A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirement for the degree of Doctor of Philosophy. Johannesburg, 2004 / Alkaline phosphatase (ALP) is a ubiquitously expressed enzyme, that has been shown to play a role in cell differentiation and organogenesis. One study has also demonstrated ALP activity in rat adipocytes. The purpose of the present study was therefore to determine whether ALP is expressed in preadipocytes and what role it may have in adipogenesis. ALP activity was detected in the murine preadipocyte cell line, 3T3-L1, and in human preadipocytes isolated from mammary tissue, and from subcutaneous abdominal fat depots. In all the cell types studied ALP activity increased in parallel with adipogenesis. In the 3T3 -L1 cell line the tissue- non -specific ALP inhibitors, levamisole and histidine inhibited ALP activity, and adipogenesis, whereas the tissue specific ALP inhibitor Phe- Gly-Gly did not inhibit ALP or adipogenesis. In human preadipocytes, histidine inhibited adipogenesis and ALP activity, whereas levamisole inhibited adipogenesis, but did not block ALP activity in intact cells. However, levamisole did inhibit ALP activity by 50% in cell extracts. Levamisole was able to inhibit adipogenesis in human preadipocytes. The tissue specific ALP inhibitor, Phe Gly Gly, did not inhibit ALP activity or adipogenesis in human preadipocytes. ALP activity and adipogenesis, were compared in preadipocytes isolated from mammary tissue taken from black (13) and white (15) female subjects. Both ALP activity and adipogenesis, were lower in white compared to black female subjects. iii Immunocytochemical, analysis of the 3T3-L1 cell line and human preadipocytes demonstrated that ALP activity was restricted to the lipid droplets of these cells. ALP activity was also measured in serum samples obtained from 100 African subjects (74 females and 26 males) of varying BMI. ALP activity was found to be higher in obese than lean subjects, whereas, the other liver enzymes or products measured in serum were not. In fact these variables correlated to varying degrees with waist-hip ratio, whereas ALP levels did not. This suggest that liver function is predominantly influenced by abdominal obesity whereas serum ALP levels are more influenced by overall body adiposity. In conclusion, ALP, may be involved in the control of adipogenesis, in the 3T3- L1 preadipocyte cell line and in human preadipocytes isolated from mammary adipose tissue and subcutaneous abdominal adipose tisssue. The presence of ALP activity in lipid droplets in 3T3-L1 cells and human preadipocytes, and the ability of ALP inhibitors to block adipogenesis strongly suggest that ALP plays a role in the control of adipogenesis. / IT2017

Flavonoids, and more specifically, red wine procyanidins, have many beneficial effectsagainst pathologies such as cardiovascular heart disease and related illnesses. Althoughadipose tissue has a central role in some of these pathologies, including obesity and diabetes,there is a lack of information about the effects of procyanidins on this tissue. This thesisaddresses this question. The effects of a grape seed procyanidin extract (GSPE) on the lipidand glucose metabolism of adipocytes were evaluated by taking the 3T3-L1 cell line as amodel of study. Results show that the GSPE has insulinomimetic effects, stimulating glucoseuptake, glycogen synthesis and trigliceride synthesis. To achieve this, the GSPE shares someof the mechanisms and intracellular mediators of the insulin-signalling pathways (such asGLUT-4 translocation, PI3K and p38 MAPK) but it must also use other, complementary,mechanisms. These results suggest that procyanidins have beneficial effects on diabetesand/or insulin resistance. This is partially proven by in vivo studies that show that GSPE hasantihyperglycemic properties on streptozotozin-induced diabetic rats. Also analyzed in thisthesis are the molecular mechanisms used by GSPE to explain the already described lipolyticeffects. Protein kinase A and PPARã are shown to be involved in these effects. Some ofthese results opened up another line of study into the effects of GSPE on the differentiationprocess of the 3T3-L1. These studies showed that procyanidins alter the differentiation ofpreadipocytes when added at the induction of differentiation. Since an increase in thenumber of adipocytes has a negative effect on obesity, this is a promising characteristic ofGSPE that should be taken into account when its possible antiobesity properties are studied. / Als flavonoides, i més concretament a les procianidines del vi negre, se'ls