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T2D is a bihormonal disorder characterised by hypoinsulinaemia and hyperglucagonaemia. The incretin GLP-1 is therapeutically attractive as, in addition to augmenting glucose-stimulated insulin secretion, it is also able to suppress glucagon secretion from alpha cells. However, the mechanism by which GLP-1 is able to modulate glucagon secretion has not been fully established. The observation that the GLP-1 receptor is expressed at extremely low levels on alpha-cells has led some groups to postulate that the effects of GLP-1 may be mediated by paracrine mechanisms. Conversely, other groups have shown that GLP-1 is able to directly regulate glucagon secretion in isolated alpha-cells, discounting the role of islet cross-talk. Further work needs to be performed to fully understand the mechanism of GLP-1 action on alpha cells. Specifically it will be important to determine if GLP-1 can mediate its effects via another receptor on alpha-cells or whether its abundantly-occurring metabolite, GLP-1 (9-36) is also able to affect glucagon secretion.

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.

OBJECTIVE: Digenic causes of human disease are rarely reported. Insulin via its receptor, which is encoded by INSR, plays a key role in both metabolic and growth signaling pathways. Heterozygous INSR mutations are the most common cause of monogenic insulin resistance. However, growth retardation is only reported with homozygous or compound heterozygous mutations. We describe a novel translocation [t(7,19)(p15.2;p13.2)] cosegregating with insulin resistance and pre- and postnatal growth deficiency. Chromosome translocations present a unique opportunity to identify modifying loci; therefore, our objective was to determine the mutational mechanism resulting in this complex phenotype. RESEARCH DESIGN AND METHODS: Breakpoint mapping was performed by fluorescence in situ hybridization (FISH) on patient chromosomes. Sequencing and gene expression studies of disrupted and adjacent genes were performed on patient-derived tissues. RESULTS Affected individuals had increased insulin, C-peptide, insulin-to-C-peptide ratio, and adiponectin levels consistent with an insulin receptoropathy. FISH mapping established that the translocation breakpoints disrupt INSR on chromosome 19p15.2 and CHN2 on chromosome 7p13.2. Sequencing demonstrated INSR haploinsufficiency accounting for elevated insulin levels and dysglycemia. CHN2 encoding beta-2 chimerin was shown to be expressed in insulin-sensitive tissues, and its disruption was shown to result in decreased gene expression in patient-derived adipose tissue. CONCLUSIONS: We present a likely digenic cause of insulin resistance and growth deficiency resulting from the combined heterozygous disruption of INSR and CHN2, implicating CHN2 for the first time as a key element of proximal insulin signaling in vivo.

OBJECTIVE: To determine the incidence of deep venous thrombosis (DVT) in patients with Parkinson disease. DESIGN: Prospective study. SETTING: Outpatient neurology clinic. PATIENTS: Eighty-one patients with Parkinson disease. OUTCOME MEASURES: Duplex ultrasonographic scans consisting of M mode images and compression images, Doppler flow assessment and augmentation of flow assessment. RESULTS: Four patients had leg DVT; in 3 of the patients the thrombi were in calf veins, whereas in 1 patient the thrombosis was in the superficial femoral vein. Of the patients with DVT, 1 (1.23%) had stage 2 Parkinson disease, 1 (1.23%) had stage 2.5, and the other 2 (2.46%) had stage 4. CONCLUSIONS: There was no statistically significant difference in the incidence of DVT among patients who were more severely disabled by Parkinson disease. However, an overall incidence of DVT of 4.9% in a group of asymptomatic patients is clinically meaningful, suggesting that patients with Parkinson disease are at risk for asymptomatic leg DVT.

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