Genômica funcional de adipócitos 3T3-L1 tratados com o extrato padronizado de Ginkgo biloba (EGb)
Data
2022-11-03
Autores
Thomaz, Fernanda Malanconi 
Orientadores
Telles, Monica Marques
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O consumo excessivo de calorias, predisposição genética e o sedentarismo são alguns dos principais fatores que contribuem para um desequilíbrio calórico, situação em que se consome mais calorias do que se gasta. Nesse cenário, adipócitos maduros aumentam de tamanho, causando hipertrofia celular que pode levar à obesidade, e inflamação do tecido adiposo, resultando em graves consequências metabólicas. Nas últimas décadas, houve uma grande expansão no estudo e na elucidação sobre o papel do tecido adiposo branco, incluindo sua importante função endócrina, a qual está relacionada à morfologia e alteração metabólica dos adipócitos. Estudos anteriores do nosso grupo demonstraram o potencial antiobesogênico e atenuador de perturbações metabólicas do extrato padronizado de Ginkgo biloba (EGb), em diferentes modelos animais, como de obesidade induzida pela dieta quanto de depleção hormonal causada pela ovariectomia. Sendo assim, esse trabalho teve por objetivo determinar o efeito do EGb sobre o metabolismo e o transcriptoma durante o processo de diferenciação de pré-adipócitos murinos 3T3-L1, bem como de avaliar o potencial do EGb de atuar na lipólise de adipócitos maduros murinos (3T3-L1) e humanos (hWA A41), a fim de serem elucidados possíveis mecanismos pelos quais o extrato modula o metabolismo dessas células. Para o estudo do potencial efeito do EGb na adipogênese, pré-adipócitos 3T3-L1 foram diferenciados durante 7 dias na ausência ou presença de EGb. A citotoxicidade do extrato foi verificada em pré-adipócitos 3T3-L1 e hWA A41 em diferentes concentrações após 48h de tratamento utilizando o método colorimétrico MTT. A análise do conteúdo lipídico dos adipócitos foi realizada por coloração com óleo vermelho O, e seu transcriptoma foi avaliado por PCR Array, utilizando placas customizadas com os genes de interesse. A análise proteica dos marcadores C/EBP, C/EBPα, perilipina-1 e FABP4 foi realizada de forma temporal, nos dias 0 (D0), 3 (D3), 5 (D5) e 7 (D7) durante o processo de diferenciação por Western Blotting. O estudo do potencial efeito do EGb na lipólise foi avaliado em adipócitos maduros 3T3-L1 diferenciados durante 7 dias e em adipócitos maduros hWA a41 diferenciados durante 19 dias, seguidos de 48h de tratamento na ausência ou presença de EGb 1.0 mg/mL. Avaliou-se a taxa de lipólise basal dessas células por meio da quantificação de glicerol. Para os adipócitos 3T3-L1, a expressão gênica dos marcadores lipolíticos Plin 1, Lipe e Pnpla2 foram avaliados por RT-PCR. Os resultados de viabilidade celular mostraram uma ausência de citotoxidade do extrato nas concentrações avaliadas para ambas as linhagens (murino e humana) de pré-adipócitos estudadas. Pré-adipócitos 3T3-L1 tratados com EGb nas concentrações 0,75 (p < 0,001) e 1,00 mg/mL (p < 0,001) apresentaram um aumento no conteúdo lipídico de 37% e 44%, respectivamente, em comparação as células não tratadas. O perfil apresentado pelo transcriptoma das células tratadas com EGb 1,0 mg/mL apresentou 42% de genes superexpressos significativamente relacionados a lipases e enzimas lipolíticas, pró-adipogênese, pró-browning, oxidação de ácidos graxos e termogênese, e citocinas, fatores de crescimento e transdução de sinal. A quantificação de proteínas-chave da via de adipogênese mostrou que o tratamento com EGb 1,0 mg/mL aumentou a expressão de C/EBPβ em 78% (p = 0.0002) no D7, bem como apresentou uma tendência de aumento da expressão de C/EBPα de 80% (p = 0,075) no D3 e de 170% (p = 0,0506) no D7, em comparação com o controle. Ao avaliar marcadores característicos de adipócitos maduros, o tratamento com EGb modulou positivamente a expressão de perilipina em aproximadamente 400% no D3 (p = 0.0039) e aproximadamente 200% no D5 (p < 0.0001), bem como elevou os níveis de FABP4 em 281% no D5 (p = 0.0007). Ao avaliar o efeito do EGb (1,0 mg/mL) em adipócitos maduros, observou-se para ambas as linhagens de células (murinas 3T3-L1 e humanas hWA A41), aumento significativo de 24% (p = 0,0102) e 26% (p < 0,0001), respectivamente, na taxa da lipólise basal em comparação com o grupo controle. Em células 3T3-L1 tratadas com EGb, os níveis de mRNA de Plin 1 e Lipe foram diminuídos em 32% (p = 0,0182) e 40% (p = 0,0047), respectivamente, em comparação as que não receberam tratamento. Assim, pode-se concluir que o EGb apresenta um efeito promissor pró-adipogênico e pró-lipolítico nos modelos celulares avaliados, sugerindo um potencial terapêutico complementar no tratamento da obesidade. No entanto, são necessários estudos adicionais para investigar com mais profundidade os mecanismos envolvidos, principalmente com foco em linhagens celulares humanas.
