UNIVERSIDADE ESTADUAL DE CAMPINAS -...
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UNIVERSIDADE ESTADUAL DE CAMPINAS INSTITUTO DE BIOLOGIA ALTERAÇÕES CARDIOVASCULARES INDUZIDAS PELO EXERCÍCIO EXTENUANTE DE LONGA DURAÇÃO EM RATOS SUPLEMENTADOS COM LEUCINA PROLONGED STRENUOUS EXERCISE INDUCED CARDIOVASCULAR CHANGES IN LEUCINE SUPPLEMENTED SWIM-TRAINED RATS GUSTAVO BARBOSA DOS SANTOS Campinas 2015
Transcript of UNIVERSIDADE ESTADUAL DE CAMPINAS -...
UNIVERSIDADE ESTADUAL DE CAMPINAS
INSTITUTO DE BIOLOGIA
ALTERAÇÕES CARDIOVASCULARES INDUZIDAS PELO
EXERCÍCIO EXTENUANTE DE LONGA DURAÇÃO EM RATOS
SUPLEMENTADOS COM LEUCINA
PROLONGED STRENUOUS EXERCISE INDUCED
CARDIOVASCULAR CHANGES IN LEUCINE SUPPLEMENTED
SWIM-TRAINED RATS
GUSTAVO BARBOSA DOS SANTOS
Campinas
2015
“Pouco conhecimento faz com que as pessoas se sintam
orgulhosas. Muito conhecimento, que se sintam humildes.
É assim que as espigas sem grãos erguem
desdenhosamente a cabeça para o céu, enquanto as
cheias as baixam para a terra, sua mãe”.
Leonardo Da Vinci
Agradecimentos
Aos meus pais, Luiz Antônio e Neide, que sempre estiveram ao meu lado, me apoiando
em todas minhas decisões, fossem elas quais fossem. Pelo exemplo de honestidade,
ética e justiça que sempre me deram. Vocês são os principais responsáveis pela
realização deste trabalho e pela pessoa que sou. Amo vocês, muito obrigado. À minha
família, em especial ao meu irmão Rafael. São poucas as pessoas com quem podemos
contar incondicionalmente e você é uma delas. Conte comigo sempre.
À Elisa Jackix (Lisa), pessoa muito importante em minha vida. É muito bom poder contar
com você sempre, saiba que é uma das maiores responsáveis por esta conquista. Muito
obrigado pela sua ajuda e compreensão durante este período. Te amo.
Aos meus grandes amigos Fernando Catanho e Renato Buscariolli. Pessoas que
contribuem muito em minha vida acadêmica e pessoal. Espero que nossa amizade
perdure para sempre, assim como nossas "boleiragens". Aos amigos de laboratório, Luís
Alberto, e Rafael Soares. Ao amigo Bernardo Neme Ide, profissional a quem admiro
muito e aprendo constantemente. Agradeço de forma especial ao Bread Leandro Gomes
da Cruz e André Gustavo de Oliveira, a amizade de vocês foi importantíssima e tornou
todo o processo mais valioso.
Registro aqui minha grande gratidão ao André Gustavo de Oliveira (Roma). Muito
obrigado pela sua paciência em ensinar os protocolos do laboratório durante os
experimentos e por ter me ajudado em todas as etapas deste trabalho. Espero poder
retribuir toda ajuda que me deu nesses quatro anos, aprendi muito com você. Muito
obrigado!
Aos amigos da AABB, Valter César, Paulo André, Fernando (Juv), Vitor (Mundinho) e
Matheus (Di) por fazerem parte da minha vida há tanto tempo e serem exemplos de
amizade e companheirismo. Agradeço de forma especial ao Rodrigo Freston pela
amizade e contribuição neste trabalho. Muito obrigado English!
Ao Prof. Dr. Miguel Arcanjo Areas, meu orientador, por tudo que passamos nesses doze
anos de convivência. Sem dúvida, aprendi muito com ele pelo lado acadêmico, mas não
se compara com o que aprendi pelo lado pessoal: com sua humildade, respeito com que
trata a todos, pela sua amizade e companheirismo. Sentirei muita falta de nossas
conversas. Muito obrigado por tudo!
À Profa. Dra. Dora Maria Grassi-Kassisse por ter aceito, mais uma vez, o convite para
fazer parte da minha banca. Sinto-me honrado por ter sua participação durante todas as
etapas da minha formação. Foi minha professora durante a graduação e mestrado, além
de ter sido membro titular tanto da banca de mestrado quanto de doutorado. É um
exemplo de profissional e ser humano, a forma gentil e educada como trata a todos é
exemplar. Muito obrigado!
