The IL-6 system is key in the development of chronic low-grade inflammation. It is known to be upregulated in response to acute exercise and lowered at rest after exercise training. IL-6 has both anti- and pro-inflammatory properties and moderation of this cytokine could alleviate chronic low-grade inflammation which is associated with obesity and Type 2 diabetes mellitus (T2DM). This thesis investigated the interplay between inflammation, glycaemic control and high intensity intermittent training (HIIT) - an exercise regimen that has been shown to yield many health benefits. There was a greater increase in IL-6 after an acute bout of HIIT than continuous moderate intensity exercise, where external work was matched (Chapter 4). Although sIL-6R and the IL-6/sIL-6R complex were both significantly increased after acute exercise there were no differences between HIIT and moderate intensity exercise. In response to 2 weeks HIIT there was a significant reduction in IL-6 and increase in IL-6R in adipose tissue in overweight and obese males (Chapter 5). It was also determined that IL-6R present in adipose tissue is at least partly composed of the membrane-bound IL-6R isoform (Chapter 6). Reductions in circulating sIL-6R, the IL-6/sIL-6R complex, MCP-1 and adiponectin, as well as a decrease in waist circumference and increase in peak oxygen uptake during exercise were also induced after 2 weeks HIIT (Chapter 5). Young adults with T2DM (< 40 y) displayed elevated levels of inflammatory proteins in comparison to lean controls, however there were no significant differences in comparison to obese controls (Chapter 7). In conclusion, the findings of this thesis demonstrate that acute and repeated bouts of HIIT have positive effects on the inflammatory profile in the circulation and adipose tissue, particularly in relation to the IL-6 system. It should be determined if HIIT is an achievable mode of exercise for patient populations, including T2DM patients, in order to downregulate the inflammatory profile.

<p>The delivery of therapeutic peptides and proteins is often challenged by a short circulation half-life, necessitating frequent injections that limit efficacy, reduce patient compliance and increase treatment cost. The covalent conjugation of therapeutic peptides and proteins, and more recently oligonucleotide-based drugs, with the “stealth” polymer poly(ethylene glycol) (PEG), termed PEGylation, is one of the most commonly used approaches to increase the in vivo half-life and reduce the immunogenicity of these therapeutic biomolecules. However, after several decades of research and clinical use, the limitations of PEGylation have begun to emerge.</p><p>Conventional methods for synthesizing peptide/protein-polymer conjugates have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of control over site and stoichiometry of conjugation, which results in heterogeneous products with significantly compromised biological activity. Additionally, anti-PEG antibodies have been induced in patients treated with PEGylated drugs and have been shown to correlate with rapid clearance of these drugs. High levels of pre-existing anti-PEG antibodies have also been found in individuals naïve to PEGylated agents, which are associated with serious first-exposure allergic reactions.</p><p>To address the synthetic limitations of PEGylation, a general approach for the high-yield synthesis of site-specific (C-terminal) and stoichiometric (1:1) peptide/protein-polymer conjugates, named sortase-catalyzed polymer conjugation, was developed. Demonstrating proof-of-concept of the approach with green fluorescent protein (GFP) as a model protein, sortase A from Staphylococcus aureus was used to site-specifically attach an initiator solely at the C-terminus of GFP, followed by in situ growth of the PEG-based brush polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) from the protein macroinitiator by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeded with high specificity and near-complete (~ 95%) product conversion. Subsequent in situ ATRP in aqueous buffer produced 1:1 stoichiometric conjugates with > 90% yield, tunable MW, low dispersity, and no denaturation of the protein. The extraordinarily high yield compares favorably to order of magnitude losses typically seen in conventional PEGylation processes.</p><p>Next, the therapeutic potential of POEGMAlation, or the conjugation of POEGMA to a peptide or protein, was demonstrated by implementing the developed sortase-catalyzed polymer conjugation strategy with exendin-4 (exendin), a therapeutic peptide for treating type 2 diabetes, to synthesize exendin-C-POEGMA conjugates with a wide and tunable range of molecular weights (MWs) and low dispersity. A single subcutaneous injection of exendin-C-POEGMA conjugates lowered blood glucose for up to 120 h in a diabetic mouse model. Most intriguingly, we showed that appending PEG as oligomeric side-chains on the conjugated POEGMA and tuning the side-chain length completely eliminated the reactivity of exendin-C-POEGMA conjugates toward patient-derived anti-PEG antibodies without compromising in vivo efficacy. Clinically, the lack of anti-PEG antigenicity of POEGMA conjugates is expected to completely eliminate serious first-exposure allergic reactions and the accelerated blood clearance of POEGMA-drug conjugates due to pre-existing anti-PEG antibodies in patients.</p><p>Collectively, these results establish POEGMAlation as a next-generation PEGylation technology that is highly useful for improving the pharmacological performance of therapeutic biomolecules while providing a timely solution to the increasing levels of pre-existing anti-PEG antibodies in patients that are seriously hindering the safety and efficacy of traditional PEGylated drugs.</p> / Dissertation

