Dietary Leucine Supplementation Minimises Tumour-induced Damage In Placental Tissues Of Pregnant, Tumour-bearing Rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The occurrence of cancer during pregnancy merges two complex, poorly understood metabolic and hormonal conditions. This association can exacerbate the conditions of both the mother and the foetus. The branched-chain amino acid leucine enhances cellular activity, particularly by increasing protein synthesis. This study aimed to analyse the modulatory effect of a leucine-rich diet on direct and indirect tumour-induced placental damage. This was accomplished by evaluating the expression of genes involved in protein synthesis and degradation and assessing anti-oxidant enzyme activity in placental tissues collected from pregnant, tumour-bearing rats. Results: Pregnant rats were either implanted with Walker 256 tumour cells or injected with ascitic fluid (to study the indirect effects of tumour growth) and then fed a leucine-rich diet. Animals in a control group underwent the same procedures but were fed a normal diet. On the 20th day of pregnancy, tumour growth was observed. Dams fed a normoprotein diet showed the greatest tumour growth. Injection with ascitic fluid mimicked the effects of tumour growth. Decreased placental protein synthesis and increased protein degradation were observed in both the tumour-bearing and the ascitic fluid-injected groups that were fed a normoprotein diet. These effects resulted in low placental DNA and protein content and high lipid peroxidation (measured by malondialdehyde content). Decreased placental protein synthesis-related gene expression was observed in the tumour group concomitant with increased expression of genes encoding protein degradation-associated proteins and proteolytic subunits. Conclusions: Consumption of a leucine-rich diet counteracted the effects produced by tumour growth and injection with ascitic fluid. The diet enhanced cell signalling, ameliorated deficiencies in DNA and protein content, and balanced protein synthesis and degradation processes in the placenta. The improvements in cell signalling included changes in the mTOR/eIF pathway. In conclusion, consumption of a leucine-rich diet improved placental metabolism and cell signalling in tumour-bearing rats, and these changes reduced the deleterious effects caused by tumour growth.1658FAPESP [2010/00209-9, 2011/08276-0, 2013/16115-1]CNPq [302863/2013-3]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Dietary leucine supplementation minimises tumour-induced damage in placental tissues of pregnant, tumour-bearing rats

Background: The occurrence of cancer during pregnancy merges two complex, poorly understood metabolic and hormonal conditions. This association can exacerbate the conditions of both the mother and the foetus. The branched-chain amino acid leucine enhances cellular activity, particularly by increasing protein synthesis. This study aimed to analyse the modulatory effect of a leucine-rich diet on direct and indirect tumour-induced placental damage. This was accomplished by evaluating the expression of genes involved in protein synthesis and degradation and assessing anti-oxidant enzyme activity in placental tissues collected from pregnant, tumour-bearing rats. Results: Pregnant rats were either implanted with Walker 256 tumour cells or injected with ascitic fluid (to study the indirect effects of tumour growth) and then fed a leucine-rich diet. Animals in a control group underwent the same procedures but were fed a normal diet. On the 20th day of pregnancy, tumour growth was observed. Dams fed a normoprotein diet showed the greatest tumour growth. Injection with ascitic fluid mimicked the effects of tumour growth. Decreased placental protein synthesis and increased protein degradation were observed in both the tumour-bearing and the ascitic fluid-injected groups that were fed a normoprotein diet. These effects resulted in low placental DNA and protein content and high lipid peroxidation (measured by malondialdehyde content). Decreased placental protein synthesis-related gene expression was observed in the tumour group concomitant with increased expression of genes encoding protein degradation-associated proteins and proteolytic subunits. Conclusions: Consumption of a leucine-rich diet counteracted the effects produced by tumour growth and injection with ascitic fluid. The diet enhanced cell signalling, ameliorated deficiencies in DNA and protein content, and balanced protein synthesis and degradation processes in the placenta. The improvements in cell signalling included changes in the mTOR/eIF pathway. In conclusion, consumption of a leucine-rich diet improved placental metabolism and cell signalling in tumour-bearing rats, and these changes reduced the deleterious effects caused by tumour growth16CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP302863/2013-32010/00209-9; 2011/08276-0; 2013/16115-

L-leucine-rich diet modulates muscle cell signalling pathway of protein metabolism in Walker 256 tumour-bearing rats

