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

Yau, Ka Long. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 97-113). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

G蛋白偶聯雌激素受體（GPER，又名GPR30）乃最近於各種動物包括小鼠、大鼠、人類及斑馬魚中發現之新型跨膜雌激素受體。 GPER表達於脂肪組織及多種器官之中，其已被證明能與雌激素結合並介導各式快速反應及基因轉錄。針對GPER於成脂作用中角色之研究將達致對雌激素作用之更全面了解，且GPER亦有望成為治療肥胖症之一種新型標靶。 / 脂肪發育調控乃一複雜且精妙之排程，而雌激素已被證明能抑制脂肪形成，是故雌激素替代療法可舒減絶經後婦女之脂肪代謝問題。此項研究發現GPER於小鼠腹部脂肪組織及小鼠前脂肪細胞系3T3-L1中均有表達，且其信使RNA量於受誘導之3T3-L1成脂作用中錄得上調。 / 3T3-L1細胞分化作用會被名為G1之特異性GPER激動劑阻撓於克隆擴增階段（MCE），此即表明GPER有參與成脂調控之可能。通過油紅O染色發現，受G1處理之3T3-L1細胞於分化後所產生之油滴量實比其對照組為低，但此一效果能被特異性GPER小干擾RNA預處理抹除。另外，本研究以流式細胞儀及西方墨點法對細胞週期及細胞週期因子進行分析後，認為激活GPER能觸發對G1期細胞週期停滯之抑制。另一方面，受G1處理並分化中之3T3-L1細胞出現蛋白激酶B磷酸化效應，意味雌激素與GPER結合對成脂作用有雙向調節之可能性。 / 總而言之，本研究結果斷定GPER能介導雌激素對脂肪組織發育之影響，並為成脂作用之負調節因子，故此，一系列成果將有助肥胖症藥物之研發。 / A novel transmembrane estrogen receptor, G-protein coupled estrogen receptor (GPER, also known as GPR30), is recently identified in various animals including mouse, rat, human and zebrafish. GPER is expressed in many organs including fatty tissues, and has been demonstrated to mediate various rapid responses and transcriptional events upon estrogen binding. The study on the role of GPER in adipogenesis would lead to a more comprehensive understanding of estrogenic actions, with the view of identifying novel therapeutic targets for the treatment of obesity. / Regulation of adipose development is a complex and subtly orchestrated process. Estrogen has been shown to inhibit adipogenesis. Estrogen replacement therapy therefore affects fat metabolism in post-menopausal women. In this study, GPER is identified in mouse abdominal fatty tissues; and there is an up-regulation of GPER in the mouse preadipocyte cell line 3T3-L1 during induced adipogenesis. / Differentiation of 3T3-L1 cells is perturbed by the selective GPER agonist G1 at mitotic clonal expansion (MCE), indicating a possible involvement of GPER in the regulation of adipogenesis. By means of Oil-Red-O staining, the production of oil droplets in the G1-treated, differentiated 3T3-L1 cells is shown to be lower than the untreated control; and such effect is reversed by a specific siRNA knockdown of GPER. FACS analysis and Western blot analysis of cell cycle factors during MCE suggest that GPER activation triggers an inhibition of cell cycle arrest at the G1 stage. On the other hand, phosphorylation of Akt in G1-treated differentiating cells implies a possibility of bi-directional estrogenic regulation of adipogenesis via GPER. / To conclude, it is postulated that GPER mediates estrogenic actions in adipose tissues as a negative regulator of adipogenesis. These results provide insights into the development of therapeutic agents for the treatment of human obesity. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yuen, Man Leuk. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 144-166). / 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 (English version) --- p.I / Abstract (Chinese version) --- p.III / Acknowledgement --- p.V / Table of Contents --- p.VII / List of Abbreviations --- p.XI / List of Tables --- p.XII / List of Figures --- p.XIII / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1. --- Obesity and adipose tissue --- p.1 / Chapter 1.1.1. --- Obesity --- p.1 / Chapter 1.1.2. --- Fat deposition --- p.3 / Chapter 1.1.3. --- Origin and development of white adipose tissue --- p.5 / Chapter 1.2. --- Adipogenesis --- p.7 / Chapter 1.2.1. --- Origins of white adipocytes --- p.7 / Chapter 1.2.2. --- Signals