Approximately 3 million people throughout the UK suffer with Type 2 diabetes mellitus (T2DM), and are 32% more likely to die early. There remains a lack of evidence for the long-term effectiveness and safety of anti-diabetic drugs in preventing diabetes-related complications, making it unclear how to optimally manage diabetes. Work to date includes observational studies subject to bias, and randomised controlled trials (RCTs), which may not reflect the ‘real world’ situation. The aim of this thesis is to combine such findings via systematic reviews, meta-analyses and retrospective cohort studies to provide more water-tight evidence of the effectiveness and safety of glucose-lowering therapies (GLT) in the long term. Firstly, a systematic review of observational studies was performed, identifying and providing a simple description of the types of biases and control measures employed in retrospective cohort studies on treatment outcomes of GLTs. Secondly, retrospective cohort studies were conducted to strengthen the evidence of the clinical effectiveness of DPP-4 inhibitors, compare their durability when combined with other anti-diabetic drugs and assess their cardiovascular safety when used in patients with T2DM, using data from The Health Improvement Network (THIN) database. Linear and logistic regression, Cox proportional hazard regression models and propensity score techniques were used to analyse routine clinical data. Thirdly, a meta-analysis was conducted on RCTs investigating the risk of bone fracture following the administration of DPP-4 inhibitor, based on data from an extensive literature search. Conducting this research has led to a better understanding of how biases may have influenced retrospective cohort studies on oral anti-diabetic drugs. An algorithm was developed to illustrate strategies for addressing biases. Potential clinical factors associated with ‘response’ to DPP-4 inhibitor treatment were found to include the addition of DPP-4 inhibitor to ongoing metformin (MET), or MET plus sulphonylurea (SU) therapy. High HbA1c at the time of treatment intensification and longer duration of diabetes were associated with the lack of HbA1c target attainment. In terms of the durability of second-line glucose-lowering agents, the co-administration of thiazolidinedione with MET was associated with the most durable glycaemic response, followed by a SU and then a DPP-4 inhibitor. Compared with a SU, adding a DPP-4 inhibitor to MET was associated with an increased need for earlier treatment intensification with a third agent. The use of statins, being a female, a smoker, having longer duration of diabetes and higher HbA1c at baseline were identified to be associated with earlier dual therapy failure. In terms of cardiovascular safety, routine clinical data showed patients who intensified MET + SU dual therapy with a DPP-4 inhibitor were associated with a decreased risk of a composite of non-fatal cardiovascular outcomes and all-cause mortality compared to those who added insulin. Furthermore, the results from meta-analysis showed DPP-4 inhibitors are not associated with increased bone fracture risks in patients with T2DM. This research is valuable in informing the choices of healthcare professionals in prescribing treatments for T2DM. For the users of this treatment, it is good news that DPP-4 inhibitors are not generally associated with fracture incidence, and that findings support the use of DPP-4 inhibitors as a second line therapeutic option, especially among non-obese patients whose glucose control remains suboptimal following MET treatment. It is recommended that treatment should be characterised on an individual basis. There remains a need for robust RCTs to investigate the influence of obesity and longer treatment durations on the efficacy of co-administering DPP-4 inhibitors to patients who are unresponsive to other oral GLTs.

It is estimated that 366 million people were living with diabetes in 2011, and this is predicted to rise to 522 million by 2030. One of the most common complications of diabetes is diabetic neuropathy, where patients experience various symptoms of neuropathic pain including mechanical allodynia. Using a high fat diet (HFD) in combination with streptozotocin (STZ) produces a model of diabetes which mimics aspects of type 2 diabetes. The aim of this thesis was to characterise pain responses in the HFD/STZ model and to explore some of the peripheral and spinal mechanisms associated with the changes in somatosensory processing. The effectiveness of a variety of drugs in alleviating/preventing neuropathic pain was also investigated in this model. The effects of the HFD/STZ model on mechanical sensitivity, and changes in metabolic parameters were investigated, and it was found to cause a robust development of mechanical hypersensitivity and a large increase in plasma glucose and a contrasting decrease in plasma insulin. The impacts of the HFD/STZ model on peripheral nerve function and pathology, and spinal mechanisms of central sensitisation, were explored to help identify the mechanisms underpinning the behavioural pain phenotype in these rats. No neuronal degeneration was detected in DRGs or the spinal cord at the timepoints investigated, and a decrease in microglial activation and GFAP immunoreactivity was observed at later timepoints (day 50). Changes in neuronal responses in the dorsal horn of the spinal cord were then investigated, and there was a trend towards a decrease in mechanically evoked responses of spinal neurones in the HFD/STZ group, but no changes in the threshold for electrical activation of C-fibres, nor any significant changes to electrically evoked responses, were observed. There was no change in spontaneous firing, possibly due to the search criteria used. The effects of different types of interventions on aberrant pain responses were also investigated. As neuropathic pain often proves intractable, one of the key objectives is to develop new drugs, or to find alternative uses of current drugs, that are able to provide symptomatic relief of pain. The gold standard treatment for pain in diabetic neuropathy, pregabalin (10mgkg-1, p.o.), was effective at alleviating established mechanical hypersensitivity at day 37 in the HFD/STZ model. A novel MAGL inhibitor, MJN110 (5mgkg-1, i.p.), was found to be as effective as pregabalin in this model, highlighting a possible role for endocannabinoid modulators in providing pain relief in diabetes. The antidiabetic pioglitazone (10mgkg-1, p.o.), however, was unable to alleviate mechanical hypersensitivity when administered at day 21 for 28 days. Two other antidiabetic drugs, linagliptin (3mgkg-1, p.o.) and metformin (200mgkg-1, p.o.), did show promise in preventing the development of mechanical hypersensitivity in this model when administered from day 4, independent of glycemic control. It is worth investigating these findings further since both of these drugs are already licensed and have undergone all necessary safety testing, and so could rapidly be put to use if effective. In conclusion, this thesis has highlighted the role that the HFD/STZ model can play in investigating underlying mechanisms of diabetic peripheral neuropathic pain, and its use in exploring potential new therapeutic options for alleviating this pain.

