This thesis sought to investigate how systemic lipids may contribute to the adipocyte derived inflammatory response and highlight how the adipocyte’s function can alter in different metabolic states which could contribute to the pathogenesis of type 2 diabetes mellitus (T2DM) and cardiovascular risk. Specifically, this thesis firstly examined the inflammatory nature of lipoprotein-associated phospholipase A2 (LpPLA2), a member of the phospholipase A2 super family of enzymes which previously has been shown to enhance Ox-LDL production in foam cells during arterial inflammation contributing to coronary artery disease. Therefore initial studies sought to (1) characterise PLA2 isoforms in lean, obese, T2DM abdominal subcutaneous (AbdSc) and omental (Om) in human adipose tissue (AT); (2) evaluate the role of lipids and inflammatory markers on circulating LpPLA2, and (3) determine the in vitro regulation of LpPLA2 in human adipocytes by its influence on LDL and Ox-LDL. AT and sera from lean, overweight, obese and T2DM subjects were taken. PLA2 gene expression was determined by microarray, RT-PCR and Western Blot. Associations between circulating LpPLA2 and metabolic parameters were investigated. The human adipocyte cell line, Chub-S7, was used to assess the effects of oxidized LDL (Ox-LDL) and LDL on PLA2 expression. LpPLA2 mRNA levels were higher in AbdSc AT than Om AT in obesity by 2-fold (P<0.05). The cPLA2 protein expression increased with obesity in AbdSc AT (P<0.01). T2DM showed increased LpPLA2 mRNA levels in AbdSc (P<0.001) and Om AT (P<0.01). Serum LpPLA2 showed positive correlations with cholesterol, TG, LDL, endotoxin and Ox-LDL (P<0.001) in non-diabetic subjects and with Ox-LDL (P<0.001), LDL (P<0.01) and cholesterol (P<0.05) in T2DM. In differentiated pre-adipocytes, activation of LpPLA2 protein expression was noted in response to LDL and Ox-LDL (P<0.001). The adipocyte appeared to be an active source of LpPLA2, altered by fat depot and metabolic state, with LpPLA2 protein expression induced by LDL and Ox-LDL, in vitro. Increased LpPLA2 protein from the adipocyte in obesity and/or T2DM could contribute to raise circulating Ox-LDL, as noted in other studies as well with increasing adiposity, which promotes further inflammation and atherosclerotic risk. Through the development of these current studies it appeared that how the adipocyte managed lipids was important to how the adipocyte may induce an inflammatory response and pathogenic factors. Therefore subsequent studies investigated how the change in metabolic state such as those derived in T2DM patients that undergo bariatric surgery may not necessarily reverse their inflammatory response. Previous studies from the team have shown that lipids may induce an innate immune response via toll like receptor (TLR) activation therefore subsequent investigations sought to consider the potential role of triglycerides (TG) as another mediator of inflammation. As such studies examined the specific impact of TG changes, pre- and post-bariatric surgery, on TLR expression in ex vivo AT and the in vitro effects of triglyceride rich lipoprotein (VLDL), on TLR expression in isolated human differentiated pre-adipocytes. Serum and AT was taken from a cohort of Obese, T2DM, female subjects prior to bariatric surgery and 6 months post-surgery. Human differentiated pre-adipocyte Chub S7 cells were again used to examine transcriptional effects of VLDL on TLR expression. Following surgical intervention, BMI (P<0.001), blood glucose (P<0.001), insulin (P<0.001), HOMA-IR (P<0.001), TG (P<0.05), Cholesterol (P<0.001) and LDL-cholesterol (P<0.05) were significantly improved. There was a significant reduction in TLR-4 mRNA post-surgery (P<0.01) irrespective of surgery type. It was also noted that subjects with the greatest drop (55.5% reduction) in TGs post-surgery (P<0.001) showed a significant correlated reduction in TLR4 mRNA expression (P<0.001). Whilst the in vitro treatment of differentiated Chub S7 cells highlighted VLDL induced TLR 4 mRNA expression (P<0.05) suggesting the inflammatory impact of lipids on adipocytes. These studies further highlighted that the reduction in AT inflammation appears dependent on how successfully subjects reduce their serum triglyceride, which appears supported by the in vitro findings. These studies suggest that bariatric surgery lead to metabolic improvement with weight loss, whilst dietary intervention is still required to ensure TGs reduce to reduce inflammation. Taken together, these studies and thesis highlight the diverse nature of lipids and their interaction with the adipocyte to impact on their inflammatory response. These data also highlight the importance to maintain a good systemic lipid profile low in TG to reduce adipocyte induced inflammation and that AT may represent an important therapeutic target to reduce inflammation, atherosclerotic risk and development of metabolic complications.