À Profa. Dra. Maria Cristina C. G. Marcondes pela gentileza de nos ceder seu laboratório
para realização de grande parte dos procedimentos experimentais. Muito obrigado!
Aos membros da banca, Prof. Dr. Carlos Alberto da Silva, Prof. Dr. Felix Guillermo Reyes
Reyes pelas correções e sugestões, contribuindo de maneira extremamente significativa
para a melhoria da qualidade desse trabalho. Ao Prof. Dr. Alexandre Gabarra de Oliveira,
por ter aceitado prontamente o convite. À Prof. Dra. Mara Patrícia Traina Chacon Mikahil,
pessoa muito importante em minha formação como educador físico, agradeço a forma tão
gentil como me recebeu em seu laboratório e o convite. À Prof. Dra. Celene Fernandes
Bernardes, pessoa que tive o prazer de conhecer há pouco tempo e, agora, tenho a
honra de tê-la em minha banca. Muito obrigado a todos.
Ao departamento de Departamento de Anatomia, Biologia Celular e Fisiologia e Biofísica
do Instituto de Biologia da Unicamp e a todos seus funcionários, assim como aos
funcionários da secretaria de pós-graduação.
À CAPES pelo apoio financeiro.
Resumo
O exercício físico regular de intensidade moderada traz benefícios incontestáveis
à saúde e, em especial, à saúde cardíaca. Já no exercício físico extenuante de longa
duração, típico de esportes de resistência (maratonas, triatlos, remo, etc.), esses efeitos
não são tão bem caracterizados. Estudos prévios têm mostrado que tais eventos podem
levar a disfunções cardíacas transitórias, denominadas “Fadiga Cardíaca”. Essas
disfunções vão desde alterações de permeabilidade da membrana dos cardiomiócitos e
da função ventricular, até elevações de biomarcadores utilizados no diagnóstico de
disfunção e dano celular cardíaco. Dessa forma, a exposição crônica à fadiga cardíaca
poderia evoluir para alterações elétricas e morfológicas semelhantes àquelas observadas
em algumas condições patológicas, como arritmias e insuficiência cardíaca. Em teoria, o
controle do turnover proteico, além da disponibilidade de substrato energético, poderia
atenuar ou impedir a fadiga cardíaca. Existe crescente evidência sobre a importância dos
BCAA (aminoácidos de cadeia ramificada), em especial da leucina, na regulação do
metabolismo proteico. Essa regulação se dá tanto via estimulação proteica quanto pela
inibição da proteólise. O entendimento dos processos funcionais e moleculares que
levam à fadiga cardíaca poderia ajudar na prevenção de distúrbios cardíacos tanto no
âmbito esportivo quanto clínico. Assim, considerando-se o potencial risco de alterações
cardíacas induzidas pelo exercício e o aumento do número de praticantes de eventos
esportivos de longa duração, este trabalho teve como objetivo determinar o efeito do
exercício físico de longa duração sobre parâmetros cardíacos funcionais e biomarcadores
de lesão cardíaca em ratos wistar machos adultos submetidos a treinamento físico de
natação. Em adição, foi avaliado o uso da suplementação de leucina como substância
auxiliar na prevenção dos prováveis efeitos adversos causados pelo exercício de longa
duração. Os animais foram divididos em quatro grupos de acordo com a dieta
(suplementado com leucina ou não) e nível de condicionamento físico (treinado ou não).
Após uma sessão de exercício à exaustão, foram avaliadas as funções cardiovasculares
pelo ECG e pressão arterial, biomarcadores plasmáticos de lesão cardíaca (IL-6, TNF-α,
Troponina I e T), além de substrato energético (glicogênio) e proteínas-chave da via de
sinalização do turnover proteico (AKT, AMPK, mTOR, 19S e 20S) e do metabolismo
oxidativo (citrato sintase) no musculo cardíaco e esquelético. Os resultados mostraram
que o exercício à exaustão elevou significativamente os biomarcadores de lesão cardíaca
e citocinas, além de causar distúrbios elétricos cardíacos e inibir a sinalização para
síntese proteica tanto no músculo cardíaco quanto esquelético. Quando combinado ao
exercício, a suplementação de leucina levou à piora dos parâmetros mencionados, além
de elevar a pressão arterial e a sinalização para degradação proteica. Embora a
suplementação de leucina tenha aumentado a concentração de glicogênio e a atividade
da citrato sintase, no músculo esquelético, isto não melhorou o desempenho dos ratos
treinados submetidos a um teste de exaustão. Dessa forma, o exercício à exaustão pode
induzir a distúrbios elétricos cardíacos e lesão no cardiomiócito. Além disso, a
suplementação de leucina, nas condições experimentais utilizadas, causou efeitos
deletérios no sistema cardiovascular dos ratos exercitados, além de não melhorar a
performance.