Type 2 diabetes mellitus is a chronic condition caused by a deficiency in the secretion of insulin from the islets of Langerhans and/or impaired insulin signalling, resulting in hyperglycaemia. The role of the endocannabinoid system is well-recognised in the CNS and immune system, but its role in glucose homeostasis is poorly understood. The aim of this study was to define the roles of cannabinoids in insulin secretion, to provide insights into their therapeutic potential (or limitation) in the treatment of type 2 diabetes. Isolated islets were used, from Wistar rats, in static incubation studies measuring changes in insulin secretion rates. The endocannabinoid anandamide (AEA) was found to inhibit insulin secretion in a glucose- and concentration-dependent manner, with an IC50 of 1.6μM (95% CI: 227nM to 4.0μM; n= 10). Upon further analysis of the concentration-response data islet sensitivity to AEA appeared to vary, with islets either appearing to be sensative (IC50 220nM; 95% CI: 21.9nM to 2.2μM; n= 5) or less sensative (IC50 12.3μM; 95% CI: 6.8μM to 19.4μM; n= 5) to AEA. Pre-incubation of islets with a fatty acid amide hydrolase inhibitor did not affect islet responsiveness to AEA. AEA-mediated inhibition of insulin secretion was not consistently affected by cannabinoid receptor 1 (CB1) or CB2 antagonism. Surprisingly, the CB1 receptor antagonist AM251 was found to inhibit insulin secretion in a glucose- and concentration-dependent (IC50 1.6μM; 95% CI: 507nM to 3.3μM; n= 6) manner. Results from this study suggest that differences in CB-receptor signalling pathways, rather than endocannabinoid metabolism, could be responsible for the variations in the potency of AEA between islet preparations. Characterisation of cannabinoid signalling in islets was hindered as the CB receptor antagonists used in this study also affected insulin secretion. This study highlights the dynamics of endocannabinoid signalling in islets, which may be linked to their physiological function.

The studies presented in this thesis aimed to investigate the effects of nutritional modulation and age-associated changes on insulin sensitivity. Four separate studies were performed; three of these had insulin sensitivity as the primary outcome. Existing studies show that ageing is associated with insulin resistance, but data may be confounded by several factors that also occur with increasing age, such as increased adiposity, skeletal muscle lipid accumulation and reduced physical activity. To elucidate this further the first study compared body composition, skeletal muscle lipid content, fat metabolism during light-intensity exercise and whole-body and skeletal muscle insulin sensitivity between 7 healthy young and 14 older males. Ageing and insulin resistance are also associated with impaired skeletal muscle protein synthesis, however the effects of insulin resistance per se on amino acid metabolism and associated insulin signalling pathways are not really known. The second study involved 8 young healthy males and aimed to explore the effect of insulin resistance on the protein synthetic response to amino acid ingestion and muscle protein signalling pathway in humans. Dietary intake has been shown to affect insulin sensitivity; however it is unclear if diet composition affects liver fat content independent of energy balance. Therefore the third study aimed to investigate the effects of hyperenergetic diets high in fat or carbohydrate on liver fat and insulin sensitivity. The study involved 23 healthy but overweight and obese males who initially consumed an isoenergetic diet for one week, and then were randomised to 2 weeks of either hyperenergetic (+25% excess) high fat or high carbohydrate diets. Liver fat content, abdominal visceral fat, skeletal muscle fat content, hepatic lipid metabolism and insulin sensitivity were assessed before and after the 2 week intervention period. Whilst dietary excess can exacerbate insulin resistance, certain micronutrients may improve insulin sensitivity. Carnitine has shown encouraging outcomes in relation to promoting fatty acid oxidation, metabolism and modulating body composition in healthy young volunteers. However the effects on older people have never been explored. This formed the basis of the fourth study that investigated the effects of 6 months of oral carnitine supplementation or placebo in 14 older (≥65 years of age) healthy males in relation to fatty acid metabolism, skeletal muscle lipid and insulin sensitivity. The main findings are summarised. Irrespective of age, adiposity and physical activity are associated with impaired fatty acid oxidation, greater skeletal muscle lipid accumulation and reduced insulin sensitivity. However ageing per se appears to increase the sympathetic response to exercise and enhance systemic fatty acid delivery and adipose tissue lipolysis. Insulin resistance induced by acute elevation of lipid was found to affect the skeletal muscle protein synthetic response to amino acid ingestion, and this impairment appeared to occur downstream from the Akt insulin signalling pathway. Energy excess per se increases liver fat content and affects liver metabolism but there were no differential effects of carbohydrate or fat on hepatic insulin sensitivity and liver fat content. Finally, oral carnitine ingestion for 6 months successfully increased skeletal muscle total carnitine content of older healthy people and resulted in increased fatty acid oxidation and intramyocellular lipid (IMCL) utilisation during light-intensity exercise, but no effect on skeletal muscle insulin sensitivity was seen. These studies have increased mechanistic insight into the associations between ageing, nutrients and insulin sensitivity, paving the way to further research.