han atribuït moltespropietats beneficioses contra diverses patologies, com les malalties cardiovasculars i altrespatologies relacionades. Tot i que el teixit adipós juga un paper important en algunesd'aquestes patologies, com la obesitat i la diabetis, la informació referent l'acció de lesprocianidines en aquest teixit és escassa. Aquesta tesis estudia els efectes de les procianidinesderivades de pinyol de raïm (GSPE) en l'adipòcit, i per a dur-ho a terme es pren com amodel d'estudi la línia cel.lular 3T3-L1. Per una banda es descriuen els efectes del GSPE enel metabolisme de lípids i glúcids de la cèl.lula adiposa. El GSPE fa un paperinsulinomimètic: estimula la captació de glucosa, la síntesi de glicògen i la síntesi de triacilglicerols. L'anàlisi dels mecanismes moleculars per exercir aquests efectes mostra que GSPEen part comparteix mecanismes i vies de senyalització propis de la insulina (translocació deGLUT-4, PI3K, p38 MAPK); tanmateix, s'observa que GSPE ha d'usar també altresmecanismes complementaris. Aquests resultats suggereixen que GSPE pot tenir efectespositius en situacions de diabetis i/o resistència a insulina, donat que a més a més, els estudisin vivo mostren que GSPE és antihiperglicèmic en condicions de diabetis induïda perestreptozotocina. En aquesta tesis també s'analitzen els mecanismes moleculars queexplicarien els efectes lipolítics de les procianidines descrits en estudis previs, i s'ha trobatque la proteina kinasa A i PPARã hi estan involucrats. Part d'aquests resultats han obert unaaltra via d'estudi sobre els efectes de la GSPE en el procés de diferenciació de la cèl.lulaadiposa on s'ha observat que el tractament amb procianidines a l'inici de la diferenciaciódificulta aquesta transformació. Donat que l'augment del nombre d'adipòcits afectanegativament la obesitat, aquest efecte de les procianidines és una característicaprometedora que caldrà tenir en compte en l'estudi del seu possible paper antiobesitat.

Obesity is a growing concern in the world today. As we ponder about the many causes of this global epidemic, we are driven to look at our food and the environmental toxicants that may contribute to obesity. Deltamethrin, being a common synthetic pyrethroid used in agriculture for pest control, is the primary insecticide this study explores to connect with obesity in 3T3-L1 adipocytes. To investigate the relationship between deltamethrin and adipogenesis, various concentrations were tested, 1nM, 10nM, 100nM, 1μM, and 10μM. The result indicated that higher concentration of deltamethrin had a direct impact on fat accumulation. These experiment results indicate that deltamethrin may potentiate adipogenesis in this model. Further in vivo studies will be needed to validate these findings and confirm the effects of deltamethrin on obesity.

White adipose tissue (WAT) stores triglycerides and is crucial to maintaining the body's energy balance. Attributed to its plasticity, WAT can undergo dynamic remodeling in response to chronic energy excess. As obesity increases, alterations in the quantity and function of progenitor and immune cells result in fibrosis and inflammation in the WAT. Hence, metabolic dysfunction becomes more severe. In a recent study, HFD feeding significantly increased the gene expressions of bone morphogenetic protein 2 (BMP2), acidic fibroblast growth factor (FGF1), and basic fibroblast growth factor (FGF2) in macrophages. It was found in a fibrotic environment that the factors stimulated the growth of progenitor cells and the expression of fibrotic genes but suppressed adipogenesis. We looked at how these ligands affect fat metabolism, including adipogenesis, fibrosis, and thermogenesis. We also found out how these ligands affect the way progenitor cells change. By conducting proliferation and differentiation experiments on the 3T3-L1 cell model in vitro with these ligands supplemented at different phases, we demonstrated these ligands' influence 3T3-L1 preadipocytes and adipocytes genotype and phenotype. Based on this research, it was found that BMP2 stimulates adipogenesis by making cells multiply and differentiate. FGF1 exhibits different phasic influences on adipogenesis. FGF1 suppresses the preadipocyte differentiation phase but promotes cell proliferation, which increases the cell confluence speed and might lead to earlier differentiation of adipocytes. FGF2 added in the proliferation phase did not have much effect on adipogenesis. FGF2 might promote preadipocyte commitment during the adipocyte-genesis stage of differentiation phase but the comprehensive effect remains ambiguous.