for adipogenesis --- p.10 / Chapter 1.2.3. --- Regulation of gene expression during adipogenesis --- p.12 / Chapter 1.2.4. --- Common adipose cell lines --- p.16 / Chapter 1.2.5. --- Mechanism of in vitro adipogenesis --- p.21 / Chapter 1.2.5.1. --- Growth arrest --- p.23 / Chapter 1.2.5.2. --- Mitotic clonal expansion --- p.23 / Chapter 1.2.5.3. --- Early and terminal differentiation --- p.24 / Chapter 1.3. --- Estrogen and adipogenesis --- p.28 / Chapter 1.4. --- G-protein coupled estrogen receptor-1 --- p.33 / Chapter 1.4.1. --- General introduction of GPER --- p.33 / Chapter 1.4.2. --- Ligands of GPER --- p.36 / Chapter 1.4.3. --- Cellular signaling of GPER --- p.38 / Chapter 1.4.4. --- Metabolic actions of GPER: A brief introduction --- p.43 / Chapter 1.4.5. --- Metabolic actions of GPER on obesity and glucose metabolism --- p.48 / Chapter 1.4.6. --- Study objectives --- p.53 / Chapter Chapter 2: --- Expression profiles and cellular localization of Gper/GPER in mouse adipose, 3T3-L1 preadipocytes and 3T3-L1 mature adipocytes --- p.54 / Chapter 2.1. --- Introduction --- p.54 / Chapter 2.1.1. --- Expression and functional roles of GPER in adipose. --- p.55 / Chapter 2.1.2. --- Swiss mouse preadipocytes 3T3-L1 --- p.57 / Chapter 2.1.3. --- Study objectives --- p.57 / Chapter 2.2. --- Materials and Methods --- p.59 / Chapter 2.2.1. --- Reagents --- p.59 / Chapter 2.2.2. --- Animal tissues --- p.59 / Chapter 2.2.3. --- Cell culture --- p.60 / Chapter 2.2.4. --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.62 / Chapter 2.2.5. --- Quantitative real-time RT-PCR (qRT-PCR) --- p.66 / Chapter 2.2.6. --- SDS-PAGE and Western blot analysis --- p.68 / Chapter 2.2.7. --- Immunofluorescence assay --- p.69 / Chapter 2.2.8. --- Statistical analysis --- p.70 / Chapter 2.3. --- Results --- p.71 / Chapter 2.3.1. --- Expression of Gper/GPER in mouse visceral adipose tissues --- p.72 / Chapter 2.3.2. --- Expression profiles of Gper/GPER in undifferentiated 3T3-L1 preadipocytes and differentiated 3T3-L1 adipocytes --- p.73 / Chapter 2.3.3. --- Cellular localization of GPER in undifferentiated 3T3-L1 preadipocytes and differentiated 3T3-L1 adipocytes --- p.75 / Chapter 2.4. --- Discussion --- p.76 / Chapter Chapter 3: --- Rapid cellular responses induced by GPER activation in 3T3-L1 preadipocytes --- p.78 / Chapter 3.1. --- Introduction --- p.78 / Chapter 3.1.1. --- Rapid cellular response of estrogen via GPER --- p.79 / Chapter 3.1.2. --- Study objectives --- p.81 / Chapter 3.2. --- Materials and Methods --- p.82 / Chapter 3.2.1. --- Reagents --- p.82 / Chapter 3.2.2. --- Cell culture --- p.82 / Chapter 3.2.3. --- SDS-PAGE and Western blot analysis --- p.83 / Chapter 3.2.4. --- Statistical analysis --- p.84 / Chapter 3.3. --- Results --- p.86 / Chapter 3.3.1. --- Phosphorylation of p44/42 MAPK after time-dependent activation of GPER by ICI182,780 and G1 --- p.87 / Chapter 3.3.2. --- Phosphorylation of p44/42 MAPK after dose-dependent activation of GPER by a combination of chemical agents --- p.88 / Chapter 3.4. --- Discussion --- p.89 / Chapter Chapter 4: --- GPER activation on cell viability of 3T3-L1 preadipocytes --- p.90 / Chapter 4.1. --- Introduction --- p.90 / Chapter 4.1.1. --- Cell proliferation mediated by GPER --- p.90 / Chapter 4.1.2. --- Study objectives --- p.92 / Chapter 4.2. --- Materials and Methods --- p.93 / Chapter 4.2.1. --- Reagents --- p.93 / Chapter 4.2.2. --- Cell culture --- p.93 / Chapter 4.2.3. --- MTT assay for cell viability --- p.94 / Chapter 