Obesity and the metabolic complications associated with it are increasing globally. Sedentary lifestyles, poor diet and genetic predisposition contribute to obesity. In addition, environmental chemicals such as Bisphenol A (BPA) may play a significant role. Exposure to BPA has been correlated with an array of adverse health effects on the endocrine system and whole-body homeostasis. This has resulted in manufacturers replacing it with structural analogues such as Tetra Methyl Bisphenol F (TMBPF), Bisphenol F (BPF), Bisphenol AP (BPAP), and fluorine-9-bisphenol (BHPF). BPA is a suspected obesogen as it can induce adipogenesis in human and murine preadipocytes. The effects of the BPA analogues listed above on adipogenesis have yet to be evaluated. The aim of this project is to investigate their adipogenic effects. For this purpose, we used 3T3-L1 mouse embryonic fibroblasts. This cell model can be differentiated into mature adipocytes with appropriate inducers including 3-isobutyl-1-methylxanthine (IBMX), insulin and dexamethasone, a synthetic steroid. To assess the effects of BPA analogues, the cells were treated with varying concentrations of TMBPF, BPF, BHPF, BPA, or BPAP in place of dexamethasone. The expression levels of mature adipocyte markers were assessed at mRNA and protein levels to determine the adipogenic potential of the analogues. Lipid accumulation was evaluated by Nile Red staining. A time course was performed to assess the expression levels of known transcriptional regulators of adipogenesis. The results indicate that TMBPF, BPF and BPA increase 3T3-L1 adipogenesis. BHPF and BPAP did not affect adipogenesis in this model. BPF appears to be at least as good as BPA at inducing adipogenesis. TMBPF, on the other hand, can induce adipogenesis to a greater extent than the other chemicals, including BPA, as evidenced by increased expression of adipogenic markers and lipid accumulation. Finally, key transcription factors C/ebpδ and C/ebpα, part of the adipogenic transcriptional cascade, were up-regulated at III two and six hours post-treatment by TMBPF. BPA also up-regulated C/ebpδ at two hours post-treatment. Though the adipogenic effects have become apparent for some of these analogues, the mechanism by which they elicit their effects remains to be discovered. More research is required to deduce the mechanism of action and to provide consensus on what the effects of these replacement bisphenols actually are.

Visando novos estudos com a proteína amilóide sérica A (SAA), propusemos a produção de seu recombinante humano em Escherichia coli, mais especificamente, a isoforma encontrada na fase aguda (A-SAA1) e da constitutivamente expressa (C-SAA4). Realizamos a expressão, identificação e purificação das proteínas recombinantes. Concomitantemente, também avaliamos o efeito da hipóxia na expressão e produção da proteína SAA nativa em linhagens de pré-adipócitos murinos 3T3-L1, não diferenciados e diferenciados e adipócitos humanos. Aparentemente quanto maior o grau de diferenciação celular, maior a expressão e produção da proteína. Para os adipócitos humanos, o perfil de expressão de mRNA da SAA mostra que SAA1>SAA2>SAA4 nas relações 500:150:1. Na hipóxia, há um aumento na expressão de SAA, entretanto não associamos esta expressão a um aumento da concentração da proteína. A importância do reconhecimento de que SAA pode ser umas das proteínas induzidas pela hipóxia em adipócitos é discutida em relação ao seu papel pró-inflamatório. / In order to provide more complex studies with the protein serum amyloid A (SAA), this study proposed the production of its human recombinant in bacteria (Escherichia coli), more specifically, the synthesis of the main isoform found in the acute phase (A-SAA1) and the constitutively expressed (C-SAA4). The expression, identification and purification of the recombinants proteins was performed. Concurrently, we also did a study evaluating the effect of hypoxia in the expression and protein production of native SAA in undifferentiated and differentiated murine preadipocytes 3T3-L1 and humans adipocytes. Apparently, the protein expression and production increases with the cell differentiation degree. For human adipocytes, we demonstrated the mRNA expression profile of SAA, in which SAA1 > SAA2 > SAA4 (500:150:1). In hypoxia, there is an increased expression of SAA, but we could not link it to an increase of protein concentration. The importance of recognizing that SAA may be one of the proteins induced by hypoxia in adipocytes is discussed in relation to the proinflammatory role of this protein.