Genome-wide association studies (GWAS) have made substantial progress in implicating genomic regions in type 2 diabetes (T2D) susceptibility. Whilst attributing causal mechanisms to loci has proved non trivial, these studies have provided insights into the genetic architecture underlying the disease. GWAS findings indicate a causal role for gene regulatory processes, and suggest that pancreatic beta-cells play a pivotal role in mediating common T2D association. Work presented in this thesis therefore sought to generate novel regulatory annotations from human islets, and to assess whether T2D-associated loci can be accurately fine-mapped using statistical approaches, with the aim of improving understanding of causal mechanisms underlying these associations through integration of the two approaches. Using small RNA sequencing in human islets and enriched beta-cell populations (both n=3) and mRNA sequencing in a large number of human islets (n=130), I increased the number of available human islet annotations. These studies identified high or islet-specific expression in many micro RNAs (miRNAs) without previously known roles in human islets. It also provided the largest study of quantitative trait loci (eQTLs) and allele-specific expression (ASE) in human islets to date, identifying significant eQTLs for 1,636 genes and significant ASE at 8,754 genes. There was enrichment of active islet chromatin, compared to other tissues, at the best eQTL variant for each gene, but also substantial sharing of significant eQTLs between islets and other tissues. Simulations were used to assess the utility of fine-mapping approaches for refining common disease-associated loci to smaller intervals or sets of variants likely to include the causal variant. The results demonstrated that fine-mapping can indeed refine these loci to sets or intervals of a size more amenable to functional follow-up or focussed intersection with high quality annotations. Furthermore, using an approximated Bayesian approach, I was able to refine twenty-one of the known common T2D-associated loci. Finally, using the newly generated annotations, I demonstrated enrichment of T2D association signal for regulatory RNA annotations (islet lncRNAs and miRNA target gene sets). I also identified examples in which these types of annotation overlap common and rare variation suggestive of a role in T2D pathogenesis. Using further islet annotations, I also uncovered potential causal mechanisms at four of the twentyone fine-mapped common T2D loci. These data therefore provide many novel islet regulatory annotations that can be intersected with T2D genetics, and provide a first example of how such an approach can lead to novel potential causal mechanisms underlying association loci.

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.

Depression and medical condition comorbidity can improve or hinder depression care. Comorbid MD-T2DM is particularly relevant for Black Americans due to increased T2DM risk and less mental health services access. Data from the NSAL was used to examine how co-morbid T2DM/HT and MD affect depression care. Only 19.2% of Black Americans with MD and 23.0% with comorbid MD-T2DM reported adequate treatment. Insurance status explained more variance in depression care than age, sex, and ethnicity. Respondents with MD+T2DM were no more or less likely to receive depression care than those with MD alone. However, respondents with MD+HT were more likely to report adequate treatment (OR=2.09 95% CI[1.20, 3.64]) and anti-depressant use for ≥60 days (OR=2.14 95% CI[1.15, 4.02]). Ethnicity, education and insurance status moderated the effects of HT. Yet guideline-concordant depression care is low among depressed Black Americans, including those with comorbid T2DM.

Caenorhabditis elegans has been studied as a model organism in various areas of biomedical research because it shares many conserved functions at molecular and genetic levels with humans. Specifically, it is an ideal organism to study heterogeneous metabolic syndromes such as Type 2 Diabetes Mellitus (T2DM) as C. elegans can be used to delineate molecular pathways that are at the core of its problems. A growing number of populations worldwide are faced with chronic T2DM, which also manifests several complications, such as blindness, neuropathy and cardiovascular diseases. Currently, metformin is the first-line drug of choice administered to treat T2DM. While the mechanism by which it alleviates the symptoms of diabetes is unknown, it has been found to reduce metabolic rate by partially inhibiting the mitochondrial complex I in mammals. Using C. elegans as a genetic model organism, we show that metformin reduces the mitochondrial activity through endosomal Na+/H+ exchanger, which a previous lab member has found to be a potential target of metformin. Furthermore, we show that high glucose diet−known to reduce the worm’s lifespan−alter the endosomal-lysosomal system and autophagy, providing insights to using C. elegans as a diabetic model. Based on these results, we propose that C. elegans can serve as a model organism to study T2DM as well as provide new ways to further investigate the pathophysiology of this disease.

