La prostate est entourée de tissu adipeux périprostatique (TAPP), séparée de celui-ci uniquement par une fine barrière de collagène appelée la capsule prostatique. En clinique, l'infiltration du TAPP par des cellules tumorales est l'un des principaux critères histologiques de mauvais pronostic suggérant que le TAPP (en particulier les adipocytes) pourrait être un acteur clé de l'agressivité du cancer de la prostate. Dans un premier temps, nous avons identifié une chimiokine adipocytaire (CCL7) et un de ses récepteur (CCR3) qui contribuent à la dissémination des cellules cancéreuses à l'extérieur de la prostate. En effet, à travers des approches in vitro et in vivo, nous avons montré que l'inhibition de l'axe CCR3/CCL7 diminue la dissémination locale du cancer de la prostate avec un effet plus prononcé en conditions d'obésité. De plus, l'expression de ce récepteur est associée à l'agressivité tumorale et à la rechute chez les patients. Au-delà de la dissémination locale, une des complications du cancer de la prostate les plus graves et les plus dures à traiter reste la dissémination métastatique osseuse. Fort des résultats obtenus dans la première partie de notre travail, nous avons voulu savoir si les adipocytes médullaires présents au niveau du site métastatique osseux étaient capable d'attirer les cellules tumorales par les mêmes mécanismes que le TAPP. En utilisant des échantillons d'adipocytes médullaires issus de patients lors d'approches in vitro, nous avons pu montrer que les adipocytes médullaires présentent un phénotype différent de ceux du TAPP et qu'ils sécrètent la chimiokine CCL7 en quantité bien plus élevée. Cette dernière va ainsi induire la migration des cellules tumorales prostatiques vers le site osseux suivant un axe CCR3/CCL7. En inhibant cet axe, nous avons pu montrer in vivo et in vitro le potentiel de cet axe comme cible thérapeutique, puisqu'il nous a permis d'empêcher spécifiquement la formation de métastases osseuses. Nous avons pu confirmer l'importance de l'axe CCR3/CCL7 dans le cancer de la prostate métastatique par des méta-analyses cliniques et des études sur des tumeurs de patients. Enfin, nous avons pu montrer que les modifications physiopathologiques des adipocytes comme l'obésité et le vieillissement tous deux considérés comme des facteurs de risque du cancer de la prostate, augmentent fortement la migration induite par les adipocytes médullaires. En conclusion, nos résultats montrent que les adipocytes périprostatiques et médullaires sont capables d'influencer la progression du cancer de la prostate en favorisant, via la sécrétion de chimiokines, le franchissement de la capsule prostatique puis la dissémination métastatique osseuse. L'ensemble de ces mécanismes est amplifié en conditions d'obésité ou de vieillissement ce qui permet de proposer de nouveaux mécanismes moléculaires à l'émergence de cancers plus agressifs chez ces patients. Ainsi notre travail a permis d'identifier l'axe CCR3/CCL7 comme un axe majeur dans la dissémination du cancer de la prostate induite par le tissu adipeux, intervenant aussi bien au niveau local qu'au niveau métastatique. / Prostate is surrounded by periprostatic adipose tissue (PPAT), only separated by a thin layer of collagen called prostatic barrier. Infiltration through PPAT and prostatic barrier crossing by tumor cells are known as major histological and clinical bad prognosis factors. These observations suggest a key role of PPAT (and mainly adipocytes) in prostate cancer development and invasiveness. First, we identified a chemokine secreted by adipocytes (CCL7) and its associated receptor (CCR3) that contribute to local dissemination of prostate cancer cell and lead them to leave the prostate. Thus, using in vitro and in vivo approach, we showed that the inhibition of CCR3/CCL7 axis decreases local dissemination of prostate cancer with an enhanced effect in obesity context. Moreover, CCR3 expression has been associated to tumor aggressiveness and relapse on patient samples. Beyond local dissemination, bone metastasis is one of the major and deadliest complication of prostate cancer, with only few treatments available. Regarding results obtained in our first work on local dissemination, we focused on medullary adipocytes contained in bone marrow and study whether or not they could chemoattract prostate cancer cells just as PPAT in local dissemination. Using medullary adipocytes samples from patients and in vivo approach, we showed that medullary adipocytes expressed a unique phenotype different from PPAT adipocytes and that they secrete high level of CCL7. This chemokine will lead to directed migration of prostate cancer cells towards bone metastatic site through CCR3/CCL7 axis. By inhibiting this axis, we observed a specific inhibition of bone metastases formation both in vitro and in vivo, highlighting the therapeutic potential of this axis. We confirmed the main impact of CCR3/CCL7 axis in prostate cancer metastases, using clinical meta-analysis and studies on tumors from patients. Finally, we showed that physiopathological modifications of adipocytes, such as obesity and aging, which are both considered as prostate cancer bad prognosis factors, increase the chemoattraction of prostate cancer cells induced by medullary adipocytes secretions. As a conclusion, our results highlight that periprostatic adipocytes and medullary adipocytes can influence prostate cancer progression by secreting chemokines and leading to prostatic barrier crossing and bone metastatic dissemination. These mechanisms are enhanced in obesity and aging context. This allow us to suggest a new molecular mechanisms enhancing cancer aggressiveness. Thus, our work allowed us to identify CCR3/CCL7 axis as a major axis in the dissemination of prostate cancer induced by adipocytes secretions, acting on both local and metastatic level.

