrate oxidation, most likely as a result of fat-induced upregulation of muscle PDK4 protein expression. The precise signaling mechanisms involved in the chronic regulation of PDK4 need to be determined. 2) Contrary to previous observation in non-diabetic individuals, it appears that hyperinsulinaemic conditions in patients with type 1 diabetes do not suppress the exercise-induced changes in muscle and liver glycogen stores. The underlying physiological mechanism(s) behind this apparently divergent response remains to be elucidated.

Submicroscopic structural genomic variation includes copy number variation (CNV) that can result changes in DNA dosage, and the impacts can be observed on common disease, metabolism, and heritable traits such as colour vision and rhesus blood group. Human beta-defensins form a cluster of at least seven genes on human chromosome 8p23.1, with a diploid copy number commonly ranging between 2 and 7 copies. They encode small secreted antimicrobial peptides with cytokine-like properties which are found expressed at high levels in psoriasis patients, and copy number at this locus has been found to be associated with inflammatory bowel disease, particularly colonic Crohn’s disease. The focus of this thesis has been divided into two studies; looking for the origin of diversity at the human beta-defensins copy number, and development of a multiplex PRT measurement system for accurately typing the beta-defensin region in large case association study. The origin of diversity at the human beta-defensin copy number has been followed by using segregation in CEPH families. Three out of 416 meiotic transmissions changed the copy number by simple allelic recombination between two distinct loci for these genes. Deducing haplotype copy number from microsatellite and multiallelic length polymorphism have allowed this study to map the beta-defensin repeats in two locations at the original location distally REPD and about 5 Mb away at proximally REPP. We have demonstrated our multiplex PRT system is a powerful technique to determine the association of the beta-defensin genes in Crohn’s disease even though we did not produce any convincing support for associations reported from previous studies.

Ca2+ is a ubiquitous intracellular signalling molecule that is involved in the regulation of numerous cellular functions. To date Ca2+ influx pathways present in white fat adipocytes have not been characterised. Additionally impaired [Ca2+]i management is implicated in the induction of the insulin resistant state in adipocytes. As adipocytes have a prominent role in the management of energy homeostasis, the presence of Ca2+ influx pathways was examined. Initial [Ca2+]i measurements confirmed the presence of functional Ca2+ influx and efflux pathways in adipocytes. Further [Ca2+]i measurements identified the Cav1.3 Voltage-gated Ca2+ channel (VGCC). The presence of the α1 subunit of Cav1.3 channel protein in adipocytes was confirmed by Western blotting, the expression of which was reduced in adipocyte samples sourced from Zucker obese rats. Initial [Ca2+]i imaging experiments utilising conditions of elevated extracellular K+ (50mM) did not stimulate Ca2+ influx. The plasma membrane potential (Vm) regulates many physiological processes, including cellular Ca2+ influx by VGCCs, with dysregulations in Vm underlying functional pathologies. K+ is widely believed to be the predominant ion that controls Vm for many cell types, however, whether K+ regulates adipocyte Vm is also unknown, prompting, investigation into the ionic species involved in the regulation of Vm in primary and differentiated 3T3-L1 adipocytes. As insulin and β-adrenoceptors regulate adipocyte function, their effect on Vm was also explored. The Vm of primary and 3T3-L1 adipocytes were -34.14mV (n=68) and -28.5mV (n=88) respectively. Elevation of extracellular K+ from 5.6mM to 50mM had no significant effect on the Vm of either type of adipocyte. The role of Cl- on adipocyte Vm was then investigated. Reduction of extracellular Cl- from 138 to 5mM, by equimolar substitution with Gluconate significantly depolarised the Vm of both primary and 3T3-L1 adipocytes. Patch clamp investigations also revealed a role of Na+ in adipocyte Vm. Neither insulin (100nM) or the β-adrenocpetor agonist isoprenaline (10µM) significantly changed adipocyte Vm. The role of Cl- in adipocyte Vm is indicative of the presence of Cl- channels, however electrophysiological studies failed to characterise the Cl- currents underlying adipocyte Vm. Overall, further investigations are required to characterise not only the Ca2+ influx pathways in adipocytes, and the roles thereof, but also the means by which they are regulated.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, which mediates the toxicity of dioxin and related compounds, and has an important role in development. However, a structural basis for ligand binding to the AhR remains unclear and the study was hindered by the low abundance and inherited instability of the AhR. Based on a previously defined minimal ligand-binding domain (LBD, residues 230-421), in the present study a series of truncated LBD constructs were created and expressed in insect cells (Sf9) using a baculovirus expression system. An antibody was produced to analyze the expressed. The antisera can detect as low as 0.3ng of AhR LBD from cytosol of Sf9. An in vitro [3H]TCDD binding assay was developed to characterized the expressed LBD. The assay yielded an estimate for the KD of C57Bl/6 mouse liver binding at 1.4nM. The present expression system yields soluble AhR LBD protein at ~0.15% of cytosol protein. Supplementation of the Sf9 culture medium with additional glucose resulted in an increase in the amount of soluble AhR, due to an increase in intracellular ATP level. However, cotransfection of LBD with hsp90 interaction protein p23 made no substantial change in the amount of cytosolic AhR. The soluble recombinant LBD retains functionality in the form of specific binding to dioxin, and its thermal stability was indistinguishable from that of mouse liver. However the ligand-binding activity of LBD was molybdate dependent, indicating a weaker association of mouse AhR LBD with Sf9 hsp90. A differential effect of Triton X-100 on the recombinant AhR LBD and native AhR also suggests that the interaction between AhR and Sf9 hsp90 is less stable. The study refined the minimal LBD to a region of 125 amino acids, which should be amenable for structural studies of the LBD.