Hypoxia is defined as a deficiency in the amount of oxygen reaching the tissues, and is a common problem in critically ill patients. It is not currently possible to predict how well an individual will adapt to hypoxic conditions, and patients presenting with hypoxia are often treated with supplemental oxygen. However, this blanket-treatment approach is not suitable in all cases and a more personalised approach is required. My thesis project builds on information acquired during the Caudwell Xtreme Everest (CXE 2007) expedition, where over 200 volunteers trekked to Everest Base Camp. CXE uses studies on healthy volunteers exposed to extreme environments to aid in the understanding of the complicated issues concerned with critical illness, and aims to use these findings to improve the treatment of critically ill patients, without putting them directly at risk. My thesis project has combined physiological information acquired during CXE with biochemical information measured in plasma samples taken during CXE. Performance at altitude has been used as a proxy for hypoxia adaptation, with individuals who show a small loss of performance at altitude compared to London assumed to be adapting better compared to individuals who show a larger loss. Analysis of the physiological and biochemical data for a core group of 24 individuals has culminated in the application of multiple linear regression to produce a number of models capable of predicting the key changes in physiological response as a function of a number of biochemical metabolites. These models have been used to identify a set of biochemical metabolites to measure in a further 190 individuals, to allow validation and training of the models on a larger sample size. These models can then be adapted for use in a critical illness environment, to allow the prediction of how well an individual will adapt to hypoxic conditions.

Glucagon-like peptide 1 is a hormone involved in the regulation of glucose. Glucose-dependent activation of brain GLP-1R stimulates GLP-1 secretion from the L-cells of the intestine in response to ingested nutrients and it enters the blood stream to direct insulin secretion and to regulate growth and apoptosis of pancreatic cells, regulate the uptake of glucagon secretion as well as other metabolic processes. GLP-1 agonists are a new class of antidiabetes agent and current research suggests a role in weight and blood pressure reduction. A primary function of GLP-1 is postulated to act as a neuropeptide in the regulation of metabolic and cardiovascular function through gut-brain regulatory feedback. As a secondary effect, GLP-1 may also increase natriuresis by direct action on sodium transport molecules in the kidney and hence play a role in long-term blood pressure homeostasis. The aims of this thesis is to 1) ascertain the effects of GLP-1 agonists on blood pressure, heart rate and body weight in T2DM patients through a systematic review and meta-analysis and 2) to explore the expression of renal GLP-1R during metabolic syndrome (obesity) and diabetes in animals and also hyperglycaemia-like conditions in isolated cells of the human renal proximal tubule and collecting duct. I also investigate for the first time GLP- 1R mediated expression of sodium transport molecules in cells of the human collecting duct. I confirm that GLP-1 agonists produce significant weight reducing effects and also a beneficial reduction in blood pressure in T2DM patients. However, there is a tendency for these agents to increase heart rate which may be related to their effects on the autonomic nervous system and direct activation of sino-atrial node GLP-1R. I show evidence that renal GLP-1R expression is inhibited during obesity and diabetes which might be a consequence of peripheral insulin signalling or regulatory feedback from pancreatic β-cells to the CNS but which needs to be further clarified. GLP-1 was also shown to modestly up-regulate α- ENaC mRNA and protein expression, possibly as a consequence of activation by SGK-1 and PKCdependent ERK activation in HCD cells. The significance of this paradoxical finding in a clinical scenario is unknown because 1) GLP-1(7-36) should theoretically undergo enzymatic degradation in peripheral tissues and further degradation and elimination in the proximal tubule. However, potential for long-acting GLP-1 agonists to remain in the kidney for longer might mean that these agents reach the collecting duct whereby paradoxical sodium reabsorption may occur. This might also be reflected in the meta-analysis findings that exenatideLAR preparations reduce blood pressure less than shorter acting formulations. I conclude that GLP-1 signalling is dysregulated in obesity and diabetes and that treatment with GLP-1 agonists confer favourable effects on body weight and blood pressure. However, due to effects on heart rate further safety studies are required to reassure long term cardiovascular safety and autonomic mechanisms behind the cardiovascular and metabolic effects of GLP-1 needs to be explored further.