Palavras-chave: Leucina; exercício; fadiga; marcadores biológicos, metabolismo.
Abstract
Regular physical exercise of moderate intensity has unarguable benefits to health,
especially cardiac health. However, in prolonged strenuous exercise, typical of endurance
sports (marathons, triathlons, rowing, etc.); these effects are not as pronounced. Recent
studies have shown that such events may lead to transitory cardiac dysfunctions, called
"Cardiac Fatigue". These dysfunctions range from alterations to the permeability of the
membrane of the cardiomyocytes and of the ventricular function to elevations of
biomarkers used in the dysfunction diagnosis and cellular cardiac damage. Therefore,
chronic exposure to cardiac fatigue may evolve into electrical and morphological
alterations similar to the ones observed in some pathological conditions, such as
arrhythmia and heart failure. In theory, management over protein turnover, as well as the
availability of the energy substrate, may mitigate or impede cardiac fatigue. There is
growing evidence about the importance of BCAA (Branch Chain Amino Acids), especially
leucine, in the regulation of protein metabolism. This regulation occurs both through
protein stimulation and inhibition of proteolysis. Understanding of the functional and
molecular processes that lead to cardiac fatigue may help in the prevention of cardiac
disorders both in the sporting sphere and in the clinical sphere. Thus, considering the
potential risks of exercise-induced cardiac alterations and the increase in the number of
practitioners of prolonged sporting events, this work´s goal was to determine the effect of
prolonged physical exercise on functional cardiac parameters and biomarkers of cardiac
injuries on sedentary adult male wistar rats submitted to swimming training. Additionally,
the use of leucine supplementation as an auxiliary substance in the prevention of the
probable adverse effects caused by prolonged exercising was evaluated. The animals
were divided into four groups according to diet (with or without leucine supplementation)
and level of physical conditioning (with or without training). After a session of exercising
until exhaustion, they were evaluated in terms of their cardiovascular functions through
ECG and arterial pressure, biomarkers of cardiac injury (IL-6, TNF-α, Troponin I and T),
as well as energy substrate (glycogen) and key proteins of the signaling pathways of the
protein turnover (AKT, AMPK, mTOR, 19S and 20S) and of the oxidative metabolism
(citrate synthase) in the cardiac and skeletal muscle. The results show that exercising until
exhaustion significantly elevated the biomarkers of cardiac injury and cytokines, besides
causing electrical cardiac disorders and inhibiting the signaling for protein synthesis both
in the heart and skeletal muscle. When combined with exercise, leucine supplementation
led to the worsening of the parameters above, as well as elevating the arterial pressure
and the signaling for protein degradation. Although leucine supplementation has
increased the concentration of glycogen and the activity of citrate synthase in the skeletal
muscle, this has not improved the performance of the trained rats submitted to an
exhaustion test. Therefore, exercising until exhaustion may induce electrical cardiac
disorders and injury to the cardiomyocyte. Moreover, in the experimental conditions used,
leucine supplementation caused harmful effects to the cardiovascular system of the rats,
as well as not improving their performance.
Keywords for review: Leucine; exercise; fatigue; biological markers; metabolism.