Le trastuzumab est un anticorps monoclonal déjà utilisé dans le traitement du cancer du sein sur-exprimant la protéine HER2. Malgré son efficacité, la résistance est souvent apparue. Ici, nous avons étudié l'impact des cellules adipocytaires sur la cytotoxicité cellulaire dépendante de l'anticorps (ADCC), une des mécanismes d'action principaux du trastuzumab. Nous avons trouvé que les adipocytes, ainsi que les pré- adipocytes, inhibent l'ADCC médié par le trastuzumab. Nous avons montré que les factors dérivées d'adipocytes, comme des protéines ou des exosomes, causent l'inhibition d'ADCC. Aucune séquestration ou dégradation de l'anticorps a été observée. Des analyses phénotypiques n'ont pas révélé de modification des récepteurs des cellules NK, ni du niveau de HER2 sur les cellules cancéreuses en présence d'adipocytes. La pré-incubation des cellules cancéreuses avec le surnageant des adipocytes réduit la sensitivité tumorale à l'ADCC. Nous avons trouvé que le factor de croissance et de différenciation GDFI5 est rapidement induit dans les cellules cancéreuses exposées au surnageant d'adipocytes. Une diminution de l'expression de GDFI5 par les siRNA réverse l'inhibition d'ADCC induite par les adipocytes. En conclusion, nous avons démontré que les adipocytes jouent un rôle dans la résistance des cellules de cancer du sein à l'ADCC médié par le trastuzumab, et nous suggérons que cibler GDFI5 ou l'interaction entre les adipocytes et les cellules cancéreuses pourrait sensibiliser les cellules cancéreuses au traitement par l'anticorps monoclonal / Trastuzumab is a monoclonal antibody already approved in the treatment of HER2-expressing breast cancer. Despite its efficacy, resistance often occurs. Here, we investigated the impact of adipocytes on antibody dependent cellular cytotoxicity (ADCC), one of the main mechanisms of action of trastuzumab. We found that adipocytes, as well as preadipocytes, inhibited trastuzumab-mediated ADCC. We showed that adipocyte-derived factors, likely proteins or exosomes, mediated the inhibition of ADCC. No titration or degradation of the antibody was detected. Analysis of cell phenotype did not reveal any modification of NK cell receptors, nor of HER2 levels on cancer cells in the presence of adipocytes. Pre-incubation of cancer cells with adipocyte-conditioned medium reduced sensitivity of cancer cells to ADCC. We found that growth differentiation factor l5 (GDFl5) was rapidly induced in cancer cells exposed to adipocyte-conditioned medium. Down-regulation of GDFl5 by siRNA reversed the adipocyte-induced inhibition of ADCC. In conclusion, we demonstrated that adipocytes play a role in the resistance of breast cancer to trastuzumab-mediated ADCC, and suggested that targeting GDFl5 or the crosstalk between adipocytes and cancer cells may sensitize cancer cells to monoclonal antibody treatment

The prevalence of type II diabetes is increasing at an alarming rate due to the western world lifestyle. Type II diabetes is characterized by an insulin resistance distinguished by impaired glucose uptake in adipose and muscle tissues. The molecular mechanisms behind the insulin recistance and also the knowledge considering normal insulin signalling in fat cells, especially in humans, are still unclear. Insulin receptor substrate (IRS) is known to be important for medating the insulin-induced signal from the insulin receptor into the cell. We developed and optimized a method for transfection of primary human adipocytes by electroporation. By recombinant expression of proteins, we found a proper IRS to be crucial for both mitogenic and metabolic signalling in human adipocytes. In human, but not rat, primary adipocytes we found IRS1 to be located at the plasma membrane in non-insulin stimulated cells. Insulin stimulation resulted in a two-fold increase of the amount of IRS1 at the plasma membrane in human cells, compared with a 12-fold increase in rat cells. By recombinant expression of IRS1 we found the species difference between human and rat IRS1 to depend on the IRS proteins and not on properties of the host cell. The adipocytes function as an energy store, critical for maintaining the energy balance, and obesity strongly correlates with insulin resistance. The insulin sensitivity of the adipocytes with regard to the size of the cells was examined by separating small and large cells from the same subject. We found no increase of the GLUT4 translocation to the plasma membrane following insulin stimulation in the large cells, whereas there was a two-fold increase in the small cells. This finding supports the idea of a causal relationship between the enlarged fat cells and reduced insulin sensitivity found in obese subjects. The insulin receptor is located and functional in a specific membrane structure, the caveola. The morphology of the caveola and the localization of the caveolar marker proteins caveolin-1 and -2 were examined. Caveolae were shown to be connected to the exterior by a narrow neck. Caveolin was found to be located at the neck region of caveolae, which imply importance of caveolin for maintaining and sequestering caveolae to the plasma membrane. In conclusion, the transfection technique proved to be highly useful for molecular biological studies of insulin signal transduction and morphology in primary adipocytes.