4.2.4. --- Statistical analysis --- p.95 / Chapter 4.3. --- Results --- p.96 / Chapter 4.3.1. --- Cell viability of 3T3-L1 after dose-dependent activation of GPER by 17β-estradiol, ICI182,780 and G1 --- p.97 / Chapter 4.4. --- Discussion --- p.99 / Chapter Chapter 5: --- GPER-mediated estrogenic action on lipid accumulation in the mature 3T3-L1 adipocytes --- p.101 / Chapter 5.1. --- Introduction --- p.101 / Chapter 5.1.1. --- Induction of differentiation in Swiss mouse preadipocyte 3T3-L1 --- p.101 / Chapter 5.1.2. --- Study objectives --- p.102 / Chapter 5.2. --- Materials and Methods --- p.103 / Chapter 5.2.1. --- Reagents --- p.103 / Chapter 5.2.2. --- Cell culture --- p.103 / Chapter 5.2.3. --- Oil-Red-O staining and measurement of absorbance --- p.105 / Chapter 5.2.4. --- Knockdown of Gper/GPER by siRNA --- p.107 / Chapter 5.2.5. --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.110 / Chapter 5.2.6. --- SDS-PAGE and Western blot analysis --- p.110 / Chapter 5.2.7. --- Statistical analysis --- p.110 / Chapter 5.3. --- Results --- p.112 / Chapter 5.3.1. --- GPER activation on 3T3-L1 differentiation --- p.114 / Chapter 5.3.2. --- Knockdown of Gper/GPER in Swiss mouse preadipocyte 3T3-L1 --- p.114 / Chapter 5.3.3. --- Phosphorylation of p44/42 MAPK in Gper/GPER-knockdown 3T3-L1 after time-dependent activation of GPER by G1 --- p.117 / Chapter 5.3.4. --- Action of drugs on differentiation of Gper/GPER-knockdown 3T3-L1 --- p.117 / Chapter 5.4. --- Discussion --- p.118 / Chapter Chapter 6: --- Role of GPER in regulating cell cycle progression during mitotic clonal expansion (MCE) stage in adipogenesis of 3T3-L1 --- p.120 / Chapter 6.1. --- Introduction --- p.120 / Chapter 6.1.1. --- Differentiation stages of Swiss mouse preadipocyte 3T3-L1 --- p.121 / Chapter 6.1.2. --- Apoptosis and cell cycle progression --- p.122 / Chapter 6.1.3. --- Study objectives --- p.126 / Chapter 6.2. --- Materials and Methods --- p.127 / Chapter 6.2.1. --- Reagents --- p.127 / Chapter 6.2.2. --- Cell culture --- p.127 / Chapter 6.2.3. --- Oil-Red-O staining and measurement of absorbance --- p.129 / Chapter 6.2.4. --- Trypan blue exclusion assay for cell viability determination --- p.129 / Chapter 6.2.5. --- SDS-PAGE and Western blot analysis --- p.131 / Chapter 6.2.6. --- Flow cytometry for analysis of cell cycle progression --- p.132 / Chapter 6.2.7. --- Statistical analysis --- p.133 / Chapter 6.3. --- Results --- p.134 / Chapter 6.3.1. --- Temporal effect of GPER activation on differentiation progress of Swiss mouse preadipocyte 3T3-L1 --- p.137 / Chapter 6.3.2. --- Effect of GPER activation on cell viability during adipogenesis --- p.139 / Chapter 6.3.3. --- Effect of GPER activation on apoptosis during adipogenesis --- p.139 / Chapter 6.3.4. --- Effect of GPER activation on cell cycle distribution during induced adipogenesis --- p.140 / Chapter 6.3.5. --- Effect of GPER activation on expression of cell cycle markers during induced adipogenesis --- p.142 / Chapter 6.3.6. --- Activation of PI3K/Akt pathway by GPER stimulation during induced adipogenesis --- p.143 / Chapter 6.4. --- Discussion --- p.144 / Chapter Chapter 7: --- Conclusions and Future Perspectives --- p.148 / References --- p.155

