Ativação da via MAPK/ERK e Integrina αvβ3 pela ação da triiodotironina (T3) na modulação da expressão gênica de adipocinas e modificação do perfil lipídico em adipócitos, 3T3-L1.
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Data
2019-02-27
Autores
Mathias, Lucas Solla
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Universidade Estadual Paulista (Unesp)
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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 ensaios Adipo Red e Oil Red, avaliação da liberação de triacilglicerol (TGL) por meio do método enzimático-colorimétrico, a mensuração do produto da lipólise pelo ensaio de glicerol; dano de DNA pela quantificação da 8-Hidroxideoxiguanosina(8O-OH-dG) por ensaio ELISA. Utilizou-se ANOVA complementada com o teste de Tukey, para dados com normalidade e teste de Friedman, complementado pelo teste de Dunn, para dados com ausência de normalidade. Resultados expressos em média ± desvio padrão e mediana (IQ 25% - IQ 75%), respectivamente. Significância dada para p<0,05. Resultados: O T3 elevou a expressão de mRNA e proteica de leptina com relação ao C, e com a inibição da integrina αvβ3 e posterior tratamento com o hormônio (Tetrac+T3), houve diminuição da expressão em ambos casos. Já a expressão de mRNA de adiponectina não foi alterada pelo T3 quando comparado ao C, contudo o hormônio aumentou a expressão proteica dessa adipocina (1,7 ± 0,15, p<0,01) em relação ao C (1 ± 0,03, p<0,01), este aumento foi abolido pelos inibidores associados ao T3, PD +T3 (0,59 ± 0,11, p<0,01) e Tetrac+T3 (0,59 ± 0,1, p<0,01). Embora o T3 não tenha alterado a expressão proteica de PPARy, acúmulo lipídico, dosagem de TGL, liberação de glicerol e dano de DNA em relação ao C, os grupos PD+T3 e Tetrac+T3, apresentaram redução de todos os parâmetros mencionados anteriormente, quando comparados ao T3 sozinho. Conclusão: O T3 age nos adipócitos por meio da via MAPK/ERK, para modular a expressão proteica de adiponectina e pela integrina αvβ3 para alterar a expressão gênica de leptina. Além disso, a integridade da via MAPK/ERK é necessária para que o T3 não altere o perfil lipídico, mantendo a homeostase dos adipócitos.
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 AdipoRed and Oil Red assays, triacylglycerol release (TGL) evaluation by the enzymatic-colorimetric method, the measurement of the lipolysis product by the glycerol test; DNA damage by quantification of 8-Hydroxyideoxyguanosine (80-OHdG) per ELISA assay. ANOVA supplemented with the Tukey test was used for data with normality and Friedman's test, complemented by the Dunn test, for data with no 8 normality. Results expressed in mean ± standard deviation and median (IQ 25% - IQ 75%), respectively. Significance given for p <0.05. Results: T3 increased the expression of mRNA and leptin protein with respect to C, and with inhibition of αvβ3 integrin and subsequent treatment with the hormone, Tetrac + T3, there was a decrease in expression in both cases, indicating that T3 binding the integrin is important for gene modulation of leptin. However, the expression of adiponectin mRNA was not altered by T3 when compared to C, however the hormone increased the protein expression of this adipocin (1.7 ± 0.15, p <0.01) in relation to C (1 ± 0, (P <0.01), and Tetrac + T3 (0.59 ± 0.1, p <0.01) were abolished by the inhibitors associated with T3, PD + T3 (0.59 ± 0.11, p < p <0.01), indicating activation of the T3 pathways for post-transcitional action of adiponectin. Although T3 did not alter the PPARγ protein expression, lipid accumulation, TGL dosage, glycerol release and DNA damage in relation to C, the groups PD + T3 and Tetrac + T3 showed reduction of all parameters mentioned previously, when compared to T3. Conclusion: T3 acts on adipocytes via the MAPK / ERK pathway to modulate the protein expression of adiponectin and αvβ3 integrin to alter the gene expression of leptin. In addition, the integrity of the MAPK / ERK pathway is required for T3 to not alter the lipid profile, maintaining adipocyte homeostasis
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 AdipoRed and Oil Red assays, triacylglycerol release (TGL) evaluation by the enzymatic-colorimetric method, the measurement of the lipolysis product by the glycerol test; DNA damage by quantification of 8-Hydroxyideoxyguanosine (80-OHdG) per ELISA assay. ANOVA supplemented with the Tukey test was used for data with normality and Friedman's test, complemented by the Dunn test, for data with no 8 normality. Results expressed in mean ± standard deviation and median (IQ 25% - IQ 75%), respectively. Significance given for p <0.05. Results: T3 increased the expression of mRNA and leptin protein with respect to C, and with inhibition of αvβ3 integrin and subsequent treatment with the hormone, Tetrac + T3, there was a decrease in expression in both cases, indicating that T3 binding the integrin is important for gene modulation of leptin. However, the expression of adiponectin mRNA was not altered by T3 when compared to C, however the hormone increased the protein expression of this adipocin (1.7 ± 0.15, p <0.01) in relation to C (1 ± 0, (P <0.01), and Tetrac + T3 (0.59 ± 0.1, p <0.01) were abolished by the inhibitors associated with T3, PD + T3 (0.59 ± 0.11, p < p <0.01), indicating activation of the T3 pathways for post-transcitional action of adiponectin. Although T3 did not alter the PPARγ protein expression, lipid accumulation, TGL dosage, glycerol release and DNA damage in relation to C, the groups PD + T3 and Tetrac + T3 showed reduction of all parameters mentioned previously, when compared to T3. Conclusion: T3 acts on adipocytes via the MAPK / ERK pathway to modulate the protein expression of adiponectin and αvβ3 integrin to alter the gene expression of leptin. In addition, the integrity of the MAPK / ERK pathway is required for T3 to not alter the lipid profile, maintaining adipocyte homeostasis
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Palavras-chave
Adiponectina, Leptina, Triiodotironina, MAPK/ERK, Integrina αvβ3, 3T3-L1, Adiponectin, Leptin, integrin αvβ3