A bifenila policlorada n° 126 (3,3\',4,4\',5-Penta-cloro-bifenil, PCB126) é um poluente orgânico persistente (POP), tóxico para os seres vivos. Recentemente, a associação entre a exposição aos POPs e doenças metabólicas têm sido descrita. No entanto, os mecanismos desta toxicidade não estão esclarecidos. Desta forma, este trabalho visou elucidar os efeitos da exposição ao PCB126 sobre pré-adipócitos da linhagem 3T3-L1, diferenciados ou não, focando nos mecanismos de morte celular, proliferação, diferenciação e metabolismo. O PCB 126 foi diluído em DMSO (0,13%). Células 3T3-L1 foram mantidas em meio Dulbecco\'s modified Eagle\'s (DMEM, 5% de CO2 a 37ºC) e sua diferenciação foi realizada pela incubação com solução contendo coquetel adipogênico durante 10 dias. As células foram incubadas com diferentes concentrações de PCB126, DMSO (0,13%) ou DMEM. Células não diferenciadas foram empregadas em ensaios de viabilidade, morte, proliferação, ciclo celular, fragmentação do DNA (citometria de fluxo) e ensaios de produção de espécies reativas de oxigênio (ROS, espectrofotometria de fluorescência). Células diferenciadas foram empregadas para quantificação dos mediadores inflamatórios (ELISA), acúmulo de triglicérides intracelulares (Oil red, microscopia de luz), produção de óxido nítrico (NO, reação de Greiss), expressão do receptor de aril hidrocarboneto (AhR, WB) e do proliferador de peroxissoma (PPAR-γ, WB). Os resultados obtidos mostraram que a exposição ao PCB126 (10, 30 ou 100µM; 24, 48 ou 72 h) não alterou a viabilidade e proliferação celular; reduziu a porcentagem de células em apoptose (30, 100µM; 24 ou 72 h); não alterou o ciclo celular (10, 30 ou 100µM; 24 ou 72 h); induziu a fragmentação do DNA (10, 30 ou 100µM; 24h); não induziu a geração de ROS (30, 100µM; 24 ou 72 h); induziu a produção de NO (300µM; 24 ou 72h); não alterou a secreção do fator quimiotáxico para monócito (MCP-1); aumentou a secreção do fator de necrose tumoral (TNF-α; 100 ou 300µM, 72 h) e da interleucina 12 (IL-12; 300µM, 72 h); reduziu a secreção de interleucina 1β (IL-1β, 300 µM, 72 h) e de interleucina 10 (IL-10; 300µM; 24h, 100 ou 300µM; 48 h; 100µM, 72 h); aumentou a expressão de PPARγ (100 ou 300µM, 24h; 300µM, 72h) e de AhR (300µM, 72h); aumentou a concentração de triglicérides intracelulares (100 ou 300µM; 24, 48 ou 72h). Em conjunto, os resultados obtidos mostram as ações diretas do PCB126 em células 3T3-L1 envolvidas na adipogênese, o que reforça, embora que em ensaios in vitro, o papel desta classe de poluentes na obesidade. / The polychlorinated biphenyl (PCB) 126 is a persistent organic pollutant and toxic to human being. The association between exposition to POPs and metabolic diseases has been described. Nevertheless this toxicity mechanism is not yet elucidated. This way this work aim to elucidate the exposure over pre adipocytes 3T3-L1 line to PCB 126, differentiated or not, focused on cellular death mechanisms, proliferation, differentiation and metabolism. The PCB126 was diluted in DMSO (0,13%). 3T3-L1 cells were maintained in Dulbecco´s modified Eagle´s medium (DMEM, 5% CO2 at 37º) and the differentiation process was performed by incubation with adpogenic cocktail solution by 10 days. The cells were incubated with different concentration of PCB126, DMSO (0,13%) or DMEM. Cells not differentiated were used on viability assays, death, proliferation, cell cycle and DNA fragmentation (flow cytometry) and reactive oxygen species production assay (ROS, fluorescence spectrophotometry). Differentiated cells were used to quantify inflammatory mediators (ELISA), Intracellular triglycerides accumulation (Oil red staining, light microscopy), nitric oxide production (NO, Greiss reaction), aril hydrocarbon receptor expression (AhR, Western Blot) and Peroxisome proliferator receptor gamma (PPAR-y, Western Blot). The obtained results showed that exposure to PCB126 (10, 30 or 100 µM; 24, 48 or 72 hours) did not interfere on cellular viability and proliferation; reduced the cells percentage in apoptosis (30, 100 µM; 24 or 72h); did not interfere on cellular cycle (10, 30 or 100 µM; 24 or 72h); induced DNA fragmentation (10, 30 or 100 µM; 24h); did not induced ROS generation (30, 100 µM; 24 or 72h); induced the production of NO (300 µM; 24 or 72h); did not alter the secretion of monocyte chemoattractant protein (MCP-1); enhanced the secretion of tumoral necrosis factor (TNF-α; 100 or 300 µM; 72h) and interleukin 12 (IL12; 300 µM; 72h); reduced secretion of interleukin 1β (IL1β 300 µM; 72h) and interleukin 10 (IL10; 300 µM; 24h; 100 or 300 µM; 48h; 100 µM; 72h); increased PPAR-y expression (100 or 300 µM; 24h; 300 µM; 72h) and AhR (300 µM, 24h); increased intracellular triglycerides concentration (100 or 300 µM, 24, 48 or 72h). In summary the results show direct actions of PCB126 in 3T3-L1 cells evolved on adipogenesis, reinforcing, although in vitro, the role of this pollutant class on obesity.