The regulation of metabolic gene expression is fundamental to maintaining energy balance. Changes in substrate availability can alter metabolic gene expression, in order to modify the utilisation of nutrients appropriately. Metabolic gene expression can also be dysregulated in disease states. The work in this thesis examines several situations in which energy balance or metabolic substrate supply was altered, and investigates how metabolic gene expression adapted and contributed to the phenotypes observed following these interventions. All experiments in this work looked at metabolic regulation from a human perspective; with either biopsy material or cells cultured from biopsy. These experiments included; 1. The influence of postprandial fat-oxidising capacity during a calorie restricted diet of either high- or low-fat content in obese subjects. 2. Transcriptional profiling of adipose tissue in obese subjects with high- or low-postprandial fat oxidising capacity 3. High-glucose treatment of primary human myotubes (as a model of hyperglycaemia). 4. Increased PDC activation and hence carbohydrate oxidation in vivo, through administration of dichloroacetate. Postprandial fat-oxidising capacity did not affect weight-loss during a calorie restricted diet, and there was no affect of diet composition. However, changes in metabolic gene expression were observed between groups over the course of the 10-week intervention. The groups which showed the greatest changes in gene expression were the low fat-oxidisers on a high fat diet and the high fat-oxidisers on a low fat diet, possibly due to a mismatch between diets and fat oxidising capacity, which required greater adaptation. Covariate analysis revealed interactions between gene expression and other phenotypic parameters. SREBP-1c showed a relationship with FFA concentrations and insulin-resistance, whilst HSL and apM1 were associated with FFA concentrations and Insulin resistance respectively, which underlines the importance of looking for underlying structures in data. Transcriptional profiling of adipose tissue in obese subjects with high- or low-postprandial fat oxidising capacity, revealed significant differences in the expression of metabolic genes, and highlighted the importance of several transcripts; including RXRA, SREBP-1c and GLUT4 in determining the phenotype of adipose tissue. The major differences observed in gene expression between high and low fat oxidizers indicated that genes involved glucose metabolism and lipogenesis rather than beta-oxidation were the major processes that differed between the two groups. Genomic data indicated that the expression of these genes was not influenced to a major degree by polymorphisms within the population. High-glucose treatment of primary myotubes, demonstrated the significance of ChREBP and some of its targets, in inducing the expression of lipogenic enzymes, which may be linked to the accumulation of intramyocellular triglyceride. However, these data also indicated the potential for the cell to initiate protective mechanisms, of substrate handling and lipid clearance, in response to carbohydrate oversupply. Conversely, increasing PDC activity and hence carbohydrate oxidation without altering substrate availability via infusion of dichloroacetate, did not alter the expression of metabolic genes in skeletal muscle. This reflects a capacity to deal with acute changes in the activity in metabolic genes without altering their expression. In conclusion, the studies from this thesis show that important differences in metabolic gene expression can be observed during situations where energy balance and substrate availability are altered. However, flexibility within the metabolic networks means that acute changes can be countered without the need for induction or suppression of metabolic genes, and that during chronic alterations in nutrient supply, rapid adaptations and protective mechanisms are activated.