Lista de Abreviaturas Akt, Proteína Quinase B;
AMPK, proteína quinase ativada por AMP;
BCAA, Aminoácidos de Cadeia Ramificada (Branched Chain Amino Acids)
CS, Citrato sintase;
cTnI, Troponina cardíaca I;
cTnT, Troponina cardíaca T;
ECG, Eletrocardiograma;
IL-6, interleucina 6;
mTOR, alvo da rapamicina em mamíferos;
PGC-1α, coativador-1 alfa do receptor ativado por proliferador de peroxissoma
TNF-α, fator de necrose tumoral-α;
C, Grupo controle sedentário;
T, Grupo treinado;
CL, Grupo sedentário, suplementado com leucina;
TL, Grupo treinado, suplementado com leucina;
Sumário
Resumo .............................................................................................................. 8
Abstract ............................................................................................................ 10
1. Introdução ................................................................................................. 14
1.1. Fadiga Cardíaca em atletas de endurance ......................................... 14
1.2. Fadiga periférica em exercício prolongados ........................................ 15
1.3. Biomarcadores de lesão cardíaca e degradação muscular proteica ... 16
1.4. Biomarcadores de fadiga durante o exercício ..................................... 17
1.5. Leucina ................................................................................................ 18
2. Objetivos ................................................................................................... 21
3. Resultados e Discussão ............................................................................ 22
3.1. Artigo 1 ................................................................................................... 23
3.2. Artigo 2 .................................................................................................. 45
4. Conclusão ................................................................................................. 64
5. Atividade concomitante à tese .................................................................. 65
6. Referências Bibliográficas ......................................................................... 66
7. Anexos ...................................................................................................... 73
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1. Introdução
1.1. Fadiga Cardíaca em atletas de endurance
Atletas de endurance são tidos como exemplo de saúde cardiovascular, por
serem capazes de gerar altas taxas de captação de oxigênio em virtude de um
extraordinário sistema de transporte de oxigênio desenvolvido por estes indivíduos em
consequência do treinamento (BHELLA; LEVINE, 2010). O aumento do
condicionamento cardiorrespiratório e o exercício físico regular estão associados à
redução da mortalidade e de várias doenças cardíacas, além da melhora do
desempenho físico (TRIVAX et al., 2010). Por estes motivos, eventos esportivos de
longa duração como corridas de rua, maratonas e triátlons, tiveram sua popularidade
muito aumentada nos últimos anos, como parte de um estilo de vida saudável.
Uma das principais adaptações cardiovasculares do atleta de endurance que
permite tal desempenho aeróbio é o aumento do volume sistólico alcançado por um
coração hipertrofiado, mais complacente e que relaxa mais rapidamente. Embora
essas adaptações possibilitem melhor desempenho físico e cardiovascular, há quem
questione se estas não poderiam trazer danos cardíacos em longo prazo. Assim,
existem relatos de que o exercício de longa duração traz consequências adversas
para o sistema cardiovascular (BHELLA; LEVINE, 2010; LAKHAN; HARLE, 2008;
LINDSAY; DUNN, 2007; SHAVE et al., 2010; TRIVAX et al., 2010).
Para descrever essas alterações cardíacas transitórias no desempenho
ventricular esquerdo após eventos de longa duração foi criado o termo “Fadiga
Cardíaca”. Esse termo refere-se às elevações dos biomarcadores cardíacos após
exercícios de longa duração, às alterações de permeabilidade na membrana dos
cardiomiócitos e da função ventricular esquerda que, em longo prazo, podem evoluir
para alterações morfológicas cardíacas (desvio do eixo elétrico cardíaco, hipertrofia
ventricular esquerda, etc.) que se assemelham àquelas observadas em algumas
condições patológicas (BHELLA; LEVINE, 2010; PELLICCIA et al., 2010).
Vários estudos (BHELLA; LEVINE, 2010; SAHLEN et al., 2010; TRIVAX et al.,
2010) mostram elevação de troponinas I e T e creatina quinase (biomarcadores
plasmáticos cardíacos específicos, utilizados no diagnóstico de lesão e/ou doença
cardíaca) após eventos de resistência, consistentes com os valores encontrados em
certas patologias cardíacas. Além disso, a ocorrência da “Fadiga Cardíaca” tem sido
relatada como consequência de exercícios prolongados de resistência, com duração
entre 3 a 17 horas (como no caso de corridas de triátlon Iron-man) (PELLICCIA et al.,
2010). Assim, o questionamento sobre a possibilidade destes episódios de lesão
cardíaca sutis, causados pelo exercício, induzir, em longo prazo, alterações
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permanentes na morfologia e função ventricular esquerda, se faz pertinente (GEORGE
et al., 2008).
Apesar desses eventos cardiovasculares serem bastante incomuns em atletas, o
risco em corredores menos treinados parece ser significativamente maior. Já foi
demonstrado que a fadiga cardíaca e o aumento de biomarcadores plasmáticos de
lesão cardíaca ocorrem, em maior magnitude, em indivíduos menos treinados
(SAHLEN et al., 2010).
Muitas são as causas atribuídas à ocorrência da fadiga cardíaca em atletas de
resistência: disfunção do ventrículo direito pelo aumento da pré e pós-carga; dilatação
das câmaras cardíacas e consequente perda da integridade das junções
intracelulares; depleção de substrato energético e desidratação (BHELLA; LEVINE,
2010; DAWSON et al., 2003; PELLICCIA et al., 2010; SAHLEN et al., 2010; TRIVAX
et al., 2010).
