Type 2 diabetes is a complex disease resulting from a combination of the inability of target cells to recognise and respond to insulin and a defect in the ability of the ~-cells to secrete insulin. Both genetic and environmental factors contribute to the risk, presentation and progression of the disease. Knowledge of the underlying architecture and mechanisms remain incomplete; however recent advancements in genotyping technology have allowed us to define potential type 2 diabetes genes through genome wide association (GWA) studies of very large populations, to compliment data from the more traditional candidate gene studies. The overall aim of this thesis was to assess common genetic variation within a healthy population of Europeans and to explore whether this alters their susceptibility to developing type 2 diabetes. The Relationship between Insulin Sensitivity and Cardiovascular disease (RISC) cohort of DNA was used to identify novel SNPs in the Perilipin/Pexlla gene region, and to validate potential type 2 diabetes susceptibility alleles identified via GWA scans. TaqMan and Sequenom platforms were used in house to genotype the Perilipin/ Pexlla gene region ht-SNPs in RISC, and deep sequencing was subsequently used to try to identify functional variants. The remaining GWA scan identified genes were genotyped by an external facility. Associations with measures of ~-cell function, insulin sensitivity and body composition were assessed. Expression studies on the Perilipin and Pexlla genes were performed on human islets and MIN6 cells. mRNA and protein levels of the expressed gene were then assessed and the impact of altered glucose and fatty acid levels on Perilipin or Pexlla expression was investigated. Two ht-SNPs were identified in the Perilipin/Pexlla gene region that were both associated with measures of ~-cell function. We confirmed Pexlla mRNA expression in both human islets and MIN6 cells, however Perilipin mRNA expression was not detectable. Exposure to low glucose and elevated palmitate levels both up-regulated Pexlla mRNA expression, but Perilipin remained undetectable. We showed that variation at the CDKALl, HHEX/IDE and MTNR1B loci increases type 2 diabetes susceptibility by decreased pancreatic ~-cell function. Finally we assessed the effects of combining susceptibility loci on the risk of ~-cell dysfunction. We demonstrated that individual type 2 diabetes-risk alleles combine in an additive manner to impact upon pancreatic ~-cell function in healthy individuals. In conclusion, we have identified two novel SNPs within the Perilipin/Pexlla gene locus associated with measures of paricreatic ~-cell function in the RISC cohort. We confirmed that Pexlla is expressed in pancreatic islets and ~-cells, and further work is required to define whether variation in this gene directly affects ~-cell function. We demonstrated for the first time that variation in the novel type diabetes susceptibility genes identified by GWA scans mediate their effects through altered pancreatic ~-cell function.

The identification of new molecular targets that could lead to new therapeutics for NIDDM is an important goal, which can be achieved under defined experimental conditions with strategies that combine genomic and proteomic approaches. In the present study we have used a proteomics approach to identify such targets. Lean and obese C5? B1/6J lep/lep mice were given BRL49653, rosiglitazone, 10mg/kg diet, by dietary admixture for 7 days. Rosiglitazone normalized the impaired glucose tolerance in lep/lep mice but had no significant effect in the lean mice. Samples of liver, liver nuclei, muscle, WAT, BAT and islets were arrayed in a two-dimensional electrophoresis system that separated 2500 individual spots. Image analysis allowed the detection of 94 significant (p<0.05) differentially expressed polypeptides in obese relative to lean controls. Thirty six of these were significantly (p<0.05) modulated in obese models to the level in lean control after rosiglitazone treatment Forty three of them were identified by mass spectrometry (MALDI- and/or Q-TOF-MS). Several of these differentially expressed proteins were components of fatty acid and carbohydrate metabolism and others are new unknown proteins. The understanding of the biomolecular pathways and interactions of these new proteins will be essential in further studies to define their biological function. In conclusion, proteomic approaches should provide in the near future new clues for the elucidation of the pathogenesis of the various defects involved in type 2 diabetes and provide major insights into new more effective treatments.