The intake of water and electrolytes is inseparable from feeding by natural or artificial means and careful attention to salt and water balance is a vital component of perioperative care and of nutritional support. Nutritional support with water and sodium restriction in post-intensive care patients with oedema, dilutional hypoalbuminaemia and fluid excess of 10 L, cleared oedema over 7-10 days, with a 1 g/L rise in serum albumin for every kg loss in weight. Return of gastrointestinal function was also observed. Accordingly, 20 patients, undergoing colonic surgery, were randomised to receive standard (>3 L water and 154 mmol sodium/day) or restricted postoperative fluids (<2 L water and 77 mmol sodium/day). Solid (72.5 vs 175 min) and liquid phase (73.5 vs 110 min) gastric emptying times were significantly longer in the standard group on the 4th postoperative day and associated with a three day longer hospital stay. In volunteers receiving 2 L of 0.9% saline and 5% dextrose infusions, on separate occasions over one hour, haematocrit and serum albumin concentration fell, mainly due to dilution. While dextrose was rapidly excreted, two-thirds of the saline was retained after 6 h. Following 1 L infusions, plasma renin and angiotensin concentrations decreased more after saline than dextrose (P<0.04). Responses of aldosterone, atrial natriuretic peptide and vasopressin were not significantly different. Comparing 2 L infusions of saline and Hartmann's solution, volunteers excreted more water (median 1000 vs 450 mL) and sodium (122 vs 73 mmol) after Hartmann's. Hyperchloraemia and reduced bicarbonate were noted after saline alone. Whereas fluctuations in water balance are dealt with efficiently through osmoreceptors and vasopressin, and sodium deficiency by volume receptors and the renin angiotensin aldosterone system, the mechanism for dealing with sodium and chloride excess appears relatively inefficient. Natriuretic peptide responds to volume expansion rather than sodium gain.

The pyruvate dehydrogenase complex (PDC) is the rate limiting step in the entry of glucose derived pyruvate into the tricarboxylic acid (TCA) cycle. As such it plays an important role in the control of the use of carbohydrate as the source of oxidative energy for skeletal muscle contraction. The first experimental chapter investigates the effect of dichloroacetate pre-treatment during low-intensity (<60% VO2max) exercise, below which it is suggested that increasing PDC activation and resting acetyl group availability via dichloroacetate (DCA) pre-treatment will be ineffective at reducing non-oxidative ATP production and improving contractile function. Despite a significant increase in both PDC activation (p<0.01) and acetylcarnitine availability (p<0.01) prior to the onset of exercise following DCA infusion, there was no difference in substrate level phosphorylation detected during exercise. The following experimental chapter examines the link between blood lactate concentration and the onset of the ventilatory threshold. Infusion of DCA (50mg.kg-1) prior to the onset of incremental exercise lead to a significant reduction in resting blood lactate (p<0.05), but this was not preserved during the following bout of incremental cycling exercise commencing at 50% VO2max. There was also no alteration in the onset of the ventilatory threshold detected after DCA pre-treatment. The final experimental chapter has investigated the effect of DCA infusion (50 mg.kg-1) upon high-fat diet induced PDC inhibition during exercise. During moderate cycling exercise (75% VO2max) DCA infusion reversed the high-fat induced inhibition of PDC activation. DCA infusion reduced the metabolic inertia present at the onset of contraction, through both PDC activation and pooling of acetyl groups prior to contraction. This thesis has highlighted the role of the PDC as an important site of regulation of human skeletal muscle fuel metabolism, which may provide a novel target for the treatment of the metabolic syndrome.

Pluripotent human embryonic stem cells (hESCs) are a valuable tool for clinical therapies, drug testing and investigation of developmental pathways. Recently, over-expression of four pluripotency-associated genes (OCT4, NANOG, SOX2, and LIN28) has proven sufficient to reprogram differentiated cells into pluripotent stem cells, potentially alleviating the need for human embryos to isolate hESCs and opening new avenues for the investigation of pluripotency. This project aimed to generate an in vitro model system to study the epigenetic mechanisms regulating pluripotency transcription factors. hESCs were differentiated into fibroblasts (hESC-Fib) and subsequently reprogrammed to induced pluripotency stem cells (iPSCs) by lentiviral over-expression of human OCT4, NANOG, SOX2 and LIN28. iPSC colonies were positively identified by live staining with the surface marker TRA-1-81 and expanded in culture. They were then further differentiated into a fibroblast line to allow comparison with hESC-Fib. All cells in the model system shared the same genotype and were cultured under similar conditions, enabling unbiased analysis of epigenetic characteristics. DNA methylation analysis of key pluripotency-genes such as OCT4, SOX2, NANOG, and REX1 by bisulfite sequencing, revealed that these were hypomethylated in hESCs and iPSCs, and hypermethylated in their fibroblast derivatives. A gradual increase in the number of CpGs gaining DNA methylation was observed when hESCs and iPSCs were differentiated into fibroblasts, while TaqMan real-time PCR and fluorescence staining revealed that expression of these genes was inversely related to the levels of DNA methylation in their promoters. The master pluripotency regulators OCT4, SOX2 and NANOG all showed differential methylation in their OCT/SOX binding regions, suggesting a common regulatory mechanism between them. This is, to our knowledge, the first report for SOX2 differential methylation in human non-cancerous cells. Reactivation of REX1 was not found to be necessary for the reprogramming of hESC-Fib to iPSCs, calling for re-evaluation of its role in human pluripotency. Based on the observation that the DNA methylation levels of pluripotency genes were higher in fibroblast cell lines compared to hESCs and iPSCs, we hypothesised that reduction in DNA methylation could render differentiated cells more permissive to reprogramming. Stable knock-downs of the DNA methyltransferases (DNMTs) DNMT1 and DNMT3A were, thus, performed in hESC-Fib. Knock-down of DNMT1, the most abundant DNMT in hESC-Fib, resulted in global reduction of DNA methylation levels as determined by restriction digests with methylation specific enzymes. Reprogramming of hESC-Fib carrying a DNMT1 knock-down showed a 40% reduction in generation of iPSC colonies compared to untreated controls, perhaps owing to the delay in progression of S phase in the cell cycle caused by DNMT1 knock-down. In contrast, knock-down of DNMT3A resulted in a >80% increase in iPSC colony formation, potentially indicating differences in mechanism of action and specificity between the two DNMT enzymes. Through this study, we have gained new insights into the epigenetic mechanisms underlying cell phenotype and provided the foundation for further improving reprogramming efficiency.