The high rate of obesity is having a significant impact on human health. Understanding the underlying biological mechanisms that regulate adipogenesis and adipocyte lipid metabolism is necessary to identify novel approaches that promote weight loss. Conjugated linoleic acid (CLA) is an example of a naturally-derived product reported to exhibit an anti-obesity effect. For this thesis, it was hypothesized that the anti-obesity effects of the t10-c12 CLA isomer is due to lipid droplet dynamics alteration through activation of the Wnt/β-catenin pathway, which leads to weight loss via affecting adipogenesis and/or adipocyte death. Testing of this hypothesis was achieved by examining the effects of the most biologically active CLA isomers, cis-9, trans-11 (c9-t11), trans-10, cis-12 (t10-c12) CLA using in vitro (3T3-L1 cell line) and in vivo (mouse) models. In 3T3-L1 preadipocytes, both c9-t11 and t10-c12 CLA stimulated early stage differentiation, while t10-c12 CLA inhibited late differentiation as indicated by fewer lipid droplets, lower adipokine levels, and decreased levels of perilipin-1 and phospho-perilipin-1 compared to null. The t10-c12 CLA isomer decreased hormone-sensitive lipase (HSL) levels and inhibited lipolysis by activating protein kinase Cα (PKCα). As well, t10-c12-CLA inhibited adipocyte differentiation by stabilizing β-catenin, which sequesters peroxisome proliferator-activated receptor-γ in an inactive complex. Reduced body weight in both db/db and C57B/L6 mice fed t10-c12 CLA was due to less white and brown fat mass without changes in lean body mass or an alteration in feed intake compared to their respective control. t10-c12 CLA did not stimulate cell death in white adipose tissue. Immune cell infiltration was decreased in calorie restricted pair weight control mice, but not with CLA. t10-c12 CLA-induced weight loss did not improve hyperglycemia in db/db mice. In conclusion, the anti-adipogenic effects of t10-c12 CLA in vitro result from stabilization of β-catenin, which alters lipid droplet dynamics through HSL levels and perilipin-1 phosphorylation via the activation of PKCα. In contrast, t10-c12 CLA promotes loss of adipose tissue in vivo, possibly by activating β-catenin, but without influencing either adipogenesis or adipocyte clearance. This study suggests a novel mechanism for the anti-obesity effect of t10-c12 CLA, and highlights the possible side-effects associated with t10-c12 CLA consumption. / February 2016

The endocannabinoid (EC) system has a role in metabolic homeostasis. The purpose of this study was to determine the effect of ECs and the fatty acids they are derived from on lipid metabolism and mitochondrial function in adipocytes. 3T3-L1 adipocytes on day 8 of differentiation were treated with ECs and fatty acids for 48 hours in the absence or presence of insulin and various inhibitors. Lysates were analyzed via Western immunoblotting, a lipolysis assay and Seahorse XF Analyzer for changes in protein levels, phosphorylation state, lipolysis, and oxygen consumption rate. Results showed that ECs (2-arachidonoyl glycerol) stimulated lipolysis via a novel AMPK-dependent pathway, while fatty acids had varying effects on insulin signaling and mitochondrial function . These data suggest adipose tissue EC receptors may be a suitable target for anti-obesity therapy. Further research is needed to understand how the dietary fatty acid profile may influence synthesis of ECs. / May 2016

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

As tiazolidinodionas (TZDs) são sensibilizadores de insulina utilizados no tratamento do diabetes mellitus tipo 2. Contudo, apesar dos efeitos benéficos sobre a glicemia, importantes efeitos adversos incluindo perda óssea e aumento de adiposidade são relatados com o uso clínico das TZDs. Assim, é necessário o desenvolvimento de novos derivados de TZDs com potenciais efeitos benéficos sobre a hiperglicemia e menos efeitos adversos. Neste estudo, investigamos os efeitos de 5 novos derivados de TZDs (LYSO-7, GQ-89, GQ-150, GQ-177 e SF-3) sobre a diferenciação celular de pré-osteoblastos murinos MC3T3-E1, pré-adipócitos murinos 3T3-L1 e pré-adipócitos SGBS de linhagem humana. Seus potenciais efeitos sobre a utilização de glicose, a produção de adipocinas e mediadores pró-inflamatórios também foram avaliados, utilizando linhagens murinas e humanas de adipócitos, e macrófagos THP-1 de linhagem humana. O principal achado de nosso estudo foi que os novos derivados de TZDs estimulam a utilização celular de glicose, porém não alteram o processo de diferenciação celular de