Visando novos estudos com a proteína amilóide sérica A (SAA), propusemos a produção de seu recombinante humano em Escherichia coli, mais especificamente, a isoforma encontrada na fase aguda (A-SAA1) e da constitutivamente expressa (C-SAA4). Realizamos a expressão, identificação e purificação das proteínas recombinantes. Concomitantemente, também avaliamos o efeito da hipóxia na expressão e produção da proteína SAA nativa em linhagens de pré-adipócitos murinos 3T3-L1, não diferenciados e diferenciados e adipócitos humanos. Aparentemente quanto maior o grau de diferenciação celular, maior a expressão e produção da proteína. Para os adipócitos humanos, o perfil de expressão de mRNA da SAA mostra que SAA1>SAA2>SAA4 nas relações 500:150:1. Na hipóxia, há um aumento na expressão de SAA, entretanto não associamos esta expressão a um aumento da concentração da proteína. A importância do reconhecimento de que SAA pode ser umas das proteínas induzidas pela hipóxia em adipócitos é discutida em relação ao seu papel pró-inflamatório. / In order to provide more complex studies with the protein serum amyloid A (SAA), this study proposed the production of its human recombinant in bacteria (Escherichia coli), more specifically, the synthesis of the main isoform found in the acute phase (A-SAA1) and the constitutively expressed (C-SAA4). The expression, identification and purification of the recombinants proteins was performed. Concurrently, we also did a study evaluating the effect of hypoxia in the expression and protein production of native SAA in undifferentiated and differentiated murine preadipocytes 3T3-L1 and humans adipocytes. Apparently, the protein expression and production increases with the cell differentiation degree. For human adipocytes, we demonstrated the mRNA expression profile of SAA, in which SAA1 > SAA2 > SAA4 (500:150:1). In hypoxia, there is an increased expression of SAA, but we could not link it to an increase of protein concentration. The importance of recognizing that SAA may be one of the proteins induced by hypoxia in adipocytes is discussed in relation to the proinflammatory role of this protein.

Os hormônios tireoidianos atuam sobre o metabolismo dos diversos tecidos do organismo e participam da regulação do consumo de glicose pelas células. Estudos já evidenciaram que o T3 atua, dependendo do tipo celular, aumentando a expressão de algumas isoformas dos transportadores de glicose (GLUTs) e a translocação do GLUT4 para a membrana plasmática, melhorando, também, a captação de glicose (CGlic) em poucos minutos. Os mecanismos envolvidos nessas ações do T3, contudo, ainda não estão bem esclarecidos. O objetivo do presente estudo foi investigar as possíveis vias de sinalização envolvidas na ação aguda do T3 sobre a CGlic em células adiposas 3T3-L1. Nossos dados demonstraram que o T3 promove aumento na CGlic, com pico aos 10 min, retornando ao nível do controle após 30 min de incubação das células com o hormônio. Sugerimos que essa ação depende, ao menos, de duas vias de sinalização. Uma delas envolve a ativação das proteínas Src, PI3K e Akt. A outra, aparentemente, é iniciada a partir da membrana plasmática via integrina aVb3 / Thyroid hormones act on the metabolism of many tissues and participate in the regulation of glucose consumption by cells. Studies from this and other laboratories have demonstrated in muscle and adipose cells that T3 increases, in a short period (minutes), the expression of some glucose transporter (GLUTs) isoforms and GLUT4 translocation to the plasma membrane leading to an improvement of glucose uptake. However, the mechanisms involved in these T3 actions are still not clear. The aim of this study was to investigate the possible signaling pathways involved in the acute T3 action on glucose uptake in 3T3-L1 adipocytes. Our results have shown that T3 increases glucose uptake with a peak at 10 min returning to the control level after 30 min of the cell incubation with the hormone. We suggest that this action depends on at least two parallel signaling pathways. One involves the activation of Src, PI3K and Akt proteins, while the other involves another mechanism triggered by T3, apparently, from the plasma membrane at aVb3 integrin.