PROX1 étant un facteur de susceptibilité au diabète de type 2 (DT2), nousavons réalisé des études génétiques et moléculaires afin de comprendre son rôledans l’étiologie du DT2.Nous avons analysé l’impact de 80 SNPs de PROX1 sur des phénotypescliniques associés au DT2 dans l’étude HELENA (n=1155 adolescents) et montréque trois SNPs (rs340838, rs340837 et rs340836) sont associés à l’insulinémie àjeun. Nous avons évalué la fonctionnalité de 9 SNPs (les 3 SNPs associés et 6 SNPsen déséquilibre de liaison) en utilisant un gène rapporteur Luciférase dans descellules HepG2 et MIN6. Les allèles associés à la diminution de l’insulinémie desSNPs rs340874, rs340873 et rs340835 sont associés à une diminution del’expression du gène rapporteur Luciférase, suggérant que l’expression de PROX1est diminuée chez les individus porteurs des allèles à risque.Nous avons aussi montré que l’inhibition de l’expression de Prox1 par siRNAsdans les cellules INS-1E engendrait une diminution de 1,7 fois de la sécrétiond’insuline en réponse au glucose et qu’une concentration élevée en glucose modulaitpositivement l’expression de la protéine Prox1.Des analyses transcriptomiques réalisées dans les cellules INS-1E ont permisde montrer que certains des gènes cibles de PROX1 dans les cellules bêta sont desgènes impliqués dans des voies de sécrétion d’insuline.Enfin, nous avons également observé que l’agoniste de PPARgamma, latroglitazone, diminuait l’expression de Prox1 dans les cellules INS-1E.Ces résultats suggèrent qu’une altération de l’expression de Prox1 par desvariants cis-régulateurs pourrait conduire à une sécrétion d’insuline en réponse auglucose altérée des cellules bêta, conférant ainsi une susceptibilité au DT2. / As PROX1 is a susceptibility factor for type 2 diabetes (T2D), we conductedgenetic and molecular studies to better understand the role of PROX1 in the etiology of T2D. We assessed the impact of 80 PROX1 SNPs on T2D-related biochemical traits in the HELENA study (n=1155 adolescents) and showed that 3 SNPs (rs340838, rs340837 and rs340836) were significantly associated with fasting plasma insulin levels. We evaluated the functional impact of 9 SNPs (the 3 insulin-associated SNPs plus 6 SNPs in linkage disequilibrium) using a Luciferase reporter gene expression in HepG2 and MIN6 cells. The insulin-lowering alleles of the rs340874, rs340873 and rs340835 SNPs were associated with lower Luciferase gene expression, suggesting that PROX1 expression may be lower in individuals carrying the insulin-lowering alleles. We also showed that the knock-down of Prox1 expression by siRNA in INS-1E cells resulted in a 1.7 fold reduced glucose-stimulated insulin secretion and that high concentrations of glucose positively modulated Prox1 protein expression. Then, microarray analyses performed in INS-1E cells showed that some PROX1 target genes in the _cells are implicated in insulin secretion pathways. Finally, we observed that the PPARgamma agonist, the troglitazone, decreased Prox1 expression in INS-1E cells. Altogether, these results suggest that an altered expression of Prox1 bys cisregulators variants results in an altered -cell glucose-stimulated insulin secretion andthereby confers susceptibility to T2D.