pré-osteoblastos e pré-adipócitos, quando comparados com a TZD clássica Rosiglitazona. Conforme esperado, o tratamento com Rosiglitazona na concentração de 5 μM inibiu a osteogênese de pré-osteoblastos murinos MC3T3-E1. No entanto, o tratamento com 2 novos derivados de TZDs (GQ-89 e GQ-177) na mesma concentração não afetou a diferenciação celular, sendo possível observar níveis de mineralização de matriz extracelular similares aos do grupo controle. Além disso, enquanto a GQ-89 estimulou a atividade da fosfatase alcalina, a GQ-177 não modulou sua atividade enzimática e induziu a expressão gênica de osteocalcina. Contudo, ambos inibiram a expressão de Runx2 e colágeno. Por sua vez, quando os efeitos foram avaliados sobre a diferenciação de adipócitos, foi possível observar que ao contrário do efeito pró-adipogênico constatado com a Rosiglitazona na concentração de 1 μM, as TZDs GQ-150, GQ-177, LYSO-7 e SF-3 foram incapazes de induzir o acúmulo lipídico em pré-adipócitos murinos e humanos. Além disso, a GQ-150 inibiu a expressão gênica de C/EBPα, assim como a expressão gênica e os níveis protéicos de CD36, enquanto que a SF-3 estimulou a expressão gênica de C/EBPα e de FABP4 e diminuiu a expressão gênica e os níveis protéicos de CD36, os quais não foram modificados pela LYSO-7 em pré-adipócitos murinos 3T3-L1. No entanto, em pré-adipócitos SGBS de linhagem humana, nenhum efeito sobre os marcadores de fenótipo adipogênico C/EBPα e FABP4 foi observado com os novos derivados de TZDs. Ademais, os novos derivados de TZDs não interferiram na via de sinalização de Wnt, não apresentaram qualquer efeito sobre a expressão de adipocinas (adiponectina, resistina e leptina) e mediadores pró-inflamatórios (IL-6, CCL2/MCP-1, TNF-α e JNK), bem como não ativaram o fator de transcrição PPARγ no ensaio de gene repórter. Por sua vez, a LYSO-7, GQ-150 e SF-3 aumentaram o consumo de glicose em presença de insulina em adipócitos 3T3-L1 e modificaram a atividade de enzimas mitocondriais em adipócitos SGBS e macrófagos THP-1. Entretanto, o efeito sensibilizador de insulina foi confirmado somente com a GQ-177 pelo aumento da captação de glicose e somente a LYSO-7 e a SF-3 foram capazes de inibir o consumo de oxigênio e modificar a taxa de glicólise em macrófagos, sugerindo que também poderiam alterar os níveis de ATP/ADP. Considerando que baixos níveis de ATP estimulam a via de sinalização de AMPK, essa via também foi investigada em nosso estudo. Entretanto, os resultados sobre a ativação de AMPK foram inconclusivos. Desse modo, nossos resultados apontam que os novos derivados de TZDs não atuam como ligantes de PPARγ, apresentam atividade sensibilizadora de insulina in vitro, e que exercem menores efeitos antiosteoblásticos e adipogênicos quando comparados com a Rosiglitazona. Mais estudos são necessários para elucidar os mecanismos responsáveis por esses efeitos, bem como para estabelecer se os novos derivados de TZDs são mais seguros in vivo, com relação ao risco de fraturas ósseas e ganho de massa adiposa. / Thiazolidinediones (TZDs) are insulin sensitizers used in the treatment of type 2 diabetes mellitus. However, despite the beneficial effects on blood glucose, significant adverse effects including bone loss and increased adiposity are reported with the clinical use of TZDs. Thus, it is necessary to develop new derivatives of TZDs with potential beneficial effects on hyperglycemia and fewer adverse effects. In this study, we investigated the effects of 5 new derivatives of TZDs (LYSO-7, GQ-89, GQ-150, GQ-177 e SF-3) on cellular differentiation in murine MC3T3-E1 preosteoblasts, murine 3T3-L1 preadipocytes, and SGBS preadipocytes from human lineage. Potential effects on glucose consumption, adipokines, and pro-inflammatory mediators were also assessed using murine and human strains of adipocytes, and macrophages from human THP-1 lineage. The main finding of this study was that new derivatives of TZDs stimulate glucose consumption, but do not change the cell differentiation process of preosteoblasts and preadipocytes compared to classical TZD Rosiglitazone. As expected, the treatmet with Rosiglitazone, at 5μM, inhibited the osteogenesis in murine MC3T3-E1 preosteoblasts. However, the treatment with 2 new derivatives of TZDs (GQ-89 and GQ-177) at the same concentration did not affect cell differentiation, and levels of mineralization of the extracellular matrix similar to the control group were observed. In addition, whereas the GQ-89 stimulated the activity of alkaline phosphatase, GQ-177 does not modulate its enzymatic activity and induced gene