Efeitos da dieta hiperlipídica sobre a expressão de receptores de leptina e sua ação sobre modificações epigenéticas em células mesenquimais multipotentes extraídas do tecido adiposo de camundongos.
Data
2022-12-20
Autores
Bispo, Andressa França de Sousa 
Orientadores
Vale, Maria Isabel Cardoso Alonso
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Uma dieta hiperlipídica leva à obesidade, uma condição que gera importantes alterações no tecido adiposo branco (TAB). As células tronco mesenquimais derivadas do tecido adiposo (Adipose derivedStromalCells – ASCs), são responsáveis por gerar novos adipócitos, expandindo o tecido adiposo. Alterações na expressão de fatores de transcrição e de marcas epigenéticas no núcleo de ASCs podem ter efeitos na proliferação e na adipogênese dessas células afetando a expansãosaudável (hiperplásica) do TAB versus expansão patológica (hipertrófica) do TAB em indivíduos obesos. A leptina é uma adipocina com efeitos no sistema nervoso central, que incluem a indução do gasto energético e da saciedade. Também, tem grande relevância na ativação de células imunes e na modulação da resposta inflamatória. Sabe-se que, na obesidade, os efeitos centrais da leptina são prejudicados, pois os aumentos no acúmulo de TAB e nos níveis de leptina são acompanhados por resistência hipotalâmica à leptina, assim, seus efeitos locais podem prevalecer e contribuir para a expansão do TAB e aumento da inflamação. Esse fenômeno tem sido considerado resistência à leptina e pode ser resultado de deficiências em vários níveis na via de sinalização da leptina, incluindo acesso reduzido do hormônio ao seu receptor devido a alterações na expressão do receptor e/ ou alterações na transdução de sinal pós-receptor. O TAB é um ambiente rico em leptina, mas as ações locais da leptina sobre a adipogênese não foram completamente investigadas. Assim, se faz necessário avaliar as contribuições da sinalização parácrina da leptina e de seus receptores na obesidade, em células mesenquimais multipotentes do TAB, objetivo do presente projeto. Considerando que as ASCs são células progenitoras que residem em um nicho altamente enriquecido em leptina (secretadas por adipócitos) no TAB de obesos, hipotetizamos que a obesidade (via ação da leptina em ASCs), levaria a importantes alterações nas enzimas promotoras de marcas epigenéticas, que culmina na queda da expressão de alguns genes comumente silenciados na obesidade, especialmente aqueles relacionados à adipogênese. Para estudarmos estas alterações avaliamos a expressão do receptor de leptina por PCR em Tempo Real, em ASCs isoladas do TAB subcutâneo e epididimal de animais controles e obesos (induzidos por dieta hiperlipídica), a fim de correlacionar os dados com demais resultados do grupo. Adicionalmente, tratamos ASCs de obesos com leptina a fim de entender seu efeito
parácrino (in vitro) sobre estas mesmas marcas. Nossos resultados mostraram que os animais obesos (induzidos pela dieta hiperlipídica DHL) apresentaram um aumento de ~30% na massa corpórea, maior glicemia de jejum (~50%) e intolerância à glicose. A expressão de ambos os receptores Lepr1 e Lepr2 foram diminuídos em ~40 e 45%, respectivamente, em ASCs de obesos em comparação aos animais controle. A leptina (como esperado) não foi expressa nestas células, tal qual LepR3. Detectamos uma diminuição na expressão da H3K27 acetilada nas ASCs de obesos, e da Acly, que parece ter um importante papel na acetilação da histona 3. Essa marca epigenética contribui para a ativação da transcrição de genes ligados a adipogênese. Adicionalmente, a expressão da Acly e da enzima demetilase Kdm6b (enzima que remove grupos metil para posterior ação de acetilases) diminuiu quando ASCs foram tratadas in vitro com leptina. Assim, sugerimos que a sinalização aumentada de leptina em ASCs diminui a expressão de ATP Citrato Liase, levando à redução de substrato para acetilação de H3K27 o que pode contribuir para o silenciamento de genes comumente observados na obesidade.