Em animais, dietas cetogênicas induzem o Browning de tecidos adiposos brancos, fenômeno caracterizado pelo de aumento de adipócitos capazes de expressar a proteína desacopladora 1 (UCP1) e outros marcadores de gordura marrom em meio a gordura branca. Estudo anterior demonstrou que o β-hidroxibutirato (βHB), principal corpo cetônico, aumenta marcadores do processo de browning em adipócitos brancos (in vitro) após 24 horas de incubação. No entanto, as doses testadas foram suprafisiológicas (50 mM) ou apenas encontradas durante a cetoacidose (25 mM). As dietas cetogênicas aumentam a cetonemia em torno de 1-3 mM. O jejum prolongado pode aumentá-lo para 4-7 mM. Uma vez que poderia ser provocada in vivo através de intervenções dietéticas, estudamos o impacto de concentrações fisiológicas de βHB no metabolismo e marcadores de Browning em adipócitos brancos / tecido adiposo em diferentes modelos: adipócitos isolados de ratos Wistar, células 3T3 -L1 e in vivo, através da suplementação de sais de βHB em ratos Wistar. Demostramos que o βHB: não induz o aparecimento diferentes marcadores de Browning (tais como o incremento: da capacidade oxidativa, da atividade de citrato sintase e de genes relacionados ao Browning) em adipócitos isolados após 24 ou 48 horas de tratamento; não exerce efeito permissivo no browning induzido por agonismo β-adrenérgico. Além disso, os adipócitos 3T3-L1 diferenciados com βHB (4mM) tiveram diminuição de 52% na expressão de Ucp1, resultado que foi reproduzido no tecido adiposo inguinal subcutâneo de ratos Wistar após a ingestão de sais DL- βHB, onde a expressão gênica de Ucp1 foi indetectável. Em conclusão, embora as causas de browning do tecido adiposo branco induzido por dietas cetogênicas permaneçam inconclusivas, nosso estudo demonstra a incapacidade de, em concentrações fisiológicas, os corpos cetônicos serem, por si, responsáveis por esse fenômeno. Pelo contrário, em algumas situações, o βHB pode prejudicar a expressão da Ucp1. / In animals, ketogenic diets induce browning of white adipose tissue, a phenomenon characterized by the increase of adipocytes capable of expressing the uncoupling protein 1 (UCP1) and other markers of brown fat in among white fat. A previous study demonstrated that β-hydroxybutyrate (βHB), the major ketone body, increases markers of the browning process in white adipocytes (in vitro) after 24 hours of incubation. However, the doses tested were supraphysiological (50 mM) or only found during ketoacidosis (25 mM). Ketogenic diets increase ketonemia by about 1-3 mM. Prolonged fasting can increase it to 4-7 mM. Since it could be elicited in vivo through dietary interventions, we studied the impact of physiological concentrations of βHB on metabolism and browning markers on white adipocytes/adipose tissue in different models: adipocytes isolated from Wistar rats, 3T3-L1 cells and in vivo, through the supplementation of βHB salts in Wistar rats. We demonstrate that βHB does not increase any browning markers (such as: oxidative capacity, citrate synthase activity, and browning related genes expression) in isolated adipocytes after 24 or 48 hours of treatment; does not exert a permissive effect on browning induced by β-adrenergic agonism. In addition, 3T3-L1 adipocytes differentiated with βHB (4mM) had a 52% decrease in Ucp1 expression, a result that was reproduced in the subcutaneous inguinal adipose tissue of Wistar rats after ingestion of DL-βHB salts, where Ucp1 expression was undetectable. In conclusion, although the causes of browning of white adipose tissue during ketogenic diets remain inconclusive, our study demonstrates the inability, in physiological concentrations, of ketone bodies themselves to be responsible for this phenomenon. In contrast, in some situations, βHB may impair the expression of Ucp1.