Ghrelin is a 28 amino-acid peptide produced predominantly by the stomach. Two main isoforms of ghrelin are currently known (octanoyl- and desoctanoyl ghrelin). It functions as a circulating orexigenic hormone In addition, it has an effect on the nervous, cardiovascular and immune system. Current data suggest that ghrelin may have beneficial anti-inflammatory effects. Chapter 3 in this thesis primarily examines the relationship between ghrelin and inflammation in Crohn’s disease (CD). Modulation of inflammation with infliximab, a powerful anti-TNFα antibody therapy, can increase total ghrelin concentration by 25%. In addition, a normal physiological post-prandial decrease in ghrelin following a meal is restored when infused with infliximab, suggesting a dysregulation of ghrelin in CD patients with active inflammation. At cellular level, there is evidence that ghrelin may have an immunosuppressive effect on activated T-lymphocytes. Chapter 4 of this thesis examines the effect of ghrelin, a manufactured agonist and des-octanoyl ghrelin on NFκB activation on a human Blymphocyte cell line. This study demonstrated that exposure to octanoyl ghrelin confers an initial increase of NFκB activation in inactivated cells of up to 50% which suggests a pro-inflammatory effect. However, NFκB activation appears to decrease at much higher concentrations of octanoyl ghrelin, which may indicate toxicity at supra-physiological levels. Ghrelin is also involved in the regulation of gastric motility and has structural similarities to motilin. Symptoms of delayed gastric emptying can occur long after cancer chemotherapy has ended. Chapter 5 of this thesis compares the contractility and pro-motility neurotransmitter expression in chemotherapy and non-chemotherapy exposed stomach tissues obtained from patients undergoing surgery for oesophagogastric cancers. Chemotherapy exposed tissues have reduced contractility to carbachol and apparent destruction of the cholinergic activity. The tendency for ghrelin receptors to increase suggests an attempt to upregulate compensating systems. In conclusion, ghrelin can be altered by inflammation and may have beneficial effects on gastric motility.

No description available.

Dementia is a set of incurable, fatal diseases characterised by irreversible degeneration of the brain. One theory of its cause is the failure of intracellular transport in the axons of the neurons that compose the brain. Kinesin is a key motor transporting vital cargo along the axon. We know that this motor is a bipedal engine stepping forward along a polypeptide track but it is too small and fast for this motion to be observed using current experimental techniques. The stepping detail is therefore open to debate. This study firstly addresses the question of how kinesin steps and secondly pilots a possible method for investigating transport disruption in silico. To investigate the detail of stepping, a program has been designed and built to simulate kinesin traversing its track along a section of axon. The motor is modelled as simple, interacting agents obeying rules abstracted from known chemical and binding properties of its components. The agent-based method has proven useful and efficient on the small scale and has potential for simulating the larger and more complex system of axonal transport. This would enable investigation of transport failure in the context of finding a cure for dementia. A new model of kinesin stepping has been formulated as a consequence of performing virtual experiments using the simulation. Analysis of in vivo and in vitro experimental studies shows that the model accounts for a wide range of published results, explaining many findings. New experiments are suggested to test the model based on its falsifiable predictions. The principal conclusion of this study is that kinesin stepping is rectified Brownian motion.