A bifenila policlorada n° 126 (3,3\',4,4\',5-Penta-cloro-bifenil, PCB126) é um poluente orgânico persistente (POP), tóxico para os seres vivos. Recentemente, a associação entre a exposição aos POPs e doenças metabólicas têm sido descrita. No entanto, os mecanismos desta toxicidade não estão esclarecidos. Desta forma, este trabalho visou elucidar os efeitos da exposição ao PCB126 sobre pré-adipócitos da linhagem 3T3-L1, diferenciados ou não, focando nos mecanismos de morte celular, proliferação, diferenciação e metabolismo. O PCB 126 foi diluído em DMSO (0,13%). Células 3T3-L1 foram mantidas em meio Dulbecco\'s modified Eagle\'s (DMEM, 5% de CO2 a 37ºC) e sua diferenciação foi realizada pela incubação com solução contendo coquetel adipogênico durante 10 dias. As células foram incubadas com diferentes concentrações de PCB126, DMSO (0,13%) ou DMEM. Células não diferenciadas foram empregadas em ensaios de viabilidade, morte, proliferação, ciclo celular, fragmentação do DNA (citometria de fluxo) e ensaios de produção de espécies reativas de oxigênio (ROS, espectrofotometria de fluorescência). Células diferenciadas foram empregadas para quantificação dos mediadores inflamatórios (ELISA), acúmulo de triglicérides intracelulares (Oil red, microscopia de luz), produção de óxido nítrico (NO, reação de Greiss), expressão do receptor de aril hidrocarboneto (AhR, WB) e do proliferador de peroxissoma (PPAR-γ, WB). Os resultados obtidos mostraram que a exposição ao PCB126 (10, 30 ou 100µM; 24, 48 ou 72 h) não alterou a viabilidade e proliferação celular; reduziu a porcentagem de células em apoptose (30, 100µM; 24 ou 72 h); não alterou o ciclo celular (10, 30 ou 100µM; 24 ou 72 h); induziu a fragmentação do DNA (10, 30 ou 100µM; 24h); não induziu a geração de ROS (30, 100µM; 24 ou 72 h); induziu a produção de NO (300µM; 24 ou 72h); não alterou a secreção do fator quimiotáxico para monócito (MCP-1); aumentou a secreção do fator de necrose tumoral (TNF-α; 100 ou 300µM, 72 h) e da interleucina 12 (IL-12; 300µM, 72 h); reduziu a secreção de interleucina 1β (IL-1β, 300 µM, 72 h) e de interleucina 10 (IL-10; 300µM; 24h, 100 ou 300µM; 48 h; 100µM, 72 h); aumentou a expressão de PPARγ (100 ou 300µM, 24h; 300µM, 72h) e de AhR (300µM, 72h); aumentou a concentração de triglicérides intracelulares (100 ou 300µM; 24, 48 ou 72h). Em conjunto, os resultados obtidos mostram as ações diretas do PCB126 em células 3T3-L1 envolvidas na adipogênese, o que reforça, embora que em ensaios in vitro, o papel desta classe de poluentes na obesidade. / The polychlorinated biphenyl (PCB) 126 is a persistent organic pollutant and toxic to human being. The association between exposition to POPs and metabolic diseases has been described. Nevertheless this toxicity mechanism is not yet elucidated. This way this work aim to elucidate the exposure over pre adipocytes 3T3-L1 line to PCB 126, differentiated or not, focused on cellular death mechanisms, proliferation, differentiation and metabolism. The PCB126 was diluted in DMSO (0,13%). 3T3-L1 cells were maintained in Dulbecco´s modified Eagle´s medium (DMEM, 5% CO2 at 37º) and the differentiation process was performed by incubation with adpogenic cocktail solution by 10 days. The cells were incubated with different concentration of PCB126, DMSO (0,13%) or DMEM. Cells not differentiated were used on viability assays, death, proliferation, cell cycle and DNA fragmentation (flow cytometry) and reactive oxygen species production assay (ROS, fluorescence spectrophotometry). Differentiated cells were used to quantify inflammatory mediators (ELISA), Intracellular triglycerides accumulation (Oil red staining, light microscopy), nitric oxide production (NO, Greiss reaction), aril hydrocarbon receptor expression (AhR, Western Blot) and Peroxisome proliferator receptor gamma (PPAR-y, Western Blot). The obtained results showed that exposure to PCB126 (10, 30 or 100 µM; 24, 48 or 72 