Análise proteômica do músculo esquelético de pacu (Piaractus mesopotamicus) após jejum prolongado e durante crescimento compensatório.
Carregando...
Data
2018-07-26
Autores
Silva-Gomes, Rafaela Nunes da
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A musculatura esquelética em peixes corresponde mais de 70% do peso corporal total desses animais e o desenvolvimento, crescimento e manutenção do fenótipo muscular envolvem o balanço dinâmico entre as vias de anabolismo e catabolismo muscular, sendo influenciado por diversos fatores, como a disponibilidade alimentar. Períodos com baixa ou nenhuma disponibilidade de alimento é frequentemente observado na natureza e talvez, por isso, garantem aos peixes uma alta capacidade adaptativa para enfrentar períodos de jejum prolongado. Com a realimentação e os níveis metabólicos restabelecidos, o animal pode apresentar um processo de crescimento acelerado, chamado crescimento compensatório. Existe um grande interesse da aquicultura nos processos de crescimento compensatório após jejum, uma vez que bem estabelecido pode gerar diminuição do custo com a alimentação e aumento dos níveis de produção. Uma ferramenta para o estudo do músculo de peixes durante períodos de jejum e realimentação é a proteômica. A proteômica permite identificar o conjunto de proteínas expressas por uma célula ou tecido em determinadas situações e modificações ambientais e possui a vantagem de caracterizar eventos pós-traducionais, algo impossibilitado pela genômica e transcriptoma. Dessa forma, nós testamos a hipótese de que juvenis de pacu (Piaractus mesopotamicus) submetidos a 30 dias de jejum e 30 e 60 dias de realimentação apresentam crescimento compensatório e alterações no perfil de expressão proteico da musculatura esquelética. Para isso dois grupos de animais foram utilizados: grupo Controle (C) alimentado continuamente por 90 dias e grupo experimental (E), submetidos a 30 dias de jejum e 60 dias de realimentação. Amostras do músculo branco foram coletadas após 30 dias de jejum e após 6, 24, 48 horas, 30 e 60 dias de realimentação. O peso corporal total, comprimento padrão, taxa de crescimento, morfologia e morfometria das fibras musculares foram analisados, assim como o proteoma do músculo branco de pacu. Nós demonstramos que os peixes apresentaram atrofia muscular após 30 dias de jejum, crescimento compensatório parcial após 30 dias de realimentação e crescimento compensatório total após 60 dias de realimentação. A análise proteômica sob estratégia de shotgun identificou 99 proteínas após jejum, sendo 21 proteínas diferencialmente expressas entre o grupo E e C e 71 proteínas após 30 dias de realimentação, com 14 proteínas diferencialmente expressas. Entre as proteínas com expressão diferencial, nós validamos a expressão gênica e proteica para parvalbumina que mostrou-se diminuída durante atrofia muscular no músculo branco de pacu após 30 dias de jejum e 30 dias de realimentação. A análise proteômica por 2D-PAGE demonstrou diminuição no número de spots proteicos identificados após jejum e aumento no número de spots proteicos após 30 e 60 dias de realimentação nos géis do grupo E. Os spots proteicos mais signficativos foram extraídos e caracterizados por espetrometria de massas. As proteínas caracterizadas foram classificadas em vias envolvidas, principalmente, no anabolismo e catabolismo muscular, enzimas do metabolismo celular, biosíntese de aminoácidos, citoesqueleto e contração muscular, que foram profundamente afetadas pelo jejum e crescimento compensatório. Além disso, o período de jejum prolongado promoveu intenso estresse celular e identificou proteínas que podem estar relacionadas com mecanismos para compensação de danos negativos. Dessa forma, esse estudo pode auxiliar na identificação de possíveis biomarcadores para monitoramento do bem-estar animal, qualidade da carne e segurança alimentar, além de melhorar o entendimento das necessidades alimentícias e do impacto da dieta administrada à produção.