Orientador: Miriane de Oliveira / Resumo: Introdução: O hormônio triiodotironina (T3) influencia o metabolismo e desenvolvimento do tecido adiposo (TA), modulando a proliferação e diferenciação de adipócitos, podendo agir sobre os reguladores do processo de adipogênese, como o receptor ativado por proliferador de peroxissomo (PPARy). O TA está envolvido na regulação da energia corporal, sintetizando e secretando substâncias denominadas adipocinas, dentre elas a adiponectina e leptina. A adiponectina está relacionada ao aumento da sensibilidade à insulina, enquanto a leptina está envolvida com o gasto energético. O T3 pode desencadear ações por ativação de vias extranucleares, dentre elas a via MAPK/ERK e integrina αVβ3. Objetivo: Verificar a ação do T3, com participação das vias extranucleares MAPK/ERK e integrina αVβ3, na modulação de adiponectina e leptina, além de avaliar os parâmetros relacionados ao perfil adipogênico e dano de DNA. Métodos: Adipócitos, 3T3-L1, foram tratados com T3 (10nM) por uma hora, na ausência ou presença dos inibidores de MAPK/ERK – PD98059 (PD, 50uM) e da integrina αvβ3 – ácido tetraiodotiroácetico (Tetrac, 10-4M). A ausência de qualquer tratamento foi considerada grupo controle (C). Após o período de tratamento foi realizado PCRq-RT para analisar a expressão de mRNA de adiponectina e leptina, e Western Blot para expressão proteica de adiponectina, leptina, PPARy, pAKT e pERK; a viabilidade celular foi realizada pelo ensaio de MTT; a quantificação do acúmulo lipídico pelos ens... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: The hormone triiodothyronine (T3) influences the metabolism and development of adipose tissue (TA), modulating the proliferation and differentiation of adipocytes, and can act on regulators of the adipogenic differentiation process, such as the peroxisome proliferator activated receptor). TA is involved in the regulation of body energy, synthesizing and secreting substances called adipokines, among them adiponectin and leptin. Adiponectin is related to increased insulin synaptic, since leptin is involved in energy expenditure. T3 can trigger actions by activation of extranuclear pathways, including MAPK / ERK and integrin α Vβ3. Objective: Given the role of T3 in TA and the importance of adipokines, the objective of this study is to verify the action of T3 with the participation of extranuclear pathways in the modulation of adiponectin and leptin and the parameters related to the adipogenic profile. Methods: Adipocytes, 3T3-L1, were treated with a physiological dose of T3 (10nM) for one hour, in the absence or presence of MAPK / ERK-PD98059 (PD) and integrin αvβ3 - tetraiodothyrocetic (Tetrac) integrin inhibitors. The absence of any treatment was considered as a control group (C). After the treatment period PCRqRT was performed to analyze the expression of leptin and adiponectin mRNA, and Western Blot for protein expression of adiponectin, leptin, PPARγ, pAKT and pERK; cell viability was performed by the MTT assay; the quantification of lipid accumulation by the... (Complete abstract click electronic access below) / Mestre

Background: The typical western diet contains foods with modest amounts of lipid oxidation products. Previous work by us and others have demonstrated that mildly oxidized lipids promote a gain in fat mass while highly oxidized lipids decrease fat mass in rodents and triglyceride (TAG) accumulation in 3T3-L1 cells. Adipocyte differentiation is regulated by a key nuclear transcription factor known as PPARγ. Objective: To investigate if the alterations in triglyceride accumulation in 3T3-L1 cells pretreated with oxidized soy oil are due to 1) a change in PPARg DNA interactions 2) changes in the expression of SREBP-1c, PPARg, and/or its target genes. Main Methods: Confluent 3T3-L1 cells were pretreated for 24hours with 0.01% soy oil (SO) which was either unheated (unheated SO) or heated for 3, (3h-SO), 6 (6h-SO), or 9hours (9h-SO). The effect of 24hour soy oil exposure was assessed at several time points throughout the differentiation process. Alterations in PPARg DNA interaction was assessed using a PPARγ transcription factor assay kit while alterations in the expression of genes upstream and downstream of PPARγ was determined by RT-PCR. Primary and secondary products of oxidation within the SO were determined by spectrophotometry. Results: The 6hr-SO contained the greatest concentration of peroxides whereas both the 6hr-SO and 9hr-SO contained a significantly higher concentration of conjugated dienes and aldehydes.Nuclear extracts from 3T3-L1 cells pretreated with 6h-SO demonstrated the greatest reduction in PPARγ DNA binding. Compared to the unheated SO and mildly oxidized 3h-SO, cells treated with the 6h-SO had a significant reduction in SREBP-1c, PPARg, LPL, and GLUT4 expression occurring early in the differentiation process. Variations in the gene expression of 6hr-SO pretreated cells persisted within partially differentiated and mature adipocytes. Conclusions: Pre-treatment of preadipocytes with soy oil heated for ³ 6h greatly decreases the activity of PPARγ in the nucleus and adipogenic gene expression . These changes seen in early differentiation seem to correlate the best with the phenotype of reduced triglyceride accumulation seen in mature adipocytes.