Excessive consumption of calories, genetic predispositions and a sedentary lifestyle are some of the main factors that contribute to a caloric imbalance, a situation in which more calories are consumed than spent. In this scenario, mature adipocytes increase in size (hypertrophy) that can lead to obesity, and inflammation of the adipose tissue causing harmful effects to the individual's health. In recent decades, there has been a great expansion in the study of the white adipose tissue, including endocrine function, which is related to the adipocyte’s morphology and metabolic alteration. Previous studies from our group have demonstrated the anti-obesogenic potential and attenuation of metabolic disturbances for the standardized extract of Ginkgo biloba (GbE) in different animal models such as diet-induced obesity and hormone depletion caused by ovariectomy. Therefore, this study aimed to determine the effect of GbE on metabolism and transcriptome during the differentiation process of 3T3- L1 murine preadipocytes, as well as to evaluate the potential of GbE to act in the lipolysis of mature adipocytes in both murine (3T3- L1) and humans (hWA A41) cell lines, in order to elucidate possible mechanisms by which the extract modulates the metabolism of these cells. To study the potential effect of GbE on adipogenesis, 3T3-L1 preadipocytes were differentiated for 7 days in the absence or presence of GbE. The cytotoxicity of the extract was verified in preadipocytes 3T3-L1 and hWA A41 at different concentrations after 48h using the MTT colorimetric method. The adipocytes lipid content analysis was performed by staining with oil red O, and their transcriptome was evaluated by Gene Expression PCR Array with customized plates. Protein analysis of markers C/EBPβ, C/EBPα, Perilipin and FABP4 was performed on days 0 (D0), 3 (D3), 5 (D5) and 7 (D7) during the differentiation process by Western Blotting. The study of the potential effect of GbE on lipolysis was evaluated in mature 3T3-L1 differentiated adipocytes for 7 days and in mature hWA a41 differentiated adipocytes for 19 days, followed by 48 h of treatment in the absence or presence of GbE 1.0 mg/mL. The basal lipolysis rate of these cells was evaluated by glycerol quantification. For 3T3-L1 adipocytes, gene expression of the lipolytic markers Plin 1, Lipe and Pnpla2 were evaluated by RT-PCR. Cell viability results showed an absence of cytotoxicity of the extract at the concentrations evaluated for both cell lines (murine and human). GbE-treated preadipocytes at 0.75 (p < 0.001) and 1.00 mg/mL (p < 0.001) showed an increase in lipid content of 37% and 44%, respectively, compared to control cells. The transcriptome profile presented of GbE-treated 1.0 mg/mL showed 42% of significantly up-regulated genes related to lipases and lipolytic enzymes, pro-adipogenesis, pro-browning, fatty acid oxidation and thermogenesis, and cytokines, growth, and signal transduction. Quantification of the key proteins of the adipogenesis pathway showed that GbE 1.0 mg/mL increased the expression of C/EBPβ by 78% (p = 0.0002) on D7, as well as showed a tendency to increase C/EBPα by 80% (p = 0.075) on D3 and by 170% (p = 0.0506) on D7, compared to the control. The evaluation of the protein levels from mature adipocytes characteristic markers showed that GbE 1.0 mg/mL increased perilipin by approximately 400% on D3 (p = 0.0039) and approximately by 200% on D5 (p < 0.0001) and raised FABP4 levels by 281% on D5 (p = 0.0007). The evaluation of GbE (1.0 mg/mL) effect on mature adipocytes, showed for both cell lines (murine 3T3-L1 and human hWA A41) a significant increase of 24% (p = 0.0102) and 26% (p < 0.0001), respectively, in the basal lipolysis rate compared to the control group. 3T3-L1 GbE-treated cells also presented mRNA levels of Plin 1 and Lipe decreased by 32% (p = 0.0182) and by 40% (p = 0.0047), respectively, when compared to control group. Thus, GbE showed a promising pro-adipogenic and pro-lipolytic effect in the evaluated cell lines, suggesting a complementary therapeutic potential in the treatment of obesity. However, further studies are needed to deeply investigate the mechanisms involved, mainly focusing on human cell lines.