Orientador: Maria Cristina Cintra Gomes MarcondesTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O câncer é uma das principais causas de morte no mundo e o quadro de caquexia provocado por alguns tipos de tumor é um dos grandes responsáveis por isso. A caquexia instaurada em pacientes com câncer, sendo mais prevalente nos tumores gastrointestinal, pulmonar, pancreático e mamário, é caracterizada, dentre outros processos, pela perda involuntária de peso, devido a constante espoliação sobre a massa magra corporal. Estudos que tenham como objetivo a manutenção da massa magra em organismos portadores de tumor caquético são importantes para contribuir com a redução de óbitos e preservar a qualidade de vida das pessoas com câncer. Nos últimos anos, a leucina tem mostrado ser eficaz na manutenção da massa magra corpórea através do estímulo de síntese protéica muscular e inibição da degradação de proteína, principalmente da massa magra corpórea. Logo, entender como a presença da leucina estimula a síntese protéica e atua de forma protetora em organismo no estado caquético tem se mostrado uma necessidade crescente. Desse modo, a proposta deste trabalho foi avaliar, ao longo do tempo, os efeitos de dieta rica em leucina sobre a sinalização de síntese e degradação protéica, envolvendo o complexo mTOR em músculos de ratos portadores do carcinossarcoma de Walker 256. Os animais foram distribuídos em grupos de acordo com a inoculação tumoral e/ou esquema nutricional com dieta rica em leucina, sendo sacrificados em três momentos diferentes do desenvolvimento tumoral (7º, 14º e 21º dias após a implantação do tumor). No músculo gastrocnêmio foram analisadas as proteínas-chave da via mTOR, como RAG A GTPase, ERK/MAP4K3, PKB/Akt, mTOR, p70S6K1, Jnk, IRS-1, STAT3, e STAT6 e, tambem, foram avaliadas as proteínas de degradação protéica pertencentes ao sistema ubiquitina-proteossomo (11S, 19S e 20S) e citocinas IL-4, IL-6, IL-10, TNF? e INF?. Os resultados mostram que o desenvolvimento tumoral reduziu a ativação de RAG-A, associada com queda de IRS-1, aumento da PKB/Akt e Erk/MAP4K3 no 21º dia e manutenção de p70S6K1; também houve aumento dos níveis de STAT-3 e STAT-6 em ratos portadores de tumor. Entretanto, a presença de leucina na dieta modulou etapas chave da via mTOR pelo desencadeamento da ativação aumentada de RAG-A e mTOR junto com a manutenção dos níveis de JNK, STAT-3 e STAT-6 no músculo durante o desenvolvimento do tumor de Walker no organismo caquético. A análise da sinalização para degradação protéica mostrou que o crescimento tumoral promoveu, simultaneamente, diminuição de proteína muscular, acentuado aumento de citocinas pró-inflamatórias (TNF?, IL-6 e IFN?) e aumento progressivo das subunidades proteossômicas (19S e 20S), sendo que a suplementação com leucina atenuou essa ativação. Os resultados obtidos apoiam o efeito benéfico do uso de leucina e esclarece as vias metabólicas utilizadas por este aminoácido, contribuindo para melhor compreensão da ação in vivo desse aminoácido sobre a caquexiaAbstract: Cancer is one of the most important causes of death worldwide and the process of cachexia caused by some types of tumour is largely responsible for this. Cancer-cachexia state established in these patients is characterized, among other processes, by involuntary loss of weight due to constant spoliation of lean body mass. Many studies aim to focus in maintenance of lean body mass in cachectic tumour-bearing host, contributing to the reduction of deaths and improving the quality of life of cancer patients. In recent years, leucine has been shown to be effective in maintaining lean body mass by stimulating muscle protein synthesis and inhibiting the proteolysis. Therefore, there are increased needs in understanding how the presence of leucine stimulates protein synthesis and acts protectively in the cachectic state. The aim of this work is to evaluate the effects of leucine-rich diet in a time-course model on signalling protein synthesis and degradation involving mTOR complex in muscles of Walker 256 carcinoma-bearing rats. Animals were divided into experimental groups based on the tumour inoculation and/or fed a nutritional supplementation diet rich in leucine. Animals were sacrificed at three different times depending on the tumour development (7, 14 and 21 days after tumour implantation), and the gastrocnemius muscles were analysed as mTOR pathway and ubiquitin-proteasome via. The results showed that the tumour development has reduced the activation of RAG-A, associated with a decrease of IRS-1, increased PKB / Akt and Erk / MAP4K3 at day 21 and maintaining p70S6K1, there has also been increasing levels of STAT-3 and STAT-6 in tumour-bearing rats. Meanwhile, the presence of leucine in the diet modulated the key steps of the mTOR pathway for triggering the increased RAG-A and mTOR activation along with the maintenance of levels of JNK, STAT-3 and STAT-6 in the muscle during tumour development in cachectic host. The gastrocnemius muscle signalling of protein degradation indicated by the ubiquitin-proteasome subunits (11S, 19S and 20S) and pro- and anti-inflammatory cytokines were marked increase of pro-inflammatory cytokines (TNF?, IL-6 and IFN?) and a progressive increase in the proteasome subunits (19S and 20S) associated with simultaneously decreased muscle protein. The supplementation with leucine attenuated these parameters, suggesting the beneficial effect of the use of leucine and clarifies the metabolic pathways used by this amino acid, contributing to better understanding of the in vivo action of this amino acid on cachexiaDoutoradoFisiologiaDoutor em Biologia Funcional e Molecula

Protein metabolism and oxidative stres in fetal muscle proceeding from tumour-bearing rats fed a leucine-rich diet