A high-fat diet leads to obesity, a condition that generates important changes in white adipose tissue (WAT). Mesenchymal stem cells derived from adipose tissue (Adipose derived Stromal Cells - ASCs) are responsible for generating new adipocytes, expanding adipose tissue. Alterations in the expression of transcription factors and epigenetic marks in the nucleus of ASCs may have effects on the proliferation and adipogenesis of these cells, affecting healthy (hyperplastic) WAT expansion versus pathological (hypertrophic) WAT expansion in obese individuals. Leptin is an adipokine with effects on the central nervous system, which include the induction of energy expenditure and satiety. Also, it has great relevance in the activation of immune cells and in the modulation of the inflammatory response. It is known that, in obesity, the central effects of leptin are impaired, as increases in WAT accumulation and in leptin levels are accompanied by hypothalamic resistance to leptin, thus, its local effects may prevail and contribute to the expansion of WAT and increased inflammation. This phenomenon has been considered leptin resistance and may be a result of deficiencies at various levels in the leptin signaling pathway, including reduced access of the hormone to its receptor due to alterations in receptor expression and/or alterations in post-receptor signal transduction. . WAT is a leptin-rich environment, but the local actions of leptin on adipogenesis have not been fully investigated. Thus, it is necessary to evaluate the contributions of leptin paracrine signaling and its receptors in obesity, in multipotent mesenchymal cells of the WAT, the objective of this project. Considering that ASCs are progenitor cells residing in a niche highly enriched in leptin (secreted by adipocytes) in the WAT of obese people, we hypothesize that obesity (via the action of leptin in ASCs) would lead to important alterations in the enzymes that promote epigenetic marks, which culminates in a drop in the expression of some genes commonly silenced in obesity, especially those related to adipogenesis. To study these alterations, we evaluated the expression of the leptin receptor by Real Time PCR, in ASCs isolated from the subcutaneous and epididymal WAT of control and obese animals (induced by a high-fat diet), in order to correlate the data with the other results of the group. Additionally, we treated obese ASCs with leptin in order to understand its paracrine effect (in vitro) on these same marks. Our results showed that obese animals (induced by the high-fat DHL diet) showed an increase of ~30% in body mass, higher fasting glycemia (~50%) and glucose intolerance. Expression of both Lepr1 and Lepr2 receptors was decreased by ~40 and 45%, respectively, in ASCs from obese compared to control animals. Leptin (as expected) was not expressed in these cells, as was LepR3. We detected a decrease in the expression of acetylated H3K27 in obese ASCs, and of Acly, which seems to play an important role in histone 3 acetylation. This epigenetic marker contributes to the activation of transcription of genes linked to adipogenesis. Additionally, the expression of Acly and the Kdm6b demethylase enzyme (an enzyme that removes methyl groups for subsequent action of acetylases) decreased when ASCs were treated in vitro with leptin. Thus, we suggest that increased leptin signaling in ASCs decreases the expression of ATP Citrate Lyase, leading to reduced substrate for H3K27 acetylation, which may contribute to the silencing of genes commonly observed in obesity.
A high-fat diet leads to obesity, a condition that generates important changes in white adipose tissue (WAT). Mesenchymal stem cells derived from adipose tissue (Adipose derived Stromal Cells - ASCs) are responsible for generating new adipocytes, expanding adipose tissue. Alterations in the expression of transcription factors and epigenetic marks in the nucleus of ASCs may have effects on the proliferation and adipogenesis of these cells, affecting healthy (hyperplastic) WAT expansion versus pathological (hypertrophic) WAT expansion in obese individuals. Leptin is an adipokine with effects on the central nervous system, which include the induction of energy expenditure and satiety. Also, it has great relevance in the activation of immune cells and in the modulation of the inflammatory response. It is known that, in obesity, the central effects of leptin are impaired, as increases in WAT accumulation and in leptin levels are accompanied by hypothalamic resistance to leptin, thus, its local effects may prevail and contribute to the expansion of WAT and increased inflammation. This phenomenon has been considered leptin resistance and may be a result of deficiencies at various levels in the leptin signaling pathway, including reduced access of the hormone to its receptor due to alterations in receptor expression and/or alterations in post-receptor signal transduction. . WAT is a leptin-rich environment, but the local actions of leptin on adipogenesis have not been fully investigated. Thus, it is necessary to evaluate the contributions of leptin paracrine signaling and its receptors in obesity, in multipotent mesenchymal cells of the WAT, the objective of this project. Considering that ASCs are progenitor cells residing in a niche highly enriched in leptin (secreted by adipocytes) in the WAT of obese people, we hypothesize that obesity (via the action of leptin in ASCs) would lead to important alterations in the enzymes that promote epigenetic marks, which culminates in a drop in the expression of some genes commonly silenced in obesity, especially those related to adipogenesis. To study these alterations, we evaluated the expression of the leptin receptor by Real Time PCR, in ASCs isolated from the subcutaneous and epididymal WAT of control and obese animals (induced by a high-fat diet), in order to correlate the data with the other results of the group. Additionally, we treated obese ASCs with leptin in order to understand its paracrine effect (in vitro) on these same marks. Our results showed that obese animals (induced by the high-fat DHL diet) showed an increase of ~30% in body mass, higher fasting glycemia (~50%) and glucose intolerance. Expression of both Lepr1 and Lepr2 receptors was decreased by ~40 and 45%, respectively, in ASCs from obese compared to control animals. Leptin (as expected) was not expressed in these cells, as was LepR3. We detected a decrease in the expression of acetylated H3K27 in obese ASCs, and of Acly, which seems to play an important role in histone 3 acetylation. This epigenetic marker contributes to the activation of transcription of genes linked to adipogenesis. Additionally, the expression of Acly and the Kdm6b demethylase enzyme (an enzyme that removes methyl groups for subsequent action of acetylases) decreased when ASCs were treated in vitro with leptin. Thus, we suggest that increased leptin signaling in ASCs decreases the expression of ATP Citrate Lyase, leading to reduced substrate for H3K27 acetylation, which may contribute to the silencing of genes commonly observed in obesity.