La mortalité cardiovasculaire représente la première cause de mortalité chez les sujets diabétiques de type 2. La dyslipidémie de ces patients caractérisée par le quatuor : hypertriglycéridémie, baisse du HDL-cholestérol, augmentation du nombre de LDL petites et denses et hyperlipidémie postprandiale, constitue un facteur de risque prépondérant. Cette dyslipidémie est en grande partie expliquée par l’accumulation sanguine des lipoparticules riches en triglycérides (TRL) d’origine hépatique (VLDL) et intestinale (chylomicrons). L’hyperproduction des chylomicrons, déjà connue pour les VLDL, est une composante nouvellement reconnue d’insulinorésistance. L’action inhibitrice aigüe de l’insuline sur la production des VLDL est absente chez le sujet diabétique de type 2. Dans notre étude, nous avons montré l’absence d’effet aigu de l’insuline sur la production des chylomicrons chez les sujets diabétiques de type 2 grâce à une étude cinétique utilisant un isotope stable (D3-leucine). / The cardiovascular mortality represents the first cause of mortality in human type 2 diabetes. The typical diabetic dyslipidemia characterized by the quartet: high triglyceride levels, low HDL-cholesterol, increased number of small and dense LDL particles and postprandial hyperlipidemia, is a major cardiovascular risk factor. This dyslipidemia is mainly explained by the accumulation of triglyceride-rich lipoproteins (TRL) from liver (VLDL) and intestine (chylomicrons).The overproduction of chylomicrons, as is known for VLDL, is a newly characteristic of insulin resistant states. The acute inhibitory effect of insulin on VLDL production is absent in type 2 diabetic patients. In our study, we have shown the absence of acute inhibitory effect of insulin on chylomicron production in type 2 diabetic patients, using a kinetic study with stable isotope (D3-leucine).

A glândula pineal é responsável pela produção circadiana do hormônio melatonina, o qual possui além da propriedade cronobiótica, ações relacionadas à neurogênese e atividade anti-inflamatória. A prevalência do distúrbio do metabolismo dos carboidratos conhecido como diabetes mellitus afeta cada vez mais a população mundial em decorrência do aumento da expectativa de vida, alimentação inadequada, obesidade e detrimento de atividade física regular, caracterizando além dos sintomas clássicos como hiperglicemia, polifagia, polidipsia e glicosúria, problemas relacionados a plasticidade cerebral, neuroinflamação e maior probabilidade do desenvolvimento de doenças neurodegenerativas. A hiperglicemia crônica influencia negativamente a síntese e secreção de melatonina, fato que contribui ainda mais para o agravamento dos sintomas do quadro de diabetes, formando um ciclo de retroalimentação positiva. Tendo em vista as recentes pesquisas que mostram que a molécula de melatonina atua como agente anti-inflamatório, neuroprotetor e indutor da neurogênese, nos propomos a investigar, com este trabalho, se a suplementação via oral de melatonina em ratos modelo de diabetes tipo 2 (Goto-Kakizaki) é capaz de amenizar os danos cerebrais provocados pela hiperglicemia crônica. Nossos resultados demonstram que a suplementação com melatonina não foi capaz de alterar a glicemia de jejum dos animais, tampouco torná-los mais sensíveis à insulina mediante teste de tolerância à glicose. Entretanto, a mesma atuou sobre o peso corporal dos animais diabéticos, fato ainda a ser elucidado do ponto de vista molecular. As diferenças encontradas no que se refere a expressão gênica e proteica de fatores estudados, se dão principalmente pelo fato do animal ser hiperglicêmico (Goto-Kakizaki) ou não (Controle), sendo que a melatonina é capaz de atuar em questões pontuais. Ainda, os animais Goto-Kakizaki não apresentam processos inflamatórios no hipocampo e hipotálamo. / The pineal gland is responsible for the circadian production of the hormone melatonin, which has besides chronobiotic properties, actions related to adultneurogenesis and anti-inflammatory activityThe prevalence of carbohydrate metabolism disorder known as diabetes mellitus increasingly affects the world population due to increased life expectancy, inadequate diet, obesity, and the detriment of regular physical activity, characterized in addition to classic symptoms such as hyperglycemia, polyphagia, polydipsia,and glycosuria. This problem related to brain plasticity, neuroinflammation, and increased probability of developing neurodegenerative diseases. Chronic hyperglycemia negatively influences the synthesis and secretion of melatonin, a fact that contributes to the worsening symptoms of diabetes, forming a positive feedback loop. Taking in count the recent studies showing that the melatonin molecule acts as an anti-inflammatory, neuroprotective and neurogenetic inducer, we propose to investigate whether oral supplementation of melatonin in type 2 diabetes mellitus (Goto-Kakizaki) can alleviate the brain damage induced by chronic hyperglycemia. Our results demonstrate that supplementation with melatonin was not able to alter the fasting glycemia of the animals, nor did it make them more sensitive to insulin through a glucose tolerance test. However, it acted on the body weight of diabetic animals, a fact still to be elucidated from the molecular point of view. The differences found in gene expression and protein expression of the studied factors are mainly because the animal is hyperglycemic (Goto-Kakizaki) or not (Control), and melatonin can act on specific questions. Furthermore, Goto-Kakizaki animals do not present an inflammatory processin the hippocampus and hypothalamus.