Alguns estudos sugerem possível relação entre a exposição crônica ao exercício
prolongado e o desenvolvimento de fibrose miocárdica (LINDSAY; DUNN, 2007;
WHYTE, G. P., 2008). Essa exposição crônica e o desenvolvimento de fibrose
poderiam deflagrar arritmias fatais (WHYTE, G. P., 2008). Ainda segundo o mesmo
autor, este fenômeno poderia estar ligado ao processo inflamatório induzido pelo
exercício observado em modelos animais.
Enquanto alguns estudos mostram que as consequências da fadiga cardíaca
são transitórias e benignas, não trazendo riscos à saúde, outros sugerem que as
consequências desse fenômeno podem levar à fibrose cardíaca (em resposta às
lesões induzidas pelo exercício), arritmias, isquemia, fibrilação e até morte súbita
(BHELLA; LEVINE, 2010; DAWSON et al., 2003; PELLICCIA et al., 2010; SAHLEN
et al., 2009; SAHLEN et al., 2010; TRIVAX et al., 2010).
1.2. Fadiga periférica em exercício prolongados
A fadiga muscular pode ser definida como qualquer perda, induzida pelo
exercício, da capacidade de produzir força de um músculo ou grupo de músculos.
Esse processo envolve todos os níveis da ativação motora, desde o disparo do
potencial de ação, pelo sistema nervoso central, até a interação actina-miosina, no
músculo. A redução progressiva, induzida pelo exercício, na ativação muscular
voluntária ou na estimulação neural da unidade motora é definida como fadiga central
(TAYLOR; TODD; GANDEVIA, 2006).
As sensações de fadiga e exaustão representam um fenômeno psicológico, que,
cedo ou tarde, induzem mudanças no comportamento. Já, as alterações físicas e
bioquímicas simultâneas que ocorrem durante o exercício, são efeitos fisiológicos. Na
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fisiologia do exercício estes efeitos são definidos como fadiga, e pode ser monitorado
objetivamente. Entretanto, a fadiga também possui um aspecto psicológico. Assim,
apesar da ativação motora durante o exercício permanecer constante, a sensação de
esforço, pode aumentar gradualmente. Eventualmente, essa sensação de esforço
pode ser tão intensa que sobrepõe a própria força de vontade para manter o exercício,
forçando o sujeito a reduzir a intensidade ou mesmo interromper o exercício. Este
momento é definido como exaustão (AMENT; VERKERKE, 2009).
Existem numerosos relatos sobre os mecanismos bioquímicos e/ou fisiológicos
de fadiga periférica: a depleção de glicogênio e fosfocreatina, diminuição no potencial
de repouso da membrana ou disfunção da bomba de cálcio no retículo
sarcoplasmático em músculos esqueléticos, e, ainda, falha de transmissão
neuromuscular (MIZUNO et al., 2008).
1.3. Biomarcadores de lesão cardíaca e degradação muscular proteica
Dentre os muitos biomarcadores plasmáticos utilizados na avaliação da lesão
cardíaca após exercício prolongado e extenuante, destacam-se as troponinas
cardíacas, por serem marcadores de dano celular cardíaco extremamente específicos,
sendo amplamente utilizadas no diagnóstico de síndromes agudas coronarianas
(SHAVE et al., 2010).
O complexo troponina é composto de três subunidades: troponina T (TnT), a
qual ancora o complexo ao filamento de tropomiosina do filamento fino da fibra
muscular; troponina C (TnC) que se liga aos íons cálcio liberados do reticulo
sarcoplasmático; troponina I (TnI) que inibe a hidrólise enzimática do ATP, que fornece
energia para a contração muscular. Apesar dos músculos esquelético e cardíaco
dividirem a mesma via de desenvolvimento, originam-se de diferentes precursores
embrionários, apresentando, consequentemente, diferentes isoformas de TnT e TnI
(músculos cardíaco - cTnT e cTnI e esquelético - eTnT e eTnI) cada um codificado por
diferentes genes, o que aumenta ainda mais a especificidade destes biomarcadores
(SHAVE et al., 2010).
Estudos tem examinado a resposta da cTn após o esforço físico, a maioria deles
em eventos atléticos competitivos que requerem, do participante, manter um elevado
nível de debito cardíaco, frequência cardíaca e pressão sistólica por várias horas. O
aumento sustentado do trabalho cardíaco estressa o miocárdio, que junto ao ambiente
fisiológico do exercício prolongado (elevação de EROs, pH alterado e aumento da
temperatura corporal) pode, teoricamente, lesar as células cardíacas (KNEBEL et al.,
2009; SHAVE et al., 2010; WHYTE, G. et al., 2008).