Obesity and type 2 diabetes (T2DM) are both inflammatory disorders with parallel escalating epidemics. Novel insights provided by the new biology suggest common pathways by which several pathogenic components of obesity affect glucose metabolism and cellular responsiveness to insulin leading eventually to the development of T2DM; inflammation is considered critical for the development of the above metabolic disorders and is directly influenced by weight gain. Adipose tissue (AT), particularly the abdominal fat depot is currently considered source of inflammatory agents that fuel whole body’s low grade inflammatory state. The HLA-B Associated Transcript 1 (BAT1) is a cellular member of the DExD/H-box RNA-helicases with essential role for cellular mRNA export, that also attains anti-inflammatory properties, as it was shown by studies investigating monocytes and T-cell lines. Furthermore, BAT1 polymorphisms were linked to predisposition to immunopathologic disorders including type 1 diabetes. These findings suggest a potential protective role of BAT1 against the obesity-associated lowgrade inflammatory state that contributes to T2DM development. The role of BAT1 in the adipocytes has not been investigated so far. Therefore, this thesis examined BAT1 expression and regulation within specific human AT depots and the adipocyte itself. Initial studies indicated BAT1 expression in ex vivo human AT but also the repressing effect of increasing adiposity and T2DM on BAT 1 expression. Remarkably, there was no difference in BAT1 expression between obese subjects and patients with T2DM indicating that BAT1 becomes suppressed with increasing adiposity and remains suppressed through to the development of T2DM and thereafter; this could in turn reduce the capacity to response to the inflammatory insults. As human AT contains many different types of cells besides adipocytes, including fibroblasts, macrophages, lymphocytes, pre-adipocytes and endothelial cells, some of which actually increase with increasing adiposity (e.g. macrophages and lymphocytes) subsequent studies determined the expression of BAT1 particularly in isolated human primary pre-adipocytes and mature adipocytes; the human pre-adipocyte cell line Chub- S7 was used for this purpose. It was shown that BAT1 (mRNA and protein) was expressed in both cell types with maximum expression in mature (lipid accumulating) adipocytes. At the stage of complete maturation, the effects of nutrients and inflammatory factors on BAT1 expression were examined. Both glucose and non-esterified fatty acids (NEFA) were shown to repress BAT 1; these findings were in keeping with the ex vivo data determined in terms of AT from obese and T2DM subjects. Furthermore, these studies indicated a synergistic action of both JNK and NFκB when used in combination to reduce BAT1 expression, indicating interconnectivity between JNK and NFκB pathways, as noted in other human AT studies examining other molecules. Regarding NEFA however, the JNK pathway seemed to mediate its repressing effect on BAT1. These studies also showed that the potent inflammatory agent lipopolysacharide (LPS) also significantly reduced BAT1 expression which was again in keeping with the previous ex vivo AT data since LPS is raised in conditions of metabolic disease. Finally, the investigation of the paracrine influences of leptin and resistin on differentiated primary adipocytes highlighted BAT 1 repression whilst adiponectin appeared to have no significant effect alone to alter BAT 1 expression or inhibit LPS-induced BAT1 repression. Taken together, BAT1 was more susceptible to the repressing effects of nutritional factors (glucose and NEFA) in excess than paracrine inflammatory or antiinflammatory adipokines. The fact that several factors modulate BAT1 expression may suggest that BAT1 represents a first line, non-selective, cellular protective agent, which is therefore influenced by several different factors through common inflammatory pathways. Thus, BAT1 suppression may be an early key event in the pathogenesis of a low chronic inflammatory state. As such BAT1 could represent an important target to manipulate to combat the low chronic inflammatory state observed in both obese and T2DM patients.

Diabetes is a chronic epidemic compounded by a burden of vascular complications including diabetic nephropathy. Diabetic nephropathy affects ~40% of patients and is characterised by increased urinary albumin excretion and decreased glomerular filtration rate. Diabetic patients exhibit ~75% decreased plasma thiamine concentration, linked to increased renal thiamine clearance. In streptozotocin-induced diabetic rats, decreased plasma thiamine concentration was also associated with a reduction in expression and activity of transketolase. Transketolase is a thiamine pyrophosphate-dependent enzyme and a critical component of the reductive pentose phosphate pathway, a metabolic pathway leading from glycolysis involved with the synthesis of ribose sugars. It is proposed that increasing the relative flux of glucose through the pentose phosphate pathway can ameliorate hyperglycaemic damage. This thesis investigates mechanisms mediating the increased renal thiamine clearance and the effects of thiamine therapy on type 2 diabetic patients with nephropathy. The hypothesis that hyperglycaemia increases flux through the hexosamine pathway, leading to increased O-glycosylation of the transcription factor Sp1 and subsequent decreased expression of thiamine transporters is investigated. Thiamine transporters in normal human kidney sections were found to be localised to the proximal tubule. Investigations in primary cultures of human proximal tubule epithelial cells and the HK-2 cell line have shown that there is a decreased expression (-48 to -80%) and abundance (-52 to -77%) of thiamine transporters in cells cultured in high glucose concentrations (26mM) with respect to low glucose concentrations (5 mM). There is only limited evidence supporting the involvement of the hexosamine pathway in these decreases. A double-blind, placebo-controlled study investigated the effect of thiamine supplementation on type 2 diabetic patients with microalbuminuria. Thiamine therapy restored plasma thiamine concentrations from 11nM to 98nM, exceeding the published median concentration observed in normal patients (64nM). After three months, thiamine therapy, but not placebo, caused a decrease in the urinary albumin excretion rate relative to baseline (-18 mg day-1). These results show promise for thiamine as a therapy for diabetic nephropathy.