The nuclear receptor Hepatocyte Nuclear Factor 4a (HNF4a; NR2A1) regulates the transcription of many genes involved in glucose and lipid metabolism. Genetic linkage analyses have implicated HNF4a in the disease processes leading to Type 2 Diabetes Mellitus and dyslipidaemia. The aim of this study was to investigate the regulation of target genes in the metabolic pathways of glycolysis, lipogenesis and gluconeogenesis by HNF4a. Initally, the expression of HNF4a and its splice variants was investigated in three human hepatoma cell lines, HuH7, HepG2 and Hep3B, with the latter two cell lines shown to express the same range of HNF4a splice variants as human adult liver. The regulation of specific HNF4a target genes, L-PK, PEPCK and SREBP-1c, was subsequently investigated in HepG2 cells using a reporter gene approach. HNF4a was found to induce expression of reporter genes containing L-PK, PEPCK and SREBP-1c proximal promoter sequences. Insulin (1 ?M), but not high glucose (25 mM), was found to stimulate HNF4a-driven expression of the SREBP-1c reporter gene, while co-expression of HNF4a with the nuclear receptor coactivators, PGC-1a or p300, led to a reduction in SREBP-1c reporter gene expression. The changes in expression of various HNF4a target genes in response to physiological mediators of the fasting-fed cycle were characterised in HepG2 cells using a real-time quantitative PCR approach. The role of HNF4a, p300 and PGC-1a was further investigated by plasmid overexpression. HNF4a and PGC-1a were found to positively regulate PEPCK expression under cell culture conditions simulating fasting (cAMP), whilst overexpression of HNF4a, PGC1a and p300 reduced L-PK mRNA expression under fed conditions. In conclusion, the results indicate that HNF4a is a transcriptional activator of both glucagon- stimulated gluconeogenic gene expression and insulin-stimulated glycolytic and lipogenic gene expression. It is hypothesised HNF4a forms separate multi-protein complexes to differentially regulate metabolic pathways under different metabolic states.

Despite a vast amount of epidemiological data documenting the ‘epidemic’ of type 2 diabetes and extensive randomised controlled trials evidence of effective interventions, there is a contrast between this wealth of quantitative research and the relatively underdeveloped qualitative understanding of how type 2 diabetes impacts upon patients and health care professionals. We therefore seem no closer to understanding how to effectively tackle the massive burden this represents for both patients and the health service. The publications discussed here represent a body of work consisting of five first author publications (2005-2012) that explore interventions and patient and health professional perspectives regarding the prevention of and care management for type 2 diabetes. Two main methods are used in the studies represented by the papers. These are primary qualitative research (three publications) and evidence synthesis (two publications) for public health guidance. The qualitative methods used in two primary studies and one evidence synthesis allow the perspectives of service users and professionals to be assessed in relation to public health interventions and health care. The remaining evidence synthesis examines how large scale diabetes prevention interventions can be tailored for use in the community. A major theme that links the publications is transferability of care management and prevention ideals to life in the real world. The Chronic Care Model (CCM) provides an optimum framework for such adaptations. However, whilst NHS policies and the CCM focus on patient engagement, it is clear that much needs to be done in recognising and addressing the feasibility of prevention and care strategies in practice. The thesis discusses how disparities between idealism and the ‘life world’ of patients might be addressed, adding to current debates about shared understanding and the importance of taking into account behavioural influences that might undermine or enhance the achievement of shared goals.

Type 2 diabetes mellitus (T2DM) is a complex disease involving various physiological factors, hormones and metabolites. Over expression of key gluconeogenic enzymes, such as PEPCK, cause features of T2DM including obesity and insulin resistance. Previous studies showed intravenous administration of corticotropin releasing factor (CRF) and sauvagine in rats cause marked hyperglycaemia, which is adrenal dependent. The 11-β hydroxylase inhibitor metyrapone augmented this hyperglycaemia raising important questions about the role of known and unknown corticosteroids in hepatic carbohydrate metabolism. The goal of this project was to identify and characterise the gluconeogenic activity of adrenal corticosteroids secreted under basal and stimulated conditions. This was accomplished by establishing a rapid, sensitive and robust multi-well assay for measurement of PEPCK activity in the hepatocyte cell line H4-II-E-C3. A sensitive fluorescent method for assay of glucose production by these cells was also developed. Extracts of media samples from adrenal glands incubated with sauvagine and metyrapone significantly increased hepatocyte glucose production (HGP) despite low corticosterone concentrations. HPLC characterisation of these extracts revealed increases in 11-deoxycorticosterone (DOC) and other unidentified peaks. However, none of these individual fractions significantly affected HGP in culture. Commercially available corticosterone, which contains DOC as an impurity, had greater gluconeogenic effect compared to purified corticosterone alone. Based on these observations and discrepancies in the literature, the effect of DOC on hepatic carbohydrate metabolism was characterised. Surprisingly, DOC suppressed the activity of gluconeogenic enzyme PEPCK in fed rat hepatocytes and enhanced insulin 4 stimulated glycogen stores in cultured hepatocytes at higher glucose concentrations (25 mM) over a 24 hour period. In serum-starved, fasted hepatocytes these effects were not significant, suggesting the need for a detailed investigation of the signalling pathways and regulatory control of DOC on GK, GS, G6Pase and PEPCK.