expression of osteocalcin. However, both of them inhibit the expression of Runx2 and collagen. In turn, when the effects were assessed on the adipocyte differentiation, unlike the proadipogenic effect observed with Rosiglitazone at a concentration of 1 μM, the new TZDs GQ-150, GQ-177, LYSO-7 and SF-3 were unable to induce lipid accumulation in human and murine preadipocytes. In addition, GQ-150 inhibited the gene expression of C/EBPα , as well as the gene expression and protein levels of CD36, whereas SF-3 stimulated the gene expression of C/EBPα and FABP4 and decreased gene expression and protein levels of CD36, which was not modified by LYSO-7 on murine 3T3- L1 preadipocytes. However, no effect on markers of adipogenic phenotype C/EBPα and FABP4 has been observed with the novel derivatives of TZDs in human SGBS preadipocytes. Furthermore, the new derivatives of TZDs do not interfere with the Wnt signaling pathway, showed no effect on the adipokines expression (adiponectin, resistin and leptin) and proinflammatory mediators (IL-6, CCL2 / MCP-1, TNF α and JNK) and did not activate the transcription factor PPARγ in the gene reporter assay. In turn, LYSO-7, GQ-150, and SF-3 increased glucose consumption in the presence of insulin in 3T3-L1 adipocytes and modified the activity of mitochondrial enzymes in SGBS adipocytes and THP-1 macrophages. However, the effect on insulin sensitization was confirmed only to GQ-177 that increased glucose uptake and just LYSO-7 and SF-3 were able to inhibit oxygen consumption and modify the rate of glycolysis in macrophages, suggesting that they could also alter the levels of ATP/ADP. Since low levels of ATP could stimulate AMPK pathway, this signaling pathway was also investigated in our study. However, the results on the AMPK activation were inconclusive. Thus, our results demonstrate that the new derivatives of TZDs do not act as PPARγ ligands, present insulin sensitizing activity in vitro, and display minor antiosteoblastic and adipogenic effects when compared to Rosiglitazone. More studies are needed to elucidate the exact mechanisms responsible for these effects, as well as to establish whether the safety of the new TZDs with respect to the risk of bone fractures and body mass gain using in vivo models.

糖尿病是一种呈现流行趋势的代谢紊乱综合症，现如今，全球大约有3.46亿糖尿病患者， 这庞大的数字给各国的公共健康安全支出带来了严重的财政负担。 其中，二型糖尿病（T2DM）占90%。其特点是周围组织的胰岛素抵抗以及后期损伤的胰岛β细胞的功能。在饮食后，小肠会分泌两种肠促胰岛素，葡萄糖依赖性促胰岛素多肽（GIP）和胰高血糖素样肽-1（GLP-1）。两种多肽的主要功能是促进餐后胰岛细胞中胰岛素的分泌，另外他们还可以通过其自身的G蛋白偶联受体，GIPR和GLP-1R发挥其他作用，如葡萄糖依赖性的刺激胰岛素的生成，刺激胰岛β细胞的增殖，抑制细胞的凋亡等。这些功能也使肠促胰岛素成为糖尿病治疗的一种手段，比如Exendin-4和DPP4抑制剂。 然而，除了在胰岛中的作用，肠促胰岛还可能和脂质代谢相关，其中GIP和脂质代谢的报导研究的更加深入。在肥胖的状态下，血液中GIP含量高于正常水平；GIPR基因敲除老鼠和GIPR的抑制剂喂养的小鼠可以抵抗高脂饮食诱导的肥胖和2型糖尿病；GIP还可以直接调节脂肪细胞的脂肪生成和脂解。这些数据表明GIP在肥胖和糖尿病的发生过程中可能存在促进作用,这使得GIP治疗药物的开发需要谨慎的对待。 / 为了进一步研究GIP在脂肪细胞中发挥的生物学效应，在本研究中，我们利用腺病毒介导技术通过在脂肪细胞中过表达GIPR来增加GIP的活性，然后检查GIP在脂肪细胞中所起的作用。实验结果表明,GIP可以通过cAMP-PKA信号通路迅速并且长期的刺激脂肪细胞的炎症反应，增强IKKβ-NFκB信号通路和增加炎症基因的表达。更深入的机制研究表明，JNK 信号通路也参与GIP诱导的炎症反应，抑制JNK通路可以大部分恢复GIP增加的炎症因子的表达和IKKβ的磷酸化水平。由于长期的炎症反应，脂肪细胞的胰岛素信号通路受到GIP的损伤，在GIPR过表达的脂肪细胞中，胰岛素刺激的AKT磷酸化水平和葡萄糖吸收能力都被GIP降低，葡萄糖转运蛋白4（Glut-4）的表达水平也同时减少。因此，本研究结果表明GIP可能在肥胖的发展过程中，通过诱导脂肪细胞的炎症反应来损伤胰岛素敏感性而最终导致2型糖尿病的发生。 / Diabetes mellitus is a type of metabolic syndrome that has prevailed all over the world with the development of economic and over-nutrient lifestyle. It is estimated to 346 million diabetes patients in the worldwide most recently. The huge population put a major burden on the cost of public health care to all the countries. Among the types of diabetes, type 2 diabetes (T2DM) makes up 90% of recorded cases. The characteristics of T2DM are insulin resistance of peripheral tissues and impaired pancreatic cell function and mass. Two major incretins GIP (glucose-dependent insulinotropic peptide) and GLP-1 (glucagon-like peptide 1) are secreted from gut in response to food ingestion. The prominent role of GIP and GLP-1 is to stimulate glucose-dependent insulin release in pancreatic β cell. In addition, they both exert multiple biological effects via their relative G-protein coupled receptors, GIPR and GLP-1R, including glucose-stimulated insulin production, cell proliferation and anti-apoptosis in pancreatic β cells. The beneficent effects of incretins potentiate them as targets for the treatment of diabetes. GLP-1 analog, exendin-4 and DDP4 (dipeptidyl peptidase-4) inhibitors (to prevent GIP and GLP-1 from degradation) have been already used in clinical research. However, in addition to their effects on pancreatic β cell, both peptides are also related to lipid metabolism. The role of GIP has been