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Fibras musculares esqueléticas estão sujeitas ao estresse metabólico e
mecânico durante a realização de uma atividade física, o que contribui para o dano
das proteínas celulares, solicitando que estas sejam continuamente degradadas e
ressintetizadas para que possam manter sua função. O exercício físico influência, de
maneira significativa, o equilíbrio entre esses dois processos (degradação e síntese
proteica). Aumento ou diminuição na atividade física traz adaptações da fibra muscular
(hipertrofia vs. atrofia), alterando a expressão proteica e o tamanho da fibra (REID,
2005).
Do mesmo modo, as proteínas do músculo cardíaco estão em contínuo estado
dinâmico de degradação e ressíntese. Este processo é, extremamente seletivo,
rigorosamente regulado e crucial para a função celular. Proteases estão localizadas
em muitas organelas, dentre elas, lisossomos e proteassomas e possuem papel
importante na degradação de proteínas cardíacas. Enquanto os lisossomos degradam
a maioria das proteínas de membrana, a via ubiquitina-proteassoma degrada proteínas
intracelulares (ZOLK; SCHENKE; SARIKAS, 2006).
A via da ubiquitina-proteassoma é a principal reguladora da degradação proteica
nas células eucarióticas, e é a responsável por degradar a maior parte (entre 80 a
90%) das proteínas intracelulares durante o remodelamento muscular. Em resumo,
essa via reconhece proteínas mal dobradas ou com danos e as rotula através da
conjugação do polipeptídio ubiquitina. Proteínas conjugadas com ubiquitina são então
reconhecidas e degradadas por um complexo enzimático chamado 26S proteassoma
(REID, 2005; ZOLK et al., 2006). As troponinas são uma das principais proteínas
miofibrilares que são predominantemente degradadas pela via da ubiquitina-
proteassoma (ZOLK et al., 2006).
1.4. Biomarcadores de fadiga durante o exercício
A escolha mais plausível de biomarcadores de fadiga muscular acompanha os
mecanismos de fadiga e as alterações metabólicas durante o exercício. Uma vez que
não há uma única causa de fadiga muscular, também não há um biomarcador único
para avaliá-la. Além disso, a escolha dos biomarcadores de fadiga muscular é
dependente da intensidade e duração do exercício. Assim, o biomarcador de fadiga
para um exercício que dura 20 segundos (com demanda de energia anaeróbia de até
90%), será diferente do escolhido para avaliar a fadiga em um exercício com duração
acima de 180 segundos (onde a contribuição do metabolismo oxidativo é maior)
(FINSTERER, 2012).
Além da depleção de ATP e da produção de espécies reativas de oxigênio, o
exercício e a fadiga deflagram um processo inflamatório. Após o exercício, linfócitos T
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são mobilizados para o sangue. Sabe-se que o músculo em contração libera miocinas
(citocinas produzidas pelos músculos), criando um ambiente anti-inflamatório
sistêmico, modulando o processo inflamatório além de exercer efeito endócrino sobre
o tecido adiposo visceral (FINSTERER, 2012).
Embora controverso, alguns estudos apontam papel importante das citocinas na
fadiga aguda e crônica induzida pelo exercício. Outra hipótese para a causa da fadiga,
seria a sobrecarga mecânica crônica das sessões de treinamento frequentes, o que
induz a microtraumas. Estes, por sua vez, induziriam a um processo inflamatório
crônico, pela ativação de algumas citocinas, especialmente a interleucina-1 e 6 (IL-6) e
o fator de necrose tumoral-α (TNF-α) (AMENT; VERKERKE, 2009).
A IL-6 pertence a um grupo de citocinas que modula o processo inflamatório.
Pode agir tanto como pró-inflamatória quanto anti-inflamatória. Durante o exercício, a
IL-6 parece agir de maneira endócrina, mobilizando substratos energéticos ou
aumentando a captação destes (FINSTERER, 2012).
O TNF-α é uma citocina pró-inflamatória, predominantemente produzida por
macrófagos e capaz de induzir apoptose, inflamação, diferenciação e proliferação
celular, além de inibir a tumorigênese e replicação viral. Durante o exercício de longa
duração o TNF-α causa resistência à insulina transitória e aumento da lipólise
(FINSTERER, 2012). Estes efeitos são de grande importância para a continuação do
exercício, pois disponibiliza substrato energético para os músculos ativos, além de
causar efeito poupador da glicose, ajudando na manutenção da glicemia.
