Análise de pathway e network em tecido adiposo in vivo e o papel da vitamina D na adipogênese in vitro
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2016-02-26
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Universidade Estadual Paulista (Unesp)
Resumo
A prevalência de obesidade mais que dobrou entre 1980 e 2014, uma vez que mais de 1,9 bilhões de adultos são com sobrepeso, e mais de 600 milhões desses são obesos. No Brasil, de acordo com as últimas pesquisas, em 2014, 52,5% dos brasileiros são sobrepesos, enquanto 17,9% são obesos. Obesidade é o maior fator de risco para doenças não-transmissíveis, como doenças cardiovasculares, diabetes mellitus tipo 2, hipertensão e câncer. Indivíduos obesos metabolicamente saudáveis tendem a apresentar um quadro de obesidade hiperplásica, o que garante a integridade da função do tecido adiposo, e tem demonstrado ser a chave para a manutenção da homeostase corporal em casos de desequilíbrio no metabolismo energético, como na obesidade. A vitamina D, e seus metabólitos, são lipofílicos e, dessa forma, são armazenados no tecido adiposo, sendo esse evento a possível causa para baixos níveis séricos de VitD em indivíduos obesos. Por isso, o objetivo da presente tese foi realizar análises de pathway e network em tecido adiposo de indivíduos obesos, comparados com indivíduos eutróficos, e avaliar a ação da vitamina 1,25(OH)2D3 na adipogênese em modelo de tecido adiposo humano normal. No primeiro artigo, realizou-se normalização dos dados brutos de microarray para análises de pathway e integração desses em um network. Já no segundo artigo, foi realizado análise celular em tempo real (RTCA), ensaios de atividade metabólica e proliferação celular foram utilizados para mensurar os efeitos da VitD na proliferação e diferenciação adipogênica de células SGBS (síndrome de Simpson-Golabi-Behmel) tratadas com VitD 10nM e 100nM. Análises de apoptose e ciclo celular foram, ainda, realizadas, seguidas por quantificação de triglicerídeos e expressão gênica por RT-qPCR. Os resultados encontrados sustentam o papel central da via da adipogênese em indivíduos obesos, além de mostrar que essa via apresenta alterações na expressão gênica, quando comparada com dados de indivíduos não obesos, além da interação de vias da vitamina D com a via da adipogênese, em obesos. Posteriormente, foi demonstrado que a vitamina D possui ação em pré-adipócitos, em genes biomarcadores da adipogênese e no VDR, além de induzir parada de ciclo celular e diminuição de lipídios intracelulares. A partir dos resultados obtidos com o tratamento de pré-adipócitos humanos com vitamina D, sugere-se a senescência celular induzida por oncogenes, como um possível mecanismo para a ação da vitamina na parada de ciclo celular. Assim, os resultados abrem caminhos para mais investigações no que tange a ação da vitamina D na proliferação de tecido adiposo, além de servir de base para a compreensão da hiperplasia na manutenção da saúde em indivíduos obesos, e garantir os benefícios da suplementação da vitamina D em todas as populações.
The obesity prevalence has more than doubled between 1980 and 2014, and more than 1.9 billion adults are overweight and over 600 million of these are obese. In Brazil, according to the latest research, in 2014, 52.5% of Brazilians are overweight, while 17.9% are obese. Obesity is a major risk factor for noncommunicable diseases such as cardiovascular disease, type 2 diabetes mellitus, hypertension and cancer. Metabolic healthy obese individuals tend to have hyperplastic obesity frame, which ensures the integrity of adipose tissue function and has been shown to be a key to maintaining body homeostasis in cases of imbalance in energy metabolism, such as obesity. Vitamin D and its metabolites are lipophilic and, therefore, are stored in adipose tissue, being this event the possible cause for low serum vitamin D in obese individuals. Therefore, the objective of this thesis was to perform pathway and network analysis in adipose tissue of obese individuals compared to normal weight individuals, and evaluate the action of vitamin 1,25 (OH) 2D3 in adipogenesis in normal human adipose tissue model. In the first article, was done the normalization of raw data from microarray for pathway analysis and integration of these into a network. In the second article, we performed real time cellular analysis (RTCA), metabolic activity assays and cell proliferation were used to measure the effects of vitamin D in proliferation and adipogenic differentiation in SGBS cells (Simpson-Golabi-Behmel syndrome) treated with vitamin D 10 nM and 100 nM. Apoptosis and cell cycle analyzes were also performed, followed by quantitation of triglycerides and gene expression by RT-qPCR. The results support the central role of the pathway of adipogenesis in obese individuals, and show that this pathway presents changes in gene expression when compared with data from non-obese individuals, in addition to the interaction of vitamin D pathway to the adipogenesis pathway, in obese. Also, it was demonstrated that vitamin D has activity in preadipocytes, biomarker genes of adipogenesis and VDR, and induced cell cycle arrest and reduction of intracellular lipids. From the results obtained by the treatment of human preadipocytes with vitamin D, it is suggested that the cellular senescence induced by oncogenes such as a possible mechanism for the action of vitamin in cell cycle arrest. Thus, the results open the way for further investigation regarding the action of vitamin D in adipose tissue proliferation, as well as serve as a basis for understanding hyperplasia in maintaining health in obese individuals, and ensure the benefits of vitamin D supplementation in all populations.
The obesity prevalence has more than doubled between 1980 and 2014, and more than 1.9 billion adults are overweight and over 600 million of these are obese. In Brazil, according to the latest research, in 2014, 52.5% of Brazilians are overweight, while 17.9% are obese. Obesity is a major risk factor for noncommunicable diseases such as cardiovascular disease, type 2 diabetes mellitus, hypertension and cancer. Metabolic healthy obese individuals tend to have hyperplastic obesity frame, which ensures the integrity of adipose tissue function and has been shown to be a key to maintaining body homeostasis in cases of imbalance in energy metabolism, such as obesity. Vitamin D and its metabolites are lipophilic and, therefore, are stored in adipose tissue, being this event the possible cause for low serum vitamin D in obese individuals. Therefore, the objective of this thesis was to perform pathway and network analysis in adipose tissue of obese individuals compared to normal weight individuals, and evaluate the action of vitamin 1,25 (OH) 2D3 in adipogenesis in normal human adipose tissue model. In the first article, was done the normalization of raw data from microarray for pathway analysis and integration of these into a network. In the second article, we performed real time cellular analysis (RTCA), metabolic activity assays and cell proliferation were used to measure the effects of vitamin D in proliferation and adipogenic differentiation in SGBS cells (Simpson-Golabi-Behmel syndrome) treated with vitamin D 10 nM and 100 nM. Apoptosis and cell cycle analyzes were also performed, followed by quantitation of triglycerides and gene expression by RT-qPCR. The results support the central role of the pathway of adipogenesis in obese individuals, and show that this pathway presents changes in gene expression when compared with data from non-obese individuals, in addition to the interaction of vitamin D pathway to the adipogenesis pathway, in obese. Also, it was demonstrated that vitamin D has activity in preadipocytes, biomarker genes of adipogenesis and VDR, and induced cell cycle arrest and reduction of intracellular lipids. From the results obtained by the treatment of human preadipocytes with vitamin D, it is suggested that the cellular senescence induced by oncogenes such as a possible mechanism for the action of vitamin in cell cycle arrest. Thus, the results open the way for further investigation regarding the action of vitamin D in adipose tissue proliferation, as well as serve as a basis for understanding hyperplasia in maintaining health in obese individuals, and ensure the benefits of vitamin D supplementation in all populations.
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Ciclo celular, SGBS, Senescência