studied more extensively. In obese state, the circulating level of GIP is elevated. GIPR knockout (KO) mice are resistant to high fat diet (HFD) induced obesity, a similar phenotype is found in GIPR antagonist administrated HFD-mice. Moreover, GIP also directly promotes lipogenesis and lipolysis in adipocytes. The rising evidence suggests a potential role of GIP in adipocyte biology and lipid metabolism, which diminishes the enthusiasm of GIP as a candidate therapeutic reagent for T2DM. / In order to further understand the biological effects of GIP in adipocytes, here, we over-expressed GIPR in 3T3-L1 CAR adipocytes via adenovirus-mediated gene transfer technology to enhance the activity of GIP. The results demonstrate that GIP impairs the physiological functions of adipocytes as a consequence of increasing the production of inflammatory cytokines, chemokines, and phosphorylation of IkB kinase (IKK) β through activation of the cyclic AMP-protein kinase A (cAMP-PKA) pathway. Activation of Jun N-terminal Kinase (JNK) pathway is also observed in GIP-induced inflammatory responses in adipocytes. An inhibitor of JNK blocks GIP-stimulated secretion of inflammatory cytokines and chemokines, as well as phosphorylation of IKKβ. The chronic inflammatory response eventually impairs insulin signaling in adipocytes, as demonstrated by reduction of protein kinase B (PKB/AKT) phosphorylation. The subsequently physiological analysis also indicates that GIP inhibits insulin-stimulated glucose uptake, and gene expression analysis reveals a decrease of glucose transporter 4 (Glut-4) in the meanwhile. The results suggest that GIP may be one of stimuli attributable to obesity induced insulin resistance via induction of adipocyte inflammation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Nie, Yaohui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 95-111). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / INTRODUCTION --- p.1 / Chapter Part 1 --- Obesity and Type 2 diabetes --- p.1 / Chapter 1.1 --- Introduction to diabetes --- p.1 / Chapter 1.1.2 --- Physiology of adipocyte --- p.4 / Chapter 1.1.3 --- Mechanism of obesity induced diabetes --- p.10 / Chapter Part 2 --- Incretins and T2DM --- p.12 / Chapter 2.1 --- History of incretins --- p.12 / Chapter 2.2 --- Physiological actions of incretins --- p.14 / Chapter 2.3 --- Molecular mechanism of incretin actions in pancreas --- p.16 / Chapter 2.4 --- Incretins and T2DM --- p.19 / Chapter Part 3 --- Incretins and lipid metabolism --- p.23 / Objective --- p.26 / Methods and materials --- p.28 / Chapter 1 --- Cell culture --- p.28 / Chapter 1.1 --- 3T3-L1 culture and differentiation --- p.28 / Chapter 1.2 --- 3T3-L1 CAR culture and differentiation --- p.29 / Chapter 2 --- Cloning and recombinant adenovirus construction --- p.30 / Chapter 2.1 --- Plasmid construct --- p.30 / Chapter 2.2 --- Construct of recombinant adenoviruses --- p.30 / Chapter 2.3 --- Generation and infection of the adenoviruses --- p.31 / Chapter 3 --- Physiological and morphological assays --- p.32 / Chapter 3.1 --- Lipolysis assay --- p.32 / Chapter 3.2 --- TUNEL assay --- p.32 / Chapter 3.3 --- Glucose uptake --- p.33 / Chapter 3.4 --- Glut-4 localization --- p.33 / Chapter 4 --- Gene expression analysis --- p.35 / Chapter 4.1 --- Quantitative real-time PCR --- p.35 / Chapter 4.2 --- Immunoblot analysis --- p.35 / Chapter 4.3 --- ELISA assay --- p.36 / Chapter 5 --- Isolation of primary adipocytes --- p.37 / Results --- p.38 / Chapter Part 1 --- Role of GIP in 3T3-L1 cells --- p.38 / Chapter 1.1 --- Differentiation of 3T3-L1 adipocytes --- p.38 / Chapter 1.2 --- GIP slightly stimulates phosphorylation of p-CREB and lipolysis in 3T3-L1 cells. --- p.40 / Chapter 1.3 --- Analysis of gene expression in GIP-treated adipocytes --- p.42 / Chapter 1.4 --- Discussion --- p.44 / Chapter Part 2 --- Role of GIP in GIPR over-expressing 3T3-L1 CAR adipocytes --- p.46 / Chapter 2.1 --- Differentiation of 3T3-L1 CAR adipocytes --- p.46 / Chapter 2.2 --- Functional tests in GIPR over-expressing 3T3-L1 CAR adipocytes. --- p.48 / Chapter 2.3 --- Effect of GIP on cell viability --- p.50 / Chapter 2.4 --- Analysis of gene expression in GIP-treated adipocytes --- p.52 / Chapter 2.5 --- GIP activates inflammatory responses in GIPR over-expressing adipocytes --- p.54 / Chapter 2.6 --- Inhibition of IKKb pathway restores GIP-induced inflammatory responses --- p.56 / Chapter 2.7 --- Effects of GIP on adipocytes are partially dependent on the cAMP-PKA pathway --- p.58 / Chapter 2.8 --- Activation of cAMP-PKA pathway induces adipocyte