Nas últimas duas décadas, os efeitos fisiológicos das citocinas tem sido
extensivamente investigado. Foi demonstrado, ainda, que a IL-6 aumenta a sensação
de fadiga, em atletas. Além disso, várias doenças são acompanhadas pelo aumento
da concentração plasmáticas das citocinas citadas (AMENT; VERKERKE, 2009).
Assim, os efeitos das diferentes citocinas sobre a fadiga induzida pelo exercício
precisa ser melhor investigada.
1.5. Leucina
Leucina é um aminoácido indispensável, ou seja, que não pode ser produzido
pelo nosso organismo e compõe um dos três aminoácidos de cadeia ramificada
(Branched-chain amino acids – BCAA), mostrados na figura 1. Existe crescente
evidência sobre o papel dos BCAAs na regulação do processo anabólico envolvendo
tanto a síntese quanto a degradação proteica via estimulação direta da síntese
proteica e liberação de hormônios anabólicos, como a insulina e/ou através da inibição
da proteólise (DE OLIVEIRA et al., 2011; GREER et al., 2007; MANNINEN, 2006). A
Leucina possui também potencial terapêutico devido aos seus efeitos sobre a
19
manutenção da massa magra durante a perda de peso, promoção de cura de
ferimentos e anabolismo muscular na sarcopenia relacionada à idade ou a caquexia
em decorrência de câncer (DE OLIVEIRA et al., 2011).
Figura 1. Aminoácidos de cadeia ramificada – BCAA.
Estudos in vivo e in vitro verificaram que dietas hiperproteicas influenciam a
síntese proteica e regulam vários processos celulares. Dentre os aminoácidos, a
leucina é a mais eficaz em estimular a síntese proteica, reduzir a proteólise e,
portanto, favorecer o balanço nitrogenado positivo. Além disso, este aminoácido
influencia direta e indiretamente a síntese e secreção de insulina podendo, assim,
aumentar as propriedades anabólicas celulares (DE OLIVEIRA et al., 2011; RENNIE,
2007; VIANNA et al., 2010). A leucina também pode estimular a síntese proteica
durante condições catabólicas como na restrição alimentar aguda e no exercício
exaustivo (VIANNA et al., 2010).
Alguns estudos sugerem que o efeito anabólico da leucina é mais evidente em
situações catabólicas, quando as concentrações plasmáticas e intracelulares da
leucina estão baixas (GREER et al., 2007; PHILLIPS, 2004; VIANNA et al., 2010). Em
um artigo de revisão, foi demonstrado que a síntese proteica está bastante reduzida
em comparação a taxa de degradação proteica no músculo após o exercício intenso
ou uma noite de jejum (PHILLIPS, 2004). Esse estado catabólico persiste até que
quantidade adequada de proteína e, especificamente, leucina, seja consumida para
reverter este estado, aumentando as concentrações plasmáticas e intracelulares deste
aminoácido. Entretanto, embora o efeito da ingestão aguda de leucina sobre a
estimulação da síntese proteica tenha sido verificado, poucos estudos avaliaram a
eficiência da ingestão crônica de leucina (VIANNA et al., 2010).
Devido ao seu papel na regulação da síntese e degradação proteica, muitos
atletas utilizam a suplementação de leucina com a intenção de diminuir a extensão do
dano muscular, reduzir a fadiga e aumentar o desempenho. Indivíduos suplementados
20
com carboidratos e leucina mantiveram os níveis de creatina quinase sanguínea
inalterados, além de apresentarem níveis de percepção subjetiva de dor, menores
quando comparados aos indivíduos suplementados apenas com carboidratos ou
solução placebo (GREER et al., 2007).
Os BCAA também exercem ações anabólicas no metabolismo proteico cardíaco
e a sua captação pelo miocárdio é amplamente dependente da sua concentração
plasmática (BIANCHI et al., 2005). Assim, fica evidente o potencial ergogênico da
leucina, principalmente em contexto de exercício extenuante de longa duração e alta
frequência de treinamento de endurance.
21
2. Objetivos
2.1. Objetivo Geral
O objetivo geral deste trabalho foi avaliar o efeito do exercício físico de longa
duração sobre parâmetros cardiovasculares moleculares, funcionais, metabólicos e
hemodinâmicos em ratos Wistar machos adultos treinados; concomitante, ou não, à
ingestão de dieta suplementada com leucina. E, desta forma, avaliar o uso da leucina
como substância auxiliar na prevenção dos prováveis efeitos adversos causados pelo
exercício físico extenuante de longa duração.
