As keratin is the principle structure of the plantar stratum corneum, it has been postulated that changes in this protein, associated with non-enzymatic glycation, may contribute to abnormalities in the development of plantar callus in diabetes consequently leading to ulceration of plantar skin. The clinical appearance of the skin in the diabetic state has been described as being thicker and less flexible than non-diabetic skin, particularly in the hands. A group of six Type II diabetic patients and six age and sex matched controls were initially selected for the verification of the methods of analysis to be used on a larger sample group. Epidermal keratin extraction protocols were tested and their validity confirmed using immunoblot analyses. The early glycation product, furosine and the advanced glycation end-products pentosidine and carboxymethyllysine were the glycation products chosen for quantification in plantar callus tissue. The quantification of furosine and pentosidine were successfully achieved using high performance liquid chromatography assays. The qualitative detection of CML was achieved using gas chromatography/mass spectrometry (GC/MS). In response to the lack of sensitivity of the GC/MS detection method for CML, polyclonal and monoclonal antibodies were developed. Screening of these antibodies identified a lack of specificity for CML detection and quantification. In addition to the quantification of the glycation compounds in plantar epidermal tissues; the flexibility and thickness of the skin on three different sites on the feet were measured. The epidermal thickness was measured using high frequency ultrasound imaging and the mechanical properties of the epidermis were tested using vertical negative pressure methods. The selected group of Type II diabetic subjects (n=103), in the final clinical study, showed a statistically significant increase in levels of pentosidine in plantar callus specimens compared with the control group (n=87). The quantity of furosine and pentosidine measured in the callus samples did not conrelate with those measured in blood serum. The HbA1c levels showed no association between glycation of plantar epidermal proteins and glycaemic control. The echogeneic images captured by ultrasonography were thickest on the plantar metatarsal sites, followed by the medial longitudinal arch and finally the dorsal skin sites. The epidermal thickness in the PMA region was greater in diabetics in comparison to controls and an association with neuropathy was suggested. Pedal skin exhibited viscoelastic properties. The magnitude of the elastic component, measured immediately after the negative displacing pressure was removed, was significantly greater in diabetic skin than controls on all three sites on the foot. Plantar diabetic skin was significantly less plastic than non-diabetic plantar skin.

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Obesity is a major risk factor for insulin resistance and therefore type 2 diabetes. However, the precise mechanisms whereby obesity causes insulin resistance are complex and not completely understood. The adipocyte derived hormone leptin and the pancreatic derived hormone insulin act as satiety factors to the central nervous system, regulating long-term energy homeostasis. In addition both hormones have been shown to act in the central nervous system (CNS) to acutely regulate peripheral glucose homeostasis. Leptin and insulin have also been shown to regulate pancreatic islet function. A growing amount of evidence suggests there is considerable overlap in the pathways by which these hormones act to mediate their physiological effects. The insulin receptor substrate-2 (IRS-2) protein is a critical mediator of cellular responses to insulin, especially those associated with somatic growth and carbohydrate metabolism. It has been suggested that IRS-2 signalling pathways act as a potential point of convergence for insulin and leptin signalling. Much of our current understanding of both the cellular and molecular effects of leptin and insulin has been derived from studies of murine models of obesity and diabetes. In this thesis I have exploited three such models to generate mice with defective 1) IRS-2 function (IRS-2 KO) and leptin production (ob/ob) and 2) IRS-2 function and leptin receptor (db/db) function. I have also produced mice which lack IRS-2 and STAT-3 specifically in their P-cells and hypothalamus. As insulin resistance and leptin resistance coexist in obese subjects, it was hoped that such murine models would give some insights into the contribution of these processes to the obesity phenotype and its associated insulin resistance and type 2 diabetes. In particular it was hoped to address the hypothesis that a linear relationship exists between IRS-2 and STAT-3 in mediating the effects of insulin and leptin action, with IRS-2 being upstream of the events that lead to the phosphorylation of STAT-3. Considering either systemic or tissue-specific approaches, it was evident that insults to leptin signalling in combination with lrs-2 deletion results in additive effects upon growth, adiposity, glucose homeostasis and islet function. These additive effects would indicate that IRS-2 and STAT-3 mediate these effects by acting in separate signalling pathways, as opposed to converging upon a linear pathway. However cross talk between these parallel pathways is likely to be critical for the maintenance of insulin and leptin sensitivity in target tissues.

