Obesity and its associated metabolic complications are major burdens upon health systems globally highlighting the need for basic research into therapeutic targets to treat such disorders. The circulating hormone adiponectin has protective effects against several obesity-associated metabolic complications including insulin-resistance, hypertension and cardiovascular disease. Recent studies have elucidated the beneficial metabolic effects of adiponectin yet the molecular mechanisms behind its actions remain poorly characterised. The recent discovery of two receptors of adiponectin in 2003, AdipoR1 and AdipoR2, promises to further our understanding of how adiponectin mediates such effects. AdipoR1 and AdipoR2 are predicted to be seven transmembrane domain proteins (7TMs), however, they are considered unique to other 7TMs such as G-protein coupled receptors (GPCRs) since they have been shown to exhibit a reversed topology and also activate a distinct set of signalling molecules. Owing to their recent discovery and a lack of essential tools to permit basic research into adiponectin receptor (AdipoR) biology, our knowledge of these receptors remains extremely limited. The aim of this thesis was to generate tools and further characterise AdipoR structure and function in order to advance our understanding of how adiponectin’s effects are mediated. Early chapters in this thesis focus upon the generation and characterisation of fundamental molecular tools such as AdipoR mammalian expression plasmids and cell lines. Using these tools, we confirm that each AdipoR localises to the plasma membrane (PM) and does indeed possess a reversed topology to GPCRs; having an intracellular amino terminus and an extracellular carboxyl terminus. AdipoR1 and AdipoR2 are able to form both homo and hetero-multimers, as shown for numerous GPCR proteins, but neither receptor appears to be modified by N-linked glycosylation, a common modification of GPCRs. AdipoR-specific polyclonal antibodies were also generated and characterised in early chapters which allowed detection of endogenous AdipoR1 and AdipoR2 protein in multiple cell lines as well as primary human adipocytes. These studies revealed that AdipoRs are not only expressed in a variety of cell lines but also exhibit differential expression profiles suggesting AdipoR1 and AdipoR2 may mediate distinct functions of adiponectin. A major finding of this thesis was the discovery of a novel AdipoR1-interacting protein, ERp46. This is the first AdipoR1-specific interacting protein identified and only the second AdipoR-interacting protein described to date. ERp46 (also known as EndoPDI and pcTRP) has been previously described although knowledge of its function and cell biology is very limited. ERp46 co-immunoprecipitated with AdipoR1 in several different cell lines and showed no evidence of interaction with AdipoR2. Via immunofluorescence microscopy, ectopically expressed ERp46 localised to both the PM and intracellular structures, likely to be the endoplasmic reticulum (ER) whilst subcellular fractionation revealed that endogenous ERp46 localised to the ER-containing and PM-containing fractions in both HeLa and HEK cell lines. In silico analyses predict that ERp46 has a transmembrane domain and exhibits an extracellular / luminal carboxyl terminus, the latter of which was confirmed by immunofluorescence microscopy studies using epitope-tagged ERp46 constructs. Given that ERp46 primarily localises to the ER, we assessed whether ERp46 was involved in regulation of AdipoR trafficking. ShRNA knockdown of ERp46 resulted in increased AdipoR1 and AdipoR2 expression at the PM providing evidence to support a functional role in AdipoR biology. The presence of ERp46 at the PM, together with the data suggesting ERp46 regulates AdipoR subcellular localisation, led to studies investigating whether ERp46 contributes to adiponectin signalling. We demonstrate that adiponectin treatment results in the phosphorylation of key adiponectin targets (such as AMPK and p38MAPK) in HeLa cells and present data to support the requirement of ERp46 for specific adiponectin-induced signalling events. Furthermore, our studies show that ERp46 is not only involved in adiponectin-induced signalling but also in specific signalling events induced by insulin and epidermal growth factor (EGF) treatment. In summary, the work presented in this thesis confirms the findings of the initial publication in which the receptors were described and also identifies several novel features of the adiponectin receptors previously not reported. The identification of an AdipoR1-specific interacting protein that modulates both the localisation and signalling events of adiponectin is of particular interest. The data described in this thesis supports emerging evidence which suggests that AdipoR1 and AdipoR2 have distinct roles in adiponectin signalling and as such, this work has provided further insights into the mechanisms behind adiponectin action and provides a basis for future studies.

The aim of this work was to verify the hypothesis that eicosapentaenoic acid and docosahexaenoic acid favorably influence the inflammatory response in the organism through their influence on the expression of genes associated with the inflammatory process. The experiment was carried out on pigs fed with fish oil supplement and palm oil supplement. Certain experimental groups were additionally stimulated by lipopolysaccharide to induce a rapid onset of inflammatory response, with particular concern for the ability of polyunsaturated fatty acids n-3 to inhibit this process. I have been observing changes in gene expression for AdipoR1 and AdipoR2 adiponectin receptors, as well as expression in other genes related to the inflammatory process of LBP; ICAM1; IL-6; TNFalfa; IL-12. The determination of the expression of selected genes using qRT-PCR was used to process the results from the collected liver and adipose tissue samples. I statistically evaluated the expression results of the genes and displayed them as graphs. The favorable demonstrable effect of fish oil was only observed in the ADIPOR2 gene in the liver, in the non-stimulated lipopolysaccharide group (P <0.05). There was no detectable effect of fish oil on modulation of inflamation in pigs. However, we can deduce the causes of the failure from the experimental results, and in other studies we might be able to confirm the hypothesis. However, we can deduce the causes of the failure from the experimental results and we might be able to confirm the hypothesis in other studies.