Excessive consumption of calories, genetic predispositions and a sedentary lifestyle are some of the main factors that contribute to a caloric imbalance, a situation in which more calories are consumed than spent. In this scenario, mature adipocytes increase in size (hypertrophy) that can lead to obesity, and inflammation of the adipose tissue causing harmful effects to the individual's health. In recent decades, there has been a great expansion in the study of the white adipose tissue, including endocrine function, which is related to the adipocyte’s morphology and metabolic alteration. Previous studies from our group have demonstrated the anti-obesogenic potential and attenuation of metabolic disturbances for the standardized extract of Ginkgo biloba (GbE) in different animal models such as diet-induced obesity and hormone depletion caused by ovariectomy. Therefore, this study aimed to determine the effect of GbE on metabolism and transcriptome during the differentiation process of 3T3- L1 murine preadipocytes, as well as to evaluate the potential of GbE to act in the lipolysis of mature adipocytes in both murine (3T3- L1) and humans (hWA A41) cell lines, in order to elucidate possible mechanisms by which the extract modulates the metabolism of these cells. To study the potential effect of GbE on adipogenesis, 3T3-L1 preadipocytes were differentiated for 7 days in the absence or presence of GbE. The cytotoxicity of the extract was verified in preadipocytes 3T3-L1 and hWA A41 at different concentrations after 48h using the MTT colorimetric method. The adipocytes lipid content analysis was performed by staining with oil red O, and their transcriptome was evaluated by Gene Expression PCR Array with customized plates. Protein analysis of markers C/EBPβ, C/EBPα, Perilipin and FABP4 was performed on days 0 (D0), 3 (D3), 5 (D5) and 7 (D7) during the differentiation process by Western Blotting. The study of the potential effect of GbE on lipolysis was evaluated in mature 3T3-L1 differentiated adipocytes for 7 days and in mature hWA a41 differentiated adipocytes for 19 days, followed by 48 h of treatment in the absence or presence of GbE 1.0 mg/mL. The basal lipolysis rate of these cells was evaluated by glycerol quantification. For 3T3-L1 adipocytes, gene expression of the lipolytic markers Plin 1, Lipe and Pnpla2 were evaluated by RT-PCR. Cell viability results showed an absence of cytotoxicity of the extract at the concentrations evaluated for both cell lines (murine and human). GbE-treated preadipocytes at 0.75 (p < 0.001) and 1.00 mg/mL (p < 0.001) showed an increase in lipid content of 37% and 44%, respectively, compared to control cells. The transcriptome profile presented of GbE-treated 1.0 mg/mL showed 42% of significantly up-regulated genes related to lipases and lipolytic enzymes, pro-adipogenesis, pro-browning, fatty acid oxidation and thermogenesis, and cytokines, growth, and signal transduction. Quantification of the key proteins of the adipogenesis pathway showed that GbE 1.0 mg/mL increased the expression of C/EBPβ by 78% (p = 0.0002) on D7, as well as showed a tendency to increase C/EBPα by 80% (p = 0.075) on D3 and by 170% (p = 0.0506) on D7, compared to the control. The evaluation of the protein levels from mature adipocytes characteristic markers showed that GbE 1.0 mg/mL increased perilipin by approximately 400% on D3 (p = 0.0039) and approximately by 200% on D5 (p < 0.0001) and raised FABP4 levels by 281% on D5 (p = 0.0007). The evaluation of GbE (1.0 mg/mL) effect on mature adipocytes, showed for both cell lines (murine 3T3-L1 and human hWA A41) a significant increase of 24% (p = 0.0102) and 26% (p < 0.0001), respectively, in the basal lipolysis rate compared to the control group. 3T3-L1 GbE-treated cells also presented mRNA levels of Plin 1 and Lipe decreased by 32% (p = 0.0182) and by 40% (p = 0.0047), respectively, when compared to control group. Thus, GbE showed a promising pro-adipogenic and pro-lipolytic effect in the evaluated cell lines, suggesting a complementary therapeutic potential in the treatment of obesity. However, further studies are needed to deeply investigate the mechanisms involved, mainly focusing on human cell lines.