The skeletal muscle in fish corresponds more than 70% of the total body weight and the development, growth and maintenance of the muscle phenotype involves the dynamic balance between muscle anabolism and catabolism pathways, being influenced by several factors, such as food availability. Periods with low or no food availability are often observed in nature and may therefore, ensure a high adaptive capacity in fish to deal with prolonged fasting periods. With refeeding and metabolic levels restored, the animal may exhibit accelerated growth, called compensatory growth. There is a great interest of aquaculture in the processes of fasting/refeeding, once well-established can generates decrease of the feeding costs and increase of the production levels. An important tool to analyze muscle fish during fasting/refeeding is Proteomics. The proteomics allows to identify a set of proteins expressed by a cell or tissue in some situations and environmental changes and has the advantage of characterizing post-translational events, something impossible to detect by genomic and transcriptome analysis. This way, we tested the hypothesis that juveniles pacu (Piaractus mesopotamicus) submitted to 30 days of fasting and 30 and 60 days of refeeding present compensatory growth and alterations in the protein expression profile of the skeletal muscle. For this, we used two animals groups: Control group (C) fed continuously for 90 days and experimental group (E), submitted to 30 days of fasting followed by 60 days of refeeding. White muscle samples were collected after 30 days of fasting and after 6, 24, 48 hours, 30 and 60 days of refeeding. The total body weight, standard length, growth rate, morphology and morphometry of the muscle fibers were analyzed, as well as the white muscle proteome of pacu.We demonstrated that fish presented muscle atrophy after 30 days of fasting, partial compensatory growth after 30 days of refeeding and total compensatory growth after 60 days of refeeding. Proteomic analysis by shotgun aproach identified 99 proteins after fasting, being 21 differentially expressed between E and Cgroup and 71 proteins after 30 days of refeeding, being 14 differentially expressed. Among the differential expression proteins, we validated gene and protein expression for parvalbumin that was down-regulated during muscle atrophy in white muscle of pacu after 30 days of fasting and 30 days of refeeding. Proteomic analysis by 2D-PAGE demonstrated decreased in the number of protein spots identified after fasting and increased number after refeeding in the gels of E group. The most significant protein spots were extracted and characterized to mass spectrometry. The proteins characterized were classified into pathways mainly involved in muscle anabolism and catabolism, cellular metabolism enzymes, amino acid biosynthesis, cytoskeleton and muscle contraction, and they were deeply affected by fasting and compensatory growth. In addition, the prolonged fasting period promoted intense cellular stress and presented proteins that may be related to compensation mechanisms for negative damages. Thus, this study can help identify possible biomarkers for monitoring animal welfare, meat quality and food safety, as well as, improving the understanding of food needs and the diet impact into aquaculture.
The skeletal muscle in fish corresponds more than 70% of the total body weight and the development, growth and maintenance of the muscle phenotype involves the dynamic balance between muscle anabolism and catabolism pathways, being influenced by several factors, such as food availability. Periods with low or no food availability are often observed in nature and may therefore, ensure a high adaptive capacity in fish to deal with prolonged fasting periods. With refeeding and metabolic levels restored, the animal may exhibit accelerated growth, called compensatory growth. There is a great interest of aquaculture in the processes of fasting/refeeding, once well-established can generates decrease of the feeding costs and increase of the production levels. An important tool to analyze muscle fish during fasting/refeeding is Proteomics. The proteomics allows to identify a set of proteins expressed by a cell or tissue in some situations and environmental changes and has the advantage of characterizing post-translational events, something impossible to detect by genomic and transcriptome analysis. This way, we tested the hypothesis that juveniles pacu (Piaractus mesopotamicus) submitted to 30 days of fasting and 30 and 60 days of refeeding present compensatory growth and alterations in the protein expression profile of the skeletal muscle. For this, we used two animals groups: Control group (C) fed continuously for 90 days and experimental group (E), submitted to 30 days of fasting followed by 60 days of refeeding. White muscle samples were collected after 30 days of fasting and after 6, 24, 48 hours, 30 and 60 days of refeeding. The total body weight, standard length, growth rate, morphology and morphometry of the muscle fibers were analyzed, as well as the white muscle proteome of pacu.We demonstrated that fish presented muscle atrophy after 30 days of fasting, partial compensatory growth after 30 days of refeeding and total compensatory growth after 60 days of refeeding. Proteomic analysis by shotgun aproach identified 99 proteins after fasting, being 21 differentially expressed between E and Cgroup and 71 proteins after 30 days of refeeding, being 14 differentially expressed. Among the differential expression proteins, we validated gene and protein expression for parvalbumin that was down-regulated during muscle atrophy in white muscle of pacu after 30 days of fasting and 30 days of refeeding. Proteomic analysis by 2D-PAGE demonstrated decreased in the number of protein spots identified after fasting and increased number after refeeding in the gels of E group. The most significant protein spots were extracted and characterized to mass spectrometry. The proteins characterized were classified into pathways mainly involved in muscle anabolism and catabolism, cellular metabolism enzymes, amino acid biosynthesis, cytoskeleton and muscle contraction, and they were deeply affected by fasting and compensatory growth. In addition, the prolonged fasting period promoted intense cellular stress and presented proteins that may be related to compensation mechanisms for negative damages. Thus, this study can help identify possible biomarkers for monitoring animal welfare, meat quality and food safety, as well as, improving the understanding of food needs and the diet impact into aquaculture.
Descrição
Palavras-chave
atrofia muscular, crescimento muscular, biomarcador, parvalbumina, proteoma muscular