A wealth of recent evidence implicates increasing obesity levels in the rising global epidemic of type 2 diabetes. At a cellular level, high fatty acid concentrations contribute to increasing p-cell dysfunction and eventual p-cell failure. We have shown that the thiazolidinedione (TZD) rosiglitazone has direct effects on pancreatic p-cell function. The aim of this study was to determine the cell signalling molecules mediating these effects, and to test the hypothesis that rosiglitazone can protect pancreatic p-cells from the detrimental effects of free fatty acids. Utilising the mouse p-cell line MIN6, MTT assays showed that at 5 mM glucose concentrations, addition of 0.4 mM palmitate for 72 hours resulted in a 60% loss of MIN6 cell viability (P<O.O 1). However, the presence of 10 p M rosiglitazone completely abolished the effect of palmitate. To determine the cell signalling pathways involved, MIN6 cells were stimulated with 10 u M rosiglitazone for 2 hours and whole cell extracts analysed. Western blot analysis indicated that total levels of the stress activated p38 MAPK were reduced by over 50% (P<0.05) in the presence of rosiglitazone. Rosiglitazone stimulated phosphorylation of ACC (P<O.Ol), an effect inhibited by compound C, indicating that rosiglitazone activates AMPK in MIN6 cells over 2 hours. Finally, casein kinase 2 (CK2) activity assays indicated that rosiglitazone stimulates a 3-fold increase in CK2 activity (P<O.Ol). This activation was inhibited by compound C, indicating that CK2 lies downstream of AMPK in the signalling pathway stimulated by rosiglitazone. In conclusion, this study has shown that rosiglitazone modulates p38, AMPK and CK2 signalling in MIN6 cells, and can protect MIN6 cells from the detrimental effects of palmitate. Our results indicate that early rosiglitazone administration in obese patients at risk of Type 2 diabetes could well protect and preserve p-cell mass and function.

Background and Methods: The involvement of iron in the risk for, and complications of, type 2 diabetes has generated substantial interest over the past 15 years, initially sparked by an association with raised serum ferritin, and the observation that people with iron overload diseases frequently develop diabetes. Considerable advances have since been made in understanding the effect glucose has on molecules, cells, and tissues; and the role that oxidative stress plays in the development of the pathologies of long-term diabetes. Poorly liganded iron is potentially both a contributor to, and consequence of, these complications. In vitro experiments with glucose-incubated transferrin by earlier workers have demonstrated loss of function with increasing glycation, leading to the suggestion that the failure of this key iron-binding protein may contribute to diabetic pathology, via the presence of redox active non-transferrin-bound iron (NTBI). In vitro glycated transferrin is examined here by ultrafiltration, to assess loss of function and possible oxidative fragmentation. Mass spectrometry is used to identify a range of amino acid glycation sites on in vitro glycated transferrin for the first time. Finally, several groups have previously measured NTBI in people with diabetes, finding little agreement in results. NTBI is measured here in a cohort of people with type 2 diabetes, using a new adaptation of earlier NTBI assays. NTBI is also assessed in pre-dialysis chronic kidney disease (CKD) stages I to III for the first time. Results and Conclusions: Experiments with glycated transferrin in vitro demonstrate oxidative fragmentation, explaining the loss of function reported by earlier groups. In vitro glycated transferrin examined by mass spectrometry reveals a substantial number and range of amino acids subject to glycation. Comparison with in vivo glycated transferrin suggests that many of the in vitro glycation sites are not glycated in vivo, and that there are many oxidized methionine residues which are potential artefacts, or likely to be repaired by methionine sulphoxide reductases in vivo. A study of people with type 2 diabetes finds no direct association between NTBI and protein glycation. Unexpected correlations between NTBI and LDL, and LDL and haemoglobin with increasing protein glycation, are reported for the first time. NTBI is suggested to be iron sourced from haemoglobin or haem, from erythrocyte haemolysis prior to sample collection. In people with pre-dialysis CKD stages I to III no significant difference in NTBI level compared to controls is seen, or correlations with markers of renal function. No link between NTBI and kidney function at this stage of disease is indicated.

