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O papel da via de sinalização Wnt associado aos cílios primários na modulação do metabolismo de glucose
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Abstract(s)
Os cílios primários são organelos especializados, semelhantes a antenas, localizados na superfície das células eucariontes. Com a capacidade de regular inúmeras funções relevantes, como a sinalização e homeostasia celular, o seu mau funcionamento torna-se responsável por vários distúrbios e a caracterizar um grupo de doenças: as Ciliopatias. Além disso, disfunções nos cílios primários têm sido associadas a distúrbios metabólicos, incluindo resistência insulínica e o desenvolvimento da diabetes. O presente trabalho teve como objetivo avaliar o papel dos cílios primários na resposta a níveis elevados de glucose em células hTERT-RPE-1. Foi também alvo do estudo a avaliação da via de sinalização Wnt, associada aos cílios primários e sua repercussão nestas condições. Assim sendo, foi-se estudado os níveis celulares de um dos elementos presentes nesta via, a proteína mediadora de transdução da sinalização DVL1. Para alcançar as metas propostas, organizamos o projeto da seguinte forma: As células hTERT-RPE-1 permaneceram por 48 horas em meio 5 mM de glucose. Após este período as mesmas células foram expostas a diferentes concentrações de glucose (5 mM, 25 mM e 5 mM glucose +20mM de manitol). No primeiro grupo de células fez-se a indução da montagem de cílios 24 horas antes do segundo grupo de células, sendo a montagem dessas estruturas neste grupo induzida após a exposição à glucose. Para esta indução foi feita a remoção do soro do meio celular utilizado. Observamos que a presença do cílio primário poderia estar relacionada ao maior nível de stress celular, proporcionalmente a variação dos níveis de DVL1 a nível nuclear, visualizados pela técnica de Imunofluorescência Indireta. Adicionalmente, analisamos através da técnica de PCR a expressão dos genes-alvo Myc, Otx2 e Mitf, responsáveis pela ativação ou supressão de diferentes processos biológicos relacionados a via de sinalização Wnt. Este trabalho evidencia que o cílio primário tem importância como reguladora da homeostase em resposta aos altos níveis de glucose nas células hTERT-RPE-1.
ABSTRACT Primary cilia are specialized, antenna-like sensory organelles located on the surface of eukaryotic cells. With the ability to regulate numerous relevant functions, such as cell signaling and homeostasis, its malfunction becomes responsible for several disorders and characterizes a group of diseases: Ciliopathies. Furthermore, dysfunctions in primary cilia have been associated with metabolic disorders, including insulin resistance and the development of diabetes. The present work aimed to evaluate the role of primary cilia in the response to high glucose levels in hTERT-RPE-1 cells. The aim of the study was also to evaluate the Wnt signaling pathway, associated with primary cilia, and its repercussions in these conditions. Therefore, we studied the cellular levels of one of the elements present in this pathway, the DVL1 signaling transduction mediator protein. To achieve the proposed goals, we organized the project as follows: hTERT-RPE-1 cells remained for 48 hours in 5 mM glucose medium. After this period, the same cells were exposed to different concentrations of glucose (5 mM, 25 mM, and 5 mM glucose + 20 mM mannitol). In the first group of cells, cilia assembly was induced 24 hours before the second group of cells, and the assembly of these structures in this group was induced after exposure to glucose. For this induction, serum was removed from the cell medium used. We observed that the presence of the primary cilium could be related to the higher level of cellular stress, proportionally to the variation in DVL1 levels at the perinuclear and nuclear level, visualized by the Indirect Immunofluorescence technique. Additionally, we analyzed, using the PCR technique, the expression of the target genes Myc, OTX2, and Mitf-Human, responsible for the activation or suppression of different biological processes related to the Wnt signaling pathway. This work shows that the primary cilium is important as a regulator of homeostasis in response to high glucose levels in hTERT-RPE-1 cells.
ABSTRACT Primary cilia are specialized, antenna-like sensory organelles located on the surface of eukaryotic cells. With the ability to regulate numerous relevant functions, such as cell signaling and homeostasis, its malfunction becomes responsible for several disorders and characterizes a group of diseases: Ciliopathies. Furthermore, dysfunctions in primary cilia have been associated with metabolic disorders, including insulin resistance and the development of diabetes. The present work aimed to evaluate the role of primary cilia in the response to high glucose levels in hTERT-RPE-1 cells. The aim of the study was also to evaluate the Wnt signaling pathway, associated with primary cilia, and its repercussions in these conditions. Therefore, we studied the cellular levels of one of the elements present in this pathway, the DVL1 signaling transduction mediator protein. To achieve the proposed goals, we organized the project as follows: hTERT-RPE-1 cells remained for 48 hours in 5 mM glucose medium. After this period, the same cells were exposed to different concentrations of glucose (5 mM, 25 mM, and 5 mM glucose + 20 mM mannitol). In the first group of cells, cilia assembly was induced 24 hours before the second group of cells, and the assembly of these structures in this group was induced after exposure to glucose. For this induction, serum was removed from the cell medium used. We observed that the presence of the primary cilium could be related to the higher level of cellular stress, proportionally to the variation in DVL1 levels at the perinuclear and nuclear level, visualized by the Indirect Immunofluorescence technique. Additionally, we analyzed, using the PCR technique, the expression of the target genes Myc, OTX2, and Mitf-Human, responsible for the activation or suppression of different biological processes related to the Wnt signaling pathway. This work shows that the primary cilium is important as a regulator of homeostasis in response to high glucose levels in hTERT-RPE-1 cells.
Description
Mestrado em Tecnologias Clínico-Laboratoriais
Keywords
Cílio Glucose DVL1 Myc Otx2 Mitf MTCL IPL/IDI&CA2024/HyperCil_ESTeSL IPL/IDI&CA2024/ENIGMA_ESTeSL
Citation
Cunha MC. O papel da via de sinalização Wnt associada aos cílios primários na modulação do metabolismo da glucose [dissertation]. Lisboa: Escola Superior de Tecnologia da Saúde de Lisboa/Instituto Politécnico de Lisboa; 2024.