Orientador: Maria Cristina Cintra Gomes MarcondesDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Câncer acomete, aproximadamente, uma entre 3000 gravidezes e causa um terço de mortes maternas. A leucina é usada experimentalmente para minimizar as alterações do metabolismo protéico durante o crescimento tumoral. Este trabalho investigou os efeitos do crescimento tumoral sobre o metabolismo protéico e estresse oxidativo nos fetos provenientes de ratas alimentadas com dieta suplementada com leucina. Os grupos de fêmeas prenhes foram divididos da seguinte forma: controle (C), controle com dieta suplementada com leucina (L), tumor (W), tumor com dieta suplementada com leucina (WL), dieta pareada á ingestão diária das ratas do grupo W (Cp), dieta suplementada com leucina e pareada á ingestão diária das ratas do grupo WL (Lp). Após 20 dias de prenhes os músculos fetais foram analisados. Os resultados mostraram que a síntese protéica fetal foi reduzida no grupo W quando comparados aos outros grupos. Houve significativa recuperação da síntese protéica no músculo dos fetos WL sugerindo efeito positivo do uso de aminoácido de cadeia ramificada nessa situação. Ocorreu aumento significativo da degradação protéica nos fetos W. Essa perda protéica foi amenizada pelo uso da dieta suplementada com leucina indicando efeito benéfico sobre a degradação protéica no grupo WL que obteve valores similares ao observado no grupo C. A atividade da enzima glutationa-S-transferase ficou elevada no músculo de fetos WL em comparação com o grupo W. Também houve aumento na atividade da enzima fosfatase alcalina no grupo WL indicando maior atividade celular nesse grupo quando comparado ao grupo W. A presença de MDA (Malondialdeído) no músculo fetal também ficou reduzida nos fetos WL. Com a análise desses parâmetros conclui-se que a dieta suplementada com leucina pode alterar o metabolismo muscular fetal aumentando a síntese protéica e reduzindo o efeito danoso do estresse oxidativo.Abstract: Cancer occurs in approximately 1 per 3.000 pregnancies and accounts for one-third of maternal deaths. Leucine has been used experimentally to minimize the protein metabolism changes during tumor growth. The present work investigated the effects of tumor growth on muscle protein metabolism and oxidative stress in fetus pregnant rats fed a leucine-rich diet. Fetal groups from pregnant Wistar rats were: control (C), control fed leucine-rich diet (L), tumor-bearing (W), tumor-bearing fed leucine-rich diet (WL), Pair-fed control (P) and Pair-fed Leucine (PL). After 20 days the fetal muscles were analyzed. The results showed that fetal protein synthesis was decreased in W group, when compared to the other groups. The significantly recover on muscle protein synthesis in fetus of WL group suggested a positive effect of this branched-chain amino acid. There was a significant increase on protein degradation in W fetus and a protective effect of the leucine-rich diet on muscle protein waste in WL group, since it was similar to C group. The muscle enzyme glutathione-S-transferase activity was increased in WL fetus in comparison to W group. The muscle alkaline phosphatase activity increased in WL group. The malondialdehyde content (MDA) in fetal muscle was decreased in WL fetus. The leucine supplemented diet can alter the fetal muscle protein metabolism, improving the fetal muscle protein synthesis and reducing the oxidative stress.MestradoFisiologiaMestre em Biologia Funcional e Molecula

Dietary Leucine Supplementation Minimises Tumour-induced Damage In Placental Tissues Of Pregnant, Tumour-bearing Rats.

The occurrence of cancer during pregnancy merges two complex, poorly understood metabolic and hormonal conditions. This association can exacerbate the conditions of both the mother and the foetus. The branched-chain amino acid leucine enhances cellular activity, particularly by increasing protein synthesis. This study aimed to analyse the modulatory effect of a leucine-rich diet on direct and indirect tumour-induced placental damage. This was accomplished by evaluating the expression of genes involved in protein synthesis and degradation and assessing anti-oxidant enzyme activity in placental tissues collected from pregnant, tumour-bearing rats. Pregnant rats were either implanted with Walker 256 tumour cells or injected with ascitic fluid (to study the indirect effects of tumour growth) and then fed a leucine-rich diet. Animals in a control group underwent the same procedures but were fed a normal diet. On the 20(th) day of pregnancy, tumour growth was observed. Dams fed a normoprotein diet showed the greatest tumour growth. Injection with ascitic fluid mimicked the effects of tumour growth. Decreased placental protein synthesis and increased protein degradation were observed in both the tumour-bearing and the ascitic fluid-injected groups that were fed a normoprotein diet. These effects resulted in low placental DNA and protein content and high lipid peroxidation (measured by malondialdehyde content). Decreased placental protein synthesis-related gene expression was observed in the tumour group concomitant with increased expression of genes encoding protein degradation-associated proteins and proteolytic subunits. Consumption of a leucine-rich diet counteracted the effects produced by tumour growth and injection with ascitic fluid. The diet enhanced cell signalling, ameliorated deficiencies in DNA and protein content, and balanced protein synthesis and degradation processes in the placenta. The improvements in cell signalling included changes in the mTOR/eIF pathway. In conclusion, consumption of a leucine-rich diet improved placental metabolism and cell signalling in tumour-bearing rats, and these changes reduced the deleterious effects caused by tumour growth