Type 2 diabetes (T2D) and fasting plasma glucose (fpg) levels have distinct genetic components which are as yet only modestly understood. Understanding the genetics of this complex disorder and its related traits is likely to be of significant benefit to the field. Not only will it shed light on critical genes, pathways and mechanisms of regulation, but it may also contribute to the development of pharmaceutical anti-hyperglycaemic agents via the identification of key therapeutic targets. Therefore the aim of this thesis was to utilise a broad, multidisciplinary approach to study the genetics of insulin secretion and signalling. Traditionally genes which harbour rare variants causing monogenic beta-cell dysfunction have also been found to harbour common variants associated with T2D and fpg. As genome-wide association studies (GWAS) identify an increasing number of common variants and genes, they also increase the number of genes which act as monogenic candidates. I screened G6PC2, a novel fpg associated gene, in patients with monogenic forms of beta-cell dysfunction and demonstrated that rare variants in this gene are unlikely to be a common cause of monogenic beta-cell dysfunction. Although GWAS have been of considerable benefit to our understanding of complex disease genetics, they are not without their own limitations, primarily concerning signal refinement. To try to overcome this barrier for T2D and fpg signals I established a pipeline for fluorescence activated cell sorting of human islets to obtain pure beta-cells. In these cells, I performed transcript profiling of genes falling within T2D and fpg associated loci, demonstrating how this approach, alongside physiological analysis, can be of benefit for GWAS researchers and provide a starting point for fine mapping. Access to human beta-cells also enabled me to follow up one novel fpg association signal, SLC2A2. Through analysis within this metabolically relevant tissue I was able to establish that the mechanism for increased fpg levels is unlikely to be mediated via a beta-cell pathway. Although GWAS have highlighted a number of key genes associated with beta-cell dysfunction; they have been far less successful at identifying genes associated with insulin resistance, another key component of T2D pathogenesis. Additional approaches, including the study of rodent models, may be required to study this aspect of T2D. PTEN is known to negatively regulate the insulin signalling pathway and adipose tissue specific Pten-/- animals were shown to be markedly insulin sensitive. To assess the role of PTEN in human insulin sensitivity I performed mRNA expression profiling of PTEN in human adipose tissue biopsies from subjects with T2D and matched controls, demonstrating that PTEN is significantly reduced in the subcutaneous adipose tissue of the former. This response is likely to be a compensatory mechanism to counteract muscular insulin resistance although further investigation needs to be performed to determine the mechanism of compensatory downregulation. These data provide insights into a number of aspects of T2D genetics, and demonstrate how a multidisciplinary approach is of benefit to T2D genetic research.