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Type 2 diabetes has become a worldwide epidemic. It is characterised by insulin resistance in major metabolic tissues, and failure of β-pancreatic cells to compensate for this abnormality. Insulin resistance is recognised as an early event in the pathogenesis of type 2 diabetes. Although the precise factors that lead to insulin resistance have not been elucidated fully, there is strong association between insulin resistance and lipid accumulation, in particular lipotoxic fatty acid metabolites in insulin-target tissues. Most recently, evidence has been presented to link abnormal fatty acid accumulation in muscle with reduced mitochondrial activity. However it was unclear if these aberrations are causally related to the development of insulin resistance and type 2 diabetes. The two major pathophysiological abnormalities that underlie type 2 diabetes have long been viewed to require two separate pathogenic processes. The resolution of type 2 diabetes after bariatric surgery has allowed the elucidation of the sequence of events that lead to the restoration of normal metabolism, paving the way for a new understanding of type 2 diabetes as a metabolic state precipitated by a single cause of chronic excess intra-organ lipid accumulation. Magnetic resonance technique provides a non-invasive way to evaluate metabolism in both normal and pathological states. Specifically, 31P magnetic resonance spectroscopy allows the observation of real-time ATP synthesis as a direct measurement of mitochondrial activity. 13C magnetic resonance spectroscopy can be applied to assess muscle glycogen concentration. Both 1H magnetic resonance spectroscopy and magnetic resonance imaging can be used to evaluate intra-organ lipid concentration. Collectively, these innovative techniques offer safe and powerful approaches to study the role of skeletal muscle oxidative capacity and intra-organ fatty acid accumulation and metabolism in the pathogenesis of type 2 diabetes. This thesis presents data which lead to a simplified understanding of the aetiology and pathogenesis of type 2 diabetes.

In this thesis, microvascular and neuronal function in the foot skin of subjects with Type 2 diabetes was investigated to determine whether abnormalities exist, if and so to determine the role of such abnormalities in the pathogenesis of ulceration and its healing. The principle findings of the thesis are: - In the absence of macrovascular disease, impaired nerve function, (large and small nerve fibre) is associated with foot ulceration in Type 2 diabetic subjects; however, there appears to be no additional predictive value in measuring small fibre function using sophisticated methods over large fibre function assessed by measuring vibration sensation. Neither Tcp02 nor the more complicated measurements of microvascular hyperaemic responses were able to discriminate between those with and without ulceration. This would suggest that such tests may not be of benefit in identifying those at greater risk of foot ulceration. The 'LDIflare' a novel, simple, non-invasive and objective method to assess small fibre function is described. This demonstrates that C-fibre dysfunction occurs relatively early in Type 2 diabetes before it is detected by the currently available methods. Thus, the LDlflare may be of value in screening for early diabetic neuropathy. The LDlflare correlates well with skin nerve fibre density and in addition demonstrates functional changes in subjects with painful neuropathy when structural nerve fibre changes are less well marked. This suggests the LDlflare may have an advantage over skin biopsies and quantitative sensory testing in diagnosing painful diabetic neuropathy. The rate of wound closure was identical between control subjects and diabetic patients despite a significant reduction in microvascular blood flow, epidermal and dermal VEGF-A and blood vessel VEGFR-2 expression, the LDlflare response and dermal nerve fibre density. These studies are important in directing further research into these devastating and expensive complications of diabetes.