inflammation. --- p.60 / Chapter 2.9 --- cAMP-Epac pathway is not involved in GIP-induced inflammation --- p.62 / Chapter 2.10 --- GIP stimulates cell stress activated kinases --- p.64 / Chapter 2.11 --- JNK partially mediates GIP-induced adipocyte inflammation --- p.65 / Chapter 2.12 --- Inhibition of JNK pathway partially restores GIP-induced inflammatory responses --- p.67 / Chapter 2.13 --- GIP impairs insulin signaling in GIPR over-expressing 3T3-L1 CAR adipocytes via inducing inflammatory response --- p.69 / Chapter 2.14 --- GIP enhances basal glucose uptake but impairs insulin stimulated glucose uptake in 3T3-L1 CAR GIPR over-expressing adipocytes --- p.71 / Chapter 2.15 --- Discussion --- p.73 / Chapter Part 3 --- Role of GIP in primary adipocytes --- p.78 / Chapter 3.1 --- GIPR expression level in primary adipocytes --- p.78 / Chapter 3.2 --- Analysis of gene expression in primary adipocytes after GIP treatment --- p.80 / Chapter 3.3 --- Discussion --- p.81 / SUMMARY --- p.82 / Chapter Future investigation --- p.83 / Chapter Appendix 1: --- Abbreviations --- p.86 / Chapter Appendix 2: --- Protocols --- p.90 / Preparation of competent cells --- p.90 / Outlines of recombinant adenovirus preparation --- p.91 / Virus titering (TCID50) --- p.92 / Primers for real-time PCR --- p.93 / Chapter Publications and Scientfic activities --- p.94 / Thesis related publication: --- p.94 / Other pubiliations: --- p.94 / Scientific activities: --- p.94 / References --- p.95

La crête neurale (CN) dérive de la partie dorsale du tube neural des Vertébrés. Pendant le développement, ces cellules migrent et contribuent à la formation de différents tissus et organes. Le long de l'axe antéro-postérieur, la CN donne naissance aux neurones et cellules gliales du système nerveux périphérique, et aux mélanocytes. Par ailleurs, la CN céphalique est aussi à l’origine de tissus mésenchymateux qui constituent tous les os et cartilages de la face, la plus grande partie du crâne, le derme facial, et les adipocytes et cellules de muscles lisses dans la tête. Dans le tronc des Vertébrés amniotes, ces tissus dérivent du mésoderme. Chez les Vertébrés inférieurs, la CN troncale génère cependant des tissus mésenchymateux, comme les rayons des nageoires du poisson-zèbre. La question qui se pose est de savoir si la capacité de la CN à produire des cellules mésenchymateuses a été totalement perdue dans le tronc au cours de l’évolution, ou bien si elle peut encore se manifester chez les Amniotes dans des conditions spécifiques. Ce travail s’est intéressé à dévoiler le potentiel mésenchymateux de la CN troncale.Notre approche expérimentale a été d'examiner le potentiel de différenciation squelettogénique et adipogénique des cellules de la CN troncale de caille en culture in vitro, par hybridation in situ (HIS), immunocytochimie et RT-PCR. L’ostéogenèse a été initialement caractérisée par l'expression de Runx2, premier facteur de transcription des ostéoprogéniteurs, qui a été détectée par HIS à partir 5 jours de culture. Plus tard, nous avons observé la maturation des ostéoblastes, avec l’expression de la protéine collagen1, des ARNm de l'ostéopontine et de la phosphatase alcaline, jusqu’à l'étape de minéralisation de la matrice osseuse. Les cellules de CN troncale ont effectué également un processus de chondrogenèse, mis en évidence par l'expression des ARNm de Sox9, aggrecan et collagène10, et par la coloration au bleu Alcian. L'observation des zones minéralisées et des régions chondrogéniques suggère que les cellules de la CN troncale in vitro effectuent une ossification de types endochondral et intramembranaire. Dans les mêmes conditions de culture, les cellules se sont aussi différenciées en adipocytes, identifiés à partir de 10 jours de culture par le colorant Oil Red O. Les ARNm des facteurs de transcription CEBP et PPAR, essentiels pour l'adipogenèse, et de la protéine FABP4, ont été détectés par RT-PCR dès 3 jours de culture. Afin de caractériser les précurseurs des cellules osseuses et adipocytaires, nous avons examiné le potentiel de différenciation des cellules individuelles de la CN troncale. L'analyse des types cellulaires dans les cultures clonales a montré que 76% des clones contiennent des ostéoblastes Runx2-positifs. De plus, les cellules de CN troncale comprennent des progéniteurs multipotents dotés à la fois de potentiels neuraux et ostéogénique. Dans une autre condition de culture clonale, les adipocytes ont été trouvés dans la descendance de 35,3% des cellules, et environ la moitié de ces cellules possédaient aussi un potentiel glial et/ou