The purpose of this study is to determine whether or not adipocytes harvested directly from fat pads or induced from bone marrow in lean and obese mice exhibit a sex-dependent rapid response to vitamin D metabolite 1á,25(OH)2D3 and if so to elucidate if it is via an ERp60 receptor mediated signaling pathway. The role of 1á,25(OH)2D3 and specifically the membrane effect will be examined in two genetically distinct mice to see if their cells have a differing sensitivity. The results indicate that there are differing responses in adipocytes that are induced from bone marrow versus differentiated fat pad adipocytes, and the function of 1á,25(OH)2D3 is sex-specific in some cases. Additionally, all the adipocytes tested demonstrated a rapid response to 1á,25(OH)2D3; mRNA for nVDR and ERp60 were found in all cells however the only functional protein found in the plasma membrane was ERp60 indicating that it may be necessary for the rapid response whereas nVDR is not required.

Dysregulation of adipokines in obese adipose tissue contributes to inflammation and insulin resistance. Fatty acids and lipopolysaccharide (LPS) can modulate adipokine secretion, however, less is known about their effects in combination. Long-chain n-3 polyunsaturated fatty acids (PUFA) exert anti-inflammatory effects and less is known about other n-3 and n-6 PUFA, which are more prevalent in the typical diet. Co-incubation of 3T3-L1 adipocytes with LPS and long-chain n-3 PUFA decreased LPS-induced secreted MCP-1 protein. n-6 PUFA arachidonic acid and LPS synergistically increased MCP-1 and IL-6 secreted proteins. Plant-derived PUFA were relatively neutral stimuli. mRNA expression results suggest potential roles for G protein-coupled receptor 120 and toll-like receptor 2 in mediating the effects of long-chain n-3 PUFA and arachidonic acid, respectively. Overall, this thesis suggests that both n-3 and n-6 PUFA are important factors to consider in the development of nutritional strategies for improving adipose tissue inflammation associated with obesity. / NSERC CGS, Ontario Graduate Scholarship

Adipocytes express angiotensin II (AngII) receptors; however the direct effects of AngII at the adipocyte remain unclear. Knockout mouse models of renin-angiotensin system components exhibit reduced body weight, reduced adiposity, improved glucose tolerance, and improved blood pressure when fed high fat diets, which may be due to reduced action of AngII through the AT1aR in adipocytes. Additionally, hypercholesterolemic AT1aR deficient mice are protected from AngII-induced increases in atherosclerosis and abdominal aortic aneurysm (AAA) formation. We hypothesized that deficiency of AT1aR in adipocytes would reduce the development of obesity, obesity-induced disorders, and vascular diseases. To test this hypothesis, we created a mouse model of adipocyte AT1aR deficiency using the Cre/LoxP system. Adipocyte-AT1aR deficiency confers no protection from the development of obesity or obesity- associated parameters. However, low fat fed adipocyte-AT1aR deficient mice exhibit remarkable adipocyte hypertrophy and reductions in adipocyte differentiation. These results demonstrate that AngII is a stimulus for adipocyte differentiation and adipocyte hypertrophy alone is insufficient to initiate obesity- associated disorders. In hypercholesterolemic mice, adipocyte AT1aR deficiency conferred no protection from diet or AngII-induced vascular diseases. Overall these studies suggest the primary role of adipocyte AT1aRs is to promote adipocyte differentiation and the development of small adipocytes.