hours) did not interfere on cellular viability and proliferation; reduced the cells percentage in apoptosis (30, 100 µM; 24 or 72h); did not interfere on cellular cycle (10, 30 or 100 µM; 24 or 72h); induced DNA fragmentation (10, 30 or 100 µM; 24h); did not induced ROS generation (30, 100 µM; 24 or 72h); induced the production of NO (300 µM; 24 or 72h); did not alter the secretion of monocyte chemoattractant protein (MCP-1); enhanced the secretion of tumoral necrosis factor (TNF-α; 100 or 300 µM; 72h) and interleukin 12 (IL12; 300 µM; 72h); reduced secretion of interleukin 1β (IL1β 300 µM; 72h) and interleukin 10 (IL10; 300 µM; 24h; 100 or 300 µM; 48h; 100 µM; 72h); increased PPAR-y expression (100 or 300 µM; 24h; 300 µM; 72h) and AhR (300 µM, 24h); increased intracellular triglycerides concentration (100 or 300 µM, 24, 48 or 72h). In summary the results show direct actions of PCB126 in 3T3-L1 cells evolved on adipogenesis, reinforcing, although in vitro, the role of this pollutant class on obesity.

Orientador: Maria Luiza Silveira Mello / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T21:08:55Z (GMT). No. of bitstreams: 1 Felisbino_MarinaBarreto_M.pdf: 5744960 bytes, checksum: 792f3ed20bbe7d53c917089733be679f (MD5) Previous issue date: 2012 / Resumo: O ácido valproico (VPA) é um potente anti-convulsante conhecido como inibidor de deacetilases de histonas (HDACi) de classe I em diversos tipos celulares. Buscando conhecer se a estrutura cromatínica se alteraria quando da ação de HDACi, investigamos a supraorganização cromatínica de células tumorais HeLa e de células NIH 3T3, estas últimas caracterizadas por apresentarem áreas de heterocromatina conspícuas, sob tratamento com VPA. Essas informações foram associadas a da atividade enzimática de HDACs assim como do nível de acetilação das histonas H3 nesses modelos celulares tratados por VPA. As frequências de anomalias cromossômicas, morte celular e índices mitóticos também foram investigados. As células tratadas com VPA nas concentrações 0,05, 0,5 e 1,0 mM por 1-24 h foram submetidas à reação de Feulgen e analisadas através de microespectrofotometria de varredura automática e microscopia óptica. Western blots, análises enzimáticas e ensaio TUNEL também foram utilizados neste estudo. Células tratadas com tricostatina A (TSA), uma HDACi de atividade mais ampla do que o VPA, foram utilizadas como controles positivos. Em todas as condições de tratamento com VPA e TSA foi demonstrada descompactação cromatínica acompanhada de diminuição na atividade de HDACs e aumento na acetilação de histona H3. Essa alteração textural cromatínica também atingiu áreas heterocromáticas de células NIH 3T3. Nenhuma alteração nas frequências de anomalias cromossômicas, índices mitóticos e morte celular foi observada nesses modelos celulares nas condições relatadas, embora tenha ocorrido um aumento de fragmentação de DNA em células HeLa tratadas com VPA por 24 h e por TSA a partir de 4 h. Diminuição na proliferação celular nas células HeLa ocorreu apenas sob tratamento com VPA 5,0 mM por 48 h. Os resultados indicam que o VPA e a TSA promovem remodelação cromatínica em células tumorais HeLa e em células fibroblásticas NIH 3T3, que pode ser atribuída à sua ação de HDACi. Não se pôde descartar, porém, que o VPA atue sobre outras proteínas nucleares, cuja expressão poderia se apresentar diminuída sob sua ação / Abstract: Valproic acid (VPA) is a potent anticonvulsant that inhibits class I histone deacetylases (HDACi) in several cell types. Seeking to know whether the chromatin structure would change when the action of HDACi, we investigated whether VPA would affect chromatin supraorganization of tumoral HeLa cells and NIH 3T3 cells, this latter characterized by presenting areas of conspicuos heterochromatin. This information was associated with enzymatic activity of HDACs as well as the level of H3 histone acetylation in these cell models treated with