mélanogénique. Ces résultats montrent que, in vitro, les cellules de la CN troncale sont capables de se différencier non seulement dans ses dérivés traditionnels trouvés in vivo (mélanocytes, neurones et cellules gliales), mais aussi dans des phénotypes mésenchymateux, y compris adipocytes et ostéoblastes. Comme dans les cellules de la CN céphalique, ces phénotypes mésenchymateux se différencient à partir de progéniteurs multipotents. Ceci suggère que, au cours de l’évolution, les cellules souches de la CN, dotées de potentiels à la fois mésenchymateux et neuraux, ont eu l'expression de leur potentiel mésenchymateux inhibée dans le tronc. Ainsi, chez les Vertébrés amniotes, même s’il ne se manifeste pas et reste dormant in vivo, un potentiel de différenciation mésenchymateuse est bien présent dans les cellules de la CN troncale et peut être révélé in vitro. / The neural crest (NC) derives from the dorsal borders of the vertebrate neural tube. During development, the NC cells migrate and contribute to the formation of different tissues and organs. Along the anteroposterior axis, the NC gives rise to neurons and glia of the peripheral nervous system and to melanocytes. Furthermore, the cephalic NC yields mesenchymal tissues, which form all facial cartilages and bones, the large part of skull, facial dermis, fat cells and smooth muscle cells in the head. In the trunk of amniotes Vertebrates, these tissues are derived from the mesoderm, not from the NC. In lower Vertebrates, however, the trunk NC generates some mesenchymal tissues, such as in the dorsal fins of zebrafish. The question therefore is raised whether the ability of the NC to produce mesenchymal cells was totally lost in the trunk of amniote Vertebrates during evolution, or if it can still be achieved under specific conditions. This work is interested in uncovering the mesenchymal potential of the avian trunk NC, with special interest in the differentiation into osteoblasts and adipocytes.Our experimental approach was to examine the skeletogenic and adipogenic differentiation potentials of quail trunk NC cells after in vitro culture. Cell differentiation was evidenced by the analysis of lineage-specific genes and markers using in situ hybridization (ISH), immunocytochemistry and RT-PCR. The established culture conditions allowed observation of both skeletogenesis and adipogenesis. Osteogenesis was initially characterized by expression of Runx2, the first transcription factor specific of the osteoprogenitors, which was detected by ISH from 5 days of culture. Later, we observed osteoblast maturation, with the expression of collagen1 protein, osteopontin mRNA and alkaline phosphatase mRNA, until the bone matrix mineralization stage. The trunk NC cells also underwent chondrogenesis, as demonstrated by Sox9, aggrecan and collagen10 mRNA expression, and Alcian blue staining. The observation of the mineralized areas and chondrogenesis suggested that the trunk NC cells in vitro are able to perform endochondral and membranous ossifications. In same culture conditions, the cells differentiated also into adipocytes, identified from 10 days of culture by Oil Red O staining. The mRNAs of the CEBP, PPAR and FABP4 adipogenic markers were detected by RT-PCR from 3 days of culture. For the characterization of bone and adipocyte progenitors, we evaluated the differentiation potential of individual trunk NC cells. The phenotypic analysis of these clonal cultures showed that 76% of the cells generated Runx2-positive osteoblasts. Moreover, most of the clone-forming trunk NC cells were multipotent progenitors endowed with both neural and osteogenic potentials. Furthermore, in another clonal culture condition, adipocytes were found in 35.3% of the clones, and approximately half of them also contained glial and/or melanogenic cells.These results show that the trunk NC cells in vitro are able to differentiate not only in their classical derivatives found in vivo (melanocytes, neurons and glial cells), but also in mesenchymal phenotypes, including adipocytes and osteoblasts. Importantly, as in cephalic NC cells, mesenchymal phenotypes differentiated from multipotent progenitor cells, suggesting that, during evolution, the NC stem cells intended for both mesenchymal and neural fates, had the expression of their mesenchymal potential inhibited in the trunk. Thus, although at the dormant state and not expressed in vivo, a significant mesenchymal potential is present in the trunk NC cells of amniotes Vertebrates and can be disclosed in vitro

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