Obesity is the foremost contributory factor in the progression to type 2 diabetes mellitus (T2DM). Moreover, chronic inflammation, through activation of innate immunity is proposed to link obesity, insulin resistance and T2DM. Adipose tissue, traditionally considered a storage compartment for triglycerides, also functions as an active endocrine organ. Adipocyte-secreted products, termed adipokines, may link obesity-associated inflammation and insulin resistance. Adipokines exert multiple effects on insulin sensitisation, glucose homeostasis, inflammatory processes or central systems mediating energy expenditure. This thesis principally examined two adipokines; resistin and adiponectin. Resistin and components of innate immunity were assessed in human obesity. In-vitro analysis established that resistin was expressed and secreted by human adipocytes. Furthermore, key factors in the innate immune pathway were highly expressed in obese and T2DM adipose tissue. This thesis further explored the pro-inflammatory actions of resistin in adipocytes. Resistin stimulated the secretion of inflammatory cytokines from adipocytes and, the expression of key intermediates of the innate immune and insulin signalling pathways. Clinical studies entailed examination of resistin as a marker of inflammation in childhood obesity. Serum analysis revealed gender-differences in resistin levels in obese children. Furthermore, bacterial endotoxin correlated with several markers of inflammation and cardiovascular disease; suggesting endotoxin as a contributor to inflammation in childhood obesity. This thesis subsequently examined another adipokine, adiponectin; considered to have a `ying-and-yang' relationship with resistin. Studies explored a central role for adiponectin in energy homeostasis. Gelfiltration liquid chromatography established that the adiponectin trimer was predominant in human cerebrospinal fluid. Such identification of trimeric adiponectin in vivo implicates the pharmacologically generated globular adiponectin in central regulation of energy expenditure. In conclusion, resistin may serve as a pathogenic pro-inflammatory factor, exacerbating inflammation within adipose tissue; potentially contributing to the progression of obesity-driven T2DM. Alternatively, adiponectin may have favourable central actions, influencing energy expenditure through its basic trimeric form. Collectively, this thesis suggests that resistin and adiponectin, with a range of opposing properties, may substantially affect whole-body metabolism.

The acute respiratory distress syndrome (ARDS) is an important cause of respiratory failure in critically ill patients characterised by severe inflammation within the lungs. This inflammation is limited by anti-­inflammatory glucocorticoid hormones released from the hypothalamus-pituitary-adrenal (HPA) system. This thesis reports a series of investigations into glucocorticoid concentrations and glucocorticoid metabolism within the lungs of patients with ARDS. It also contains an investigation into a potential biomarker for ARDS. Our study of glucocorticoid concentrations in alveolar epithelial lining fluid showed increased cortisol concentrations within the lungs at onset of ARDS. These concentrations have a positive relationship with critical illness severity indices, but negative relationships with alveolar permeability and alveolar neutrophil counts. In peripheral tissues cortisone and cortisol are inter-converted by iso-­enzymes of 11β-­hydroxysteroid dehydrogenase (11β-­HSD). We have shown that healthy primary resident alveolar macrophages increase their production of active cortisol by the oxo-­reduction of inactive cortisone in response to inflammatory stimuli. Alveolar macrophages are responsible for the removal of spent and apoptotic inflammatory cells, failure of this process causes further inflammation. We have shown that glucocorticoids increase the rate of uptake of apoptotic cells by alveolar macrophages, and that macrophage 11β-HSD production of cortisol increases this process. We have shown however that alveolar macrophages extracted from patients with established ARDS have decreased 11β-HSD oxo-reductase activity. This decreased conversion of cortisone to cortisol will cause a diminished response to the anti-inflammatory signal of the HPA system. The implications of this are that they will have a limited capacity to up-­regulate efferocytosis and a diminished anti-­inflammatory potential. The receptor for advanced glycation end-­products (RAGE) is a potential biomarker in ARDS. We have shown that RAGE concentrations in plasma and BALF had excellent diagnostic compatibility with ARDS diagnostic criteria. The use of a threshold RAGE concentration could assure pulmonary inflammation in future investigations.