<p>Obesity is the latest epidemic of the 21^st century. Indeed, numerous studies have associated obesity with an increased risk of developing several health conditions, including cancer. Moreover, modest increases in body mass index increase the risk of developing cancer, especially cancer of the kidney. Although the mechanism mediating this increased risk is unknown, the plasma level of adiponectin is known to be inversely correlated with body weight and risk of developing kidney cancer.</p> <p>Tumour suppression via adiponectin is believed to be mediated through adiponectin receptor-1, which activates AMPK by LKB1 and suppresses pathways upregulated in cancer by inhibiting mTOR. Consistent with the anti-tumourigenic properties of this pathway, several cancers display reduced AdipoR1 and LKB1 expression and/or increased mTOR activity. In this study we identified reduced AdipoR1 and LKB1 protein expression in patients’ renal cell carcinomas and quantified the reduction in LKB1, on tissue microarrays containing 201 RCC patients, to be significant.</p> <p>Targeted knockdown of LKB1 in CRL-1932 cells (shLKB1) was accompanied by a reduction in AdipoR1, and recapitulated our observations in RCC tumours. These shLKB1 cells were unable to execute established events of adiponectin-AMPK signalling and, presented increased proliferation and invasion abilities <em>in vitro</em> and tumour growth <em>in vivo</em>. Collectively, these results suggest that a reduced plasma level of adiponectin coupled with a downregulation of AdipoR1 and LKB1 expression, disrupts the tumour-suppressive adiponectin-AMPK signalling pathway, and rationalizes the association of obesity with the development of RCC.</p> / Master of Science in Medical Sciences (MSMS)

Il est maintenant reconnu qu'un excès ou un manque de tissu adipeux peut avoir un impact négatif sur la reproduction porcine. Ainsi, une faible adiposité résulte en une augmentation du temps requis pour le premier oestrus, une diminution des tailles de portées et une augmentation de l'intervalle sevrage-oestrus. Les producteurs de porcs étant payés selon le rendement en maigre des carcasses de porcs envoyés aux abattoirs, la sélection génétique des dernières années a surtout été orientée vers la production de porcs de plus en plus maigres. Cependant, cette réduction du tissu adipeux a, du même coup, entraîné de nombreux problèmes de reproduction et un taux de réforme des truies de plus en plus élevé. Nous savons aujourd'hui que les cellules adipeuses sont le siège d'une activité métabolique intense et qu'elles sont capables de sécréter une multitude de facteurs vers la circulation sanguine. Parmi ces facteurs, nous retrouvons diverses adipokines telles la leptine et l'adiponectine, lesquelles auraient des effets bénéfiques sur les organes reproducteurs. L'adiponectine est l'adipokine la plus abondamment sécrétée par le tissu adipeux et la présence de ses récepteurs, AdipoR1 et AdipoR2, au niveau des ovaires et de l'utérus de la truie suggère un rôle pour l'adiponectine sur diverses fonctions reproductrices. Dans cette étude, nous avons voulu déterminer l'effet de l'adiponectine recombinante sur l'utérus de la truie en utilisant un modèle in vitro de culture de cellules endométriales primaires. Dans un deuxième temps, nous avons étudié la modulation de l'expression de cette adipokine et de ses récepteurs pendant le cycle oestral et au début de la gestation. Utilisant la méthode de PCR en temps réel, nous avons tout d'abord confirmé la présence des récepteurs AdipoRl, AdipoR2 et cadhérine 13 dans l'endomètre de la truie et constaté que l'expression de ces gènes est modulée pendant le cycle oestral, étant à son maximum au milieu de la phase lutéale. Nous avons aussi observé une modulation de l'expression de l'adiponectine dans le tissu adipeux sous-cutané, avec un transcrit plus abondant chez les truies hypo-prolifiques (? 10 porcelets par portée) et les truies cycliques, comparativement aux truies hyper-prolifiques (? 15 porcelets par portée) et gestantes (jour 15 de la gestation), respectivement. Dans l'endomètre, le récepteur AdipoR2 est plus fortement exprimé chez la truie gestante que chez la cyclique, alors qu'il est plus élevé chez les truies hypo-prolifiques que chez les hyper-prolifiques. Chez l'embryon de 15 jours, le profil inverse est observé, avec une quantité plus élevée de transcrits observée pour les récepteurs AdipoRl et AdipoR2 chez les embryons issus des truies hyper-prolifiques. Bien que l'expression de la cadhérine 13 soit modulée pendant le cycle oestral, la gestation (cyclique vs gestante) et la prolificité (hyper- vs hypo-prolifique) ne présentent aucun effet significatif sur son expression. Finalement, l'utilisation de culture de cellules stromales endométriales primaires traitées à l'adiponectine recombinante a révélé une augmentation de l'expression en ARN messager des gènes de la prostaglandine synthétase 2 (PTGS2) et du facteur de croissance endothéliale vasculaire (VEGF), deux gènes ayant un rôle clé en début de gestation pour la survie embryonnaire. De plus, cette modulation de l'expression génique serait médiée par l'activation des voies de signalisation de la MAPK-ERK1/2 et de la kinase activée par l'AMP (AMPK). Collectivement, ces résultats suggèrent une implication directe de l'adiponectine sur les cellules endométriales porcines et dans les processus de reproduction chez la truie, autant pendant le cycle oestral qu'en début de gestation. De plus, nos résultats identifient l'adiponectine comme un régulateur potentiel des adaptations métaboliques se produisant en début de gestation.