Glucocorticoids are widely used in clinical therapy. However, they cause adverse effects including insulin resistance and Type 2 diabetes, which are characterised by decreased glucose transport into the muscles and fat. How glucocorticoids inhibit glucose transport remains unclear. Insulin stimulates glucose uptake via the insulin receptor substrate (IRS)-1 / phosphoinositide-3-kinase (PI3K) / protein kinase B (AKT) pathway and promotes the redistribution of GLUT4 from intracellular storage compartments to the plasma membrane (PM). Insulin-stimulated phosphorylation of AKT substrate of 160 kDa (AS160), a Rab-GTPase activating protein is downstream of AKT and appears to be essential for exposure of GLUT4 at the PM and glucose uptake. This is mediated through the association of phosphorylated AS160 (at the key residue T642) with 14-3-3 in the cytosol. The mildly insulin-responsive GLUT1 mediates basal glucose uptake in adipocytes. It is also subject to regulated trafficking like GLUT4. This study aimed to determine the level at which glucocorticoids inhibit glucose uptake in adipocytes. Effects of the synthetic glucocorticoid dexamethasone (Dex) and the natural glucocorticoid cortisol, on GLUT1 and GLUT4 function were examined. Candidates for the glucocorticoid-mediated inhibition of GLUT1- and GLUT4-mediated glucose uptake were investigated. These were glycogen synthase kinase (GSK) 3β (an AKT substrate) for GLUT1-mediated glucose transport; and adaptor protein containing PH domain, PTB domain, and leucine zipper motif (APPL)-1 (an AKT-interacting protein) and AS160 for GLUT4-mediated glucose transport. Dex and cortisol significantly decreased basal glucose uptake by 50% (p<0.05) in SGBS and 3T3-L1 adipocytes. Similarly, insulin-stimulated glucose uptake was decreased by 50% (p<0.001 for SGBS; p<0.05 for 3T3-L1) and 30% (p<0.05 for both) at 1 nM and 100 nM insulin respectively. Similar results were observed with differentiated primary human preadipocytes and human adipose explants. Dex-mediated inhibition of basal glucose uptake was limited to insulin-sensitive cell types implying that glucocorticoids may regulate GLUTs at steps common to GLUT1 and GLUT4 trafficking. Dex-mediated reduction in glucose uptake correlated with the reduction in basal and insulin-stimulated expression of GLUT1 and GLUT4 to the PM without changes in total GLUT1/4 expression. Dex did not alter total expression or phosphorylation of proximal insulin-signalling molecules up to and including AKT but increased FOXO1 expression, and modified GSK3β-S9 phosphorylation. Dex did not alter total APPL1 expression or subcellular distribution. Dex significantly decreased 1nM-insulin stimulated AS160-T642 phosphorylation by 50% (p<0.05) in SGBS and 3T3-L1 adipocytes via the glucocorticoid repector (GR). This correlated with reduced AS160:14-3-3 interaction. Similar results were obtained for AS160-T642 basal phosphorylation. At 1nM insulin, AS160-T642 phosphorylation is maximal at sub-maximal glucose uptake, i.e. AS160 phosphorylation significantly contributes to glucose uptake. RU486 significantly prevented but did not fully abrogate the Dex-mediated reduction in glucose uptake suggesting additional Dex-induced defects. In conclusion, glucocorticoids inhibit glucose uptake at a level distal to AKT by GR-dependent mechanisms. A role for GSK3β or APPL1 in glucocorticoid-mediated inhibition of glucose uptake requires further investigations. FOXO1 represents a suitable candidate for mediating the Dex-induced defects. Of significance, perturbation in AS160-T642 phosphorylation contributes to Dex-mediated inhibition of glucose uptake. Thus, AS160 presents a novel therapeutic target in the improvement of glucocorticoid-mediated inhibition of glucose uptake.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.