VPA. The frequency of chromosome abnormalities and cell death and mitotic indices were also investigated. VPA-treated cells at concentration 0.05, 0.5 and 1.0 mM for 1-24 h were subjected to the Feulgen reaction and analysed by automatic scanning microspectrophotometry and optical microscopy. Western blots, enzymatic analysis and TUNEL assay were also performed in this study. Trichostatin A (TSA)-treated cells, an HDACi whose activity is broader than VPA, were used as positive control. Chromatin decondensation was demonstrated under all TSA and VPA treatments and was associated with decrease in HDAC activity and with increase in the level of H3 histone acetylation. This chromatin textural change also affected heterochromatic areas of NIH 3T3 cells. No changes in chromosome abnormalities, mitotic indices or morphologically identified cell death were found in both cellular models with the VPA treatment conditions mentioned above, although there was an increase of DNA fragmentation after a 24 h-VPA treatment and a 4 h-TSA treatment in HeLa cells. Decrease in cell proliferation in HeLa cells ocurred only under a 5.0 mM 48 h-VPA treatment. The results indicate that VPA and TSA promote chromatin remodeling in tumoral HeLa cells and fibroblastic NIH 3T3 cells, which may be attrituted to their HDACi action. It may not be discarded, however, that VPA acts on other nuclear proteins whose expression could be reducted under its action / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Many studies have shown that peroxisome-proliferator-activated receptor [gamma] (PPAR[gamma]) plays an important role in adipose tissue formation by activating genes implicated in adipogenesis. PPAR[gamma] heterodimerizes with retinoid X receptor [alpha] (RXR[alpha]), in the presence of ligand, on PPAR response elements (PPREs) in the promoter of target genes involved in adipocyte differentiation. General receptor for phosphoinositides 1 (GRP1) is a corepressor of thyroid hormone receptors (TRs), a nuclear receptor like PPAR[gamma]. GRP1 decreases TRs' transcriptional activity by lowering dimerisation on DNA. Since PPARs and TRs have important structural similarities and that GRP1 interacts with PPARs in vitro, we hypothesized that GRP1 could be a coregulator of PPARs and, thus be implicated in adipogenesis. To better understand GRP1's effect on PPAR[gamma]2, transcriptional activity assays have been done and show that increasing concentrations of GRP1 decrease the transcriptional activity of PPAR[gamma]2. We also studied GRP1 expression by Western blots of total protein extracts from 3T3-L1 cells at different times during differentiation: GRP1 is present in 3T3-L1 preadipocytes and its expression decreases during adipogenesis. According to those results, GRP1 may be a PPAR[gamma] corepressor. After those observations, GRP1 effects on adipogenesis were studied by modulating its expression with lentiviral particles. Interestingly, GRP1 knock-down before inducing 3T3-L1 differentiation, almost abrogates adipogenesis and adipocytes markers, PPAR[gamma] and aP2, while its overexpression increases lipid storage without affecting PPAR[gamma] expression. On the opposite, GRP1 modulation after differentiation induction shows that expression knock-down slightly promotes adipogenesis by increasing PPAR[gamma], aP2 and lipid accumulation and that overexpression weakly decreases lipid storage. Our results suggest that GRP1 implication during adipogenesis occurs at two distinct and precise moments. It seems to be a key factor in the early stages of adipocyte differentiation and to be implicated as a PPAR[gamma] transcriptional activity modulator as a corepressor. Future experiments will help detail modulation of protein expression and underlying mechanisms to better understand the role of GRP1 in adipogenesis and, eventually, comorbidities linked to obesity like cardiovascular diseases and type 2 diabetes mellitus.