The overall goal of this research was to investigate oxidative stress related changes to the proteomes of 3T3-L1 adipocytes and an inducible CYP2E1 expressing HepG2 cells. Enhanced oxidative stress in hypertrophic adipocytes is associated with metabolic dysregulation and insulin resistance. Because mitochondria generate reactive oxygen species (ROS), we monitored changes to the adipocyte mitochondrial proteome during differentiation and enlargement. We labeled mitochondrial extracts from 3T3-L1 cells that were 0, 4, 7, 10, 14, and 18 days post differentiation with iTRAQ, followed by MS based identification. We found citric acid cycle proteins such as pyruvate carboxylase, citrate synthase, as well as beta-oxidation enzymes; cartinine acyl transferase and long-chain enoyl-CoA hydratase up-regulated from 7 through 18 days post differentiation onset. These data indicate TCA up-regulation for enhanced metabolic and citrate output necessary for lipid synthesis in adipocytes. Paradoxically, the data also show the simultaneous increase in the fatty acid oxidation, indicating a metabolic overdrive state. Biochemical assays showing peaks in ATP and ROS generation in 3 day old adipocytes provide further evidence of this overdrive state. A second peak in ROS generation occurred in 10 day old adipocytes; concurrent ATP generation reduced to near pre-adipocyte levels and this may indicate a metabolic shift that may be responsible for increased oxidative stress in hypertrophic adipocytes. We developed a doxycycline inducible CYP2E1 expressing HepG2 cell line using the pTet-On/pRevTRE expression system to allow greater control and sensitivity in the generation CYP2E1 mediated oxidative stress. Our cell line (RD12) demonstrated stability and tight expression control. After induction, RD12 cells showed 30 percent higher CYP2E1 activity when compared to the constitutive E47 cell line. RD12 cells showed 30 percent greater toxicity than E47 cells and 25 percent less free glutathione when exposed to 20 mM acetaminophen, indicating RD12 cells are more sensitive to the effects reactive intermediates and oxidative stress generated by CYP2E1. We conducted a survey of the toxicity of dietary fatty acids (oleic, linoleic, and palmitic) on HepG2 cells to determine fatty acid doses that induced metabolic changes, but did not cause excessive cell death. The dose of 0.20 mM linoleic and palmitic acid for 48 hours produced low toxicity, but oleic acid actually produced lower toxicity than untreated cells. After exposure cells were treated with a pro-oxidant to determine which fatty acid increased the susceptibility to protein carbonylation. The carbonylated protein isolation procedure indicated the palmitic acid may induce more carbonylation than oleic acid, but greater efficiency in the isolation procedure is required for a confidant determination.

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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

The increase in the prevalence of obesity and obesity-related diseases has caused greater attention to be placed on the molecular mechanisms controlling adipogenesis. In this study, we studied the role of 5-aza-2'-deoxycytidine (5-Aza-dC), an inhibitor of DNA methylation, on adipocyte differentiation. We found that inhibiting DNA methylation by 5-Aza-dC significantly inhibited adipocyte differentiation whereas promoting osteoblastogenesis. Wnt10a was up-regulated by 5-Aza-dC treatment and it was suggested that Wnt10a might play a vital role in suppressing adipogenesis and promoting osteoblastogenesis by inhibiting DNA methylation. In 3T3-L1 cells, Wnt signaling inhibitor IWP-2 was found to reverse the inhibitory effect of 5-Aza-dC on Adipocyte differentiation, whereas in mesenchymal stem cell line, ST2 cells, IWP-2 treatment reversed the effect of 5-Aza-dC on promoting osteoblastogenesis. These studies will provide a better understanding to the cause and treatment of obesity and bone-related diseases.