Genome wide association studies (GWAS) and their subsequent meta-analysis have identified a large number of susceptibility variants for Type 2 diabetes (T2D) risk. However, the familial aggregation seen in this disease is not yet fully explained. The sibling relative risk (λ_s) due to all known variants is ~1.104 which is well below the epidemiological estimates of λ_s of ~3.0. There has therefore been great interest in the potential role of variants that would have been largely invisible to the initial wave of GWAS and linkage approaches. Low frequency (minor allele frequency 1-5%), incompletely penetrant (odds ratio 2-4) variants (LFIP), are one such group of potential susceptibility variants. The overall objective of this project (designed and implemented in 2007-2010) was to evaluate the contribution of LFIP variants to the inherited susceptibility to T2D. I tested the specific hypothesis that genes already-implicated in diabetes pathogenesis (due to an established role in monogenic or multifactorial disease) also harbour LFIP variants, and that those variants may contribute appreciably to the prediction of disease risk. Mutations in exons only encoding isoform-A of HNF1A have been demonstrated to lead to a later age of diagnosis of HNF1A-MODY. This region was therefore felt to be auspicious for harbouring LFIP variants impacting on T2D risk. I have demonstrated that such variants impacting on T2D risk are unlikely to be present in this region by use of Sanger sequencing in a sample enriched for young onset, familial T2D. The role in T2D risk of candidate LFIP variants across 5 genes (HNF1A, HNF4A, PDX1, KCNJ15 and LARS2), was evaluated by large scale association studies. For one variant, T130I of HNF4A, a modest association (p=5x10^-4) with T2D was seen in UK samples and the strength of association was marginally improved by incorporation of all previous studies of this variant in T2D in a meta-analysis (p=2.1x10^-5). This study demonstrated the difficulties encountered in confirming the association of low frequency variants to complex diseases, especially for those with modest effect sizes. At the time of project design and inception “next-generation” sequencing platforms were in their infancy and the study design I planned (that of pooled, targeted sequencing) had not been widely applied. It was therefore necessary to design and optimise protocols for sample preparation for sequencing on this platform. I used the Genome Analyzer II platform to sequence ten genes previously implicated in T2D or monogenic diabetes pathogenesis in pooled DNA samples. This approach yielded in excess of 2900 variants, a large portion being novel. As part of this project I have highlighted heuristics that can be used in the follow-up of potential susceptibility variants discovered using high throughput sequencing. I have also established protocols and pathways for sample preparation that can be utilised across several next generation sequencing platforms for future studies in the host institution and beyond.

Insulin resistance, a key risk factor for type 2 diabetes, can be defined as when cells fail to respond effectively to insulin. In striated muscle and fat, this manifests as impaired insulin-stimulated glucose uptake due to reduced plasma membrane insertion of the glucose transporter GLUT4. In cell culture models, insulin resistance induced by chronic exposure to insulin, endothelin-1 or glucosamine, is correlated with reduced immunoreactivity of the lipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in plasma membrane sheets. However, the reason for this decrease, and whether other factors that induce insulin resistance affect PtdIns(4,5)P2 levels, is unknown. Using L6 skeletal muscle myotubes and 3T3-L1 adipocytes, this project has investigated whether PtdIns(4,5)P2 levels are perturbed in insulin resistance induced by several factors, including exposure to insulin, oxidative stress, and treatment with tumour necrosis factor α, endothelin-1 or angiotensin II (Ang II).All these pre-treatments were found to abolish insulin-stimulated 3H 2-deoxy-glucose uptake, and significantly decrease PtdIns(4,5)P2 levels, measured in cell extracts by quantitative blotting using a PtdIns(4,5)P2-specific probe, developed from the PH domain of phospholipase C (PLC) δ. Importantly the ability of insulin to stimulate glucose uptake can be restored by replenishing PtdIns(4,5)P2 in L6 myotubes treated with insulin and Ang II. PtdIns(4,5)P2 levels are regulated by three families of proteins; PIP kinases, which synthesise it, phosphatases, which remove phosphate groups from the inositol headgroup, and PLCs, which hydrolyse it. Membrane preparations from Ang II- and insulin-induced insulin resistant L6 myotubes showed no differences in PtdIns(4,5)P2 production or dephosphorylation. However a significant increase in PLC activity was detected in membranes from insulin resistant cells and membrane localisation of PLCβ family members was increased in insulin resistant cells. Furthermore, studies using PLC inhibitors show a restoration of PtdIns(4,5)P2 levels in insulin resistant cells, leading to partial reversal of insulin resistance.This study therefore shows a causal link between decreased PtdIns(4,5)P2 levels and insulin resistance in L6 myotubes, and that PLCs are the reason for the PtdIns(4,5)P2 decrease in Ang II- and insulin-induced insulin resistance. PLCs, or their activation pathways, may thus be a novel target for combating insulin resistance, and preventing type 2 diabetes.