The covalent post-translational modification of selected substrates with the ubiquitin protein has emerged as a central regulatory mechanism, governing protein stability, activity and localisation, and accordingly an array of cellular processes. Ubiquitin signalling versatility arises owing to the diverse nature of (poly)ubiquitin modification, with distinct modifications subsequently transduced in a specific manner by ubiquitin-binding domains found in ubiquitin-binding proteins. In recent years the notion that ubiquitin exerts influence solely via the covalent modification of substrates has been challenged, with unanchored, or substrate-free polyubiquitin chains emerging as key regulators of cellular physiology. The investigations described in this thesis seek to exploit the inherent selectivity of natural and engineered ubiquitin-binding proteins, to afford the purification of endogenous unanchored polyubiquitin, probing the molecular composition and interactions of this biologically significant ubiquitin pool. In chapter 3 by utilising the deubiquitinating enzyme USP5, which contains multiple ubiquitin-binding domains, and is normally responsible for the selective disassembly of unanchored ubiquitin, we purify unanchored polyubiquitin from mammalian cell extracts. Subsequently, we apply both ubiquitin-selective antibodies and mass spectrometry-based analyses to examine the polyubiquitin profile of the mammalian unanchored ubiquitin pool. In chapter 4 we then assess the mechanisms of ubiquitin recognition by USP5, presenting a structural mass spectrometry-based framework to probe and quantify ubiquitin: ubiquitin-binding domain interactions. Finally in chapter 5, based on the conclusions we draw from USP5-ubiquitin recognition in chapter 4, that multiple domains in suitable arrangement yield specificity for polyubiquitin chains, we design and synthesize a synthetic protein to favour the capture of unanchored polyubiquitin chains of defined topology, namely Lys-48 linked diubiquitin (and longer polyubiquitin chains), from mammalian cell extracts. We conclude that strategies for the rational design and engineering of polyubiquitin chain-selective binding in non-biological polymers are possible, paving the way for the generation of reagents to probe the unanchored polyubiquitin chains of defined topology, and more widely the ‘ubiquitome’.

There are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O2 tension. In high O2 tension (normoxia) the PHDs hydroxylate HIFα subunits on 2 conserved proline residues inducing binding of the von-Hippel-Lindau (VHL) tumour suppressor, the recognition component of a multi-protein ubiquitin-ligase complex, initiating HIFα ubiquitylation and degradation by the 26S proteasome. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIFα or as a multi-protein complex. In this thesis, data are presented that shows that the tumour suppressor protein LIMD1 acts as a molecular scaffold simultaneously binding the PHDs and pVHL into a normoxic protein complex (normoxiplex), increasing their physical proximity in order to enable efficient and rapid sequential modifications and thus degradation of HIF1α. Data are presented which indicates that increased LIMD1 expression down regulates HIF transcriptional activity, by promoting HIF1α degradation via the oxygen dependent degradation domain in a manner dependent on hydroxylase and 26S proteasome activities. However, degradation of this domain is not wholly dependent on the well characterised proline residues subject to hydroxylation, suggesting that LIMD1 may alter proline hydroxylation specificity or modulate HIF via a different mechanism. Furthermore, endogenous depletion of LIMD1 results in the converse, leading to HIF1α stabilisation and accumulation, enhancing HIF transcriptional activity. Moreover, Limd1-/- MEFs show increased HIF transcriptional activity. One mechanism by which this is achieved involves the binding of PHD2 within the N-terminal portion of LIMD1 while allowing concurrent binding of VHL to the C-terminal zinc-finger LIM domains. However, the LIMD1 mediated mechanism regulating HIF1α independently of proline residues 402 and 564 is still unclear. Finally, data are presented that show that the LIMD1 family member proteins Ajuba and WTIP all bind specifically to VHL but differentially to PHDs 1, 2 and 3 and thus these three LIM domain containing proteins represent a new group of hypoxic regulators.