Les apolipoprotéines L (APOL) forment une famille de protéines conservées chez les mammifères. L’APOL6 murine est principalement exprimée par les adipocytes présents dans le tissu adipeux. Dans un modèle de culture d’adipocytes, l’adipogénèse a causé l’induction de l’expression de l’APOL6. Celle-ci a pu encore être modulée à la hausse par de l’IFNγ, et à la baisse par du TGFβ. Des facteurs élevant la concentration en AMP cyclique ont aussi permis de diminuer l’expression d’APOL6. In vivo, lorsque des souris APOL6 KO ont été nourries par un régime riche en graisses, elles ont pris moins de poids que les souris WT correspondantes. De plus, les adipocytes des souris APOL6 KO obèses étaient plus petits que ceux des contrôles WT. Finalement, la recherche de protéines interagissant avec l’APOL6 par immunoprécipitation a permis de mettre en évidence une majorité de protéines associées au cytosquelette d’actine. En conclusion, l’APOL6 semble être associée au cytosquelette d’actine des adipocytes et permettrait la régulation de la taille de leurs gouttelettes lipidiques. / Apolipoproteins L (APOL) are a family of conserved proteins among mammals. Murine APOL6 is mainly expressed by adipocytes in the adipose tissue. In a model of in vitro adipocyte cell culture, adipogenesis induced the expression of APOL6. This expression increased with IFNγ and decreased with TGFβ. Cyclic-AMP elevating agents also decreased the expression of APOL6. In vivo, APOL6 KO mice that were fed with a high fat diet gained less weight than their wild type (WT) counterparts. Furthermore, adipocytes from obese APOL6 KO mice were smaller than those from WT controls. Finally, immunoprecipitation experiments showed that APOL6 probably interacted with actin cytoskeleton proteins within adipocytes. In conclusion, APOL6 is likely associated with the actin cytoskeleton in adipocytes and could be involved in the regulation of the size of lipid droplets. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Plusieurs problèmes de santé peuvent être associés à un déséquilibre de l'homéostasie énergétique tels l'obésité, le diabète, les cancers et les maladies cardiovasculaires. Il est bien établi que l'angiotensine II (Ang II) joue un rôle dans le développement de ces pathologies. La plupart des effets néfastes de l'Ang II sont attribués au récepteur de type I (R-AT1) alors que l'activation du récepteur de type 2 (R-AT2) génère des effets qui s'opposent souvent à ceux du R-AT1. Des travaux suggèrent un rôle de l'Ang II dans la physiologie de l'adipocyte. Afin d'étudier l'implication du R-AT2 lors de l'adipogenèse dans des conditions normales et de perturbations de l'homéostasie, les cellules 3T3-L1 ont été choisies. Elles sont un modèle de différenciation adipocytaire utilisé depuis longtemps puisqu'elles possèdent les mêmes caractéristiques principales que les adipocytes et plusieurs résultats obtenus avec ce modèle ont été validés dans des modèles in vivo. Nos expériences de RT-PCR et d'études de liaison hormone-récepteur montrent que le R-AT2 n'est pas présent dans les préadipocytes, mais que son expression augmente au cours de la différenciation des cellules 3T3-L1. Nous avons utilisé un nouvel agoniste non-peptidique sélectif du R-AT2, le M24, afin d'éclaircir les rôles de ce récepteur lors de l'adipogenèse. L'activation du R-AT2 par le M24, en condition normale de différenciation, ne modifie pas l'expression des R-AT1 et R-AT2 ainsi que celle des marqueurs de la différenciation fonctionnelle tels aP2 et PPAR?. De plus, l'activation du R-AT2 n'affecte pas l'accumulation lipidique. Nous avons utilisé des traitements avec les acides gras, oléate et palmitate, afin de perturber l'homéostasie des cellules. Ces traitements n'ont pas modifié de manière significative les différents paramètres étudiés. Par conséquent, les effets bénéfiques potentiels de l'activation du R-AT2 par le M24, notamment sur la différenciation adipocytaire, n'ont pu être observés. D'autres études seront donc nécessaires afin d'éclaircir l'implication du R-AT2 lors de la différenciation adipocytaire des cellules 3T3-L1 et son effet bénéfique potentiel en condition de désordres métaboliques. Les causes possibles de cette absence d'effet sont discutées.