2.2. Objetivo Específico
De forma paralela, investigamos o papel da suplementação de leucina sobre
respostas moleculares e metabólicas no músculo esquelético de ratos treinados,
submetidos a uma sessão de exercício exaustivo de longa duração, concomitante ou
não, à suplementação de leucina. De forma específica, avaliamos parâmetros
metabólicos através da atividade da enzima citrato sintase, no coração e no músculo
gastrocnêmio, além da concentração de glicogênio nos dois tecidos. Avaliamos
parâmetros moleculares pelas vias de sinalização de síntese proteica, através da
concentração das proteínas Akt, mTOR e AMPK, nos tecidos citados e a sinalização
de degradação proteica através da concentração das proteínas 19s e 20s, no músculo
cardíaco. Os parâmetros cardíacos funcionais foram avaliados através do ECG, dos
biomarcadores de lesão cardíaca (troponinas T e I) e das citocinas IL-6 e TNF-α.
Avaliamos, também, parâmetros hemodinâmicos através da pressão arterial.
22
3. Resultados e Discussão
Os resultados e discussão estão apresentados na forma de dois capítulos compostos
por artigos científicos, compilando os dados referentes às respostas cardíacas e
musculares funcionais, moleculares e metabólicas induzidas pelo exercício extenuante
de longa duração em ratos treinados, submetidos ou não à dieta rica em leucina.
Encontram-se a seguir os dois artigos resultantes do desenvolvimento desse trabalho.
O primeiro artigo será submetido à revista The Journal of Physiology, já o segundo
artigo foi submetido à revista Nutrition, ambos, de acordo com a indexação Capes,
em classificação Qualis A.
23
3.1. Artigo 1 será submetido ao periódico “The Journal of Physiology”
_____________________________________________________________________
LONG-TERM LEUCINE SUPPLEMENTATION AGGRAVATES PROLONGED
STRENUOUS EXERCISE-INDUCED CARDIOVASCULAR CHANGES IN TRAINED
RATS
Authors:
Gustavo Barbosa dos Santos1, MSc
André Gustavo de Oliveira1, MSc
Luiz Alberto Ferreira Ramos1, PhD
Maria Cristina Cintra Gomes Marcondes1, PhD
Miguel Arcanjo Areas1, PhD
Affiliation:
1Department of Structural and Functional Biology, Institute of Biology, University of
Campinas (UNICAMP), Campinas, São Paulo, Brazil.
Corresponding author:
G.B. Santos, +55 (019) 3521-6196, [email protected]
Departamento de Biologia Funcional e Molecular
Instituto de Biologia - Caixa Postal 6109
Av. Bertrand Russell - Bloco O
Universidade Estadual de Campinas - UNICAMP
CEP-13083-865
Campinas - SP
Keywords for review: Leucine; prolonged exercise; electrocardiogram; cardiac
biomarkers.
Total word count (excluding references and figure captions): 3948
Number of Figures: 6
24
Key points Summary
Prolonged endurance exercise does not seem to exceed cardiac energetic
capacity, hence does not represent an energy threat to this organ, at least in
trained subjects.
Prolonged endurance exercise may induce, in susceptible individuals, a state of
cardiac electrical instability, which has been associated with ventricular
arrhythmias and cardiac sudden death. This situation may be worsened when
combined with leucine supplementation, which led to increased blood pressure
and cardiac injury.
Leucine supplementation failed to prevent cardiac fatigue symptoms, and may
also aggravate prolonged strenuous exercise-induced cardiovascular
disturbances in trained rats.
Abstract Aim: Observational studies have raised concerns that prolonged strenuous exercise
training may be associated with increased risk of cardiac arrhythmia and even primary
cardiac arrest or sudden death. It has been demonstrated that leucine can reduce
prolonged exercise-induced muscle damage and accelerate the recovery process. The
aim of this study was to investigate the effects of prolonged strenuous endurance
exercise on cardiovascular parameters and biomarkers of cardiac injury in trained adult
male rats and assess the use of leucine as an auxiliary substance to prevent the likely
cardiac adverse effects caused by strenuous exercise.
Methods: Twenty-four male Wistar rats were randomly allocated to receive a balanced
control diet (18% protein) or a leucine-rich diet (15% protein with 3% leucine) for 6
weeks. The rats were submitted to 1 hour of swimming exercise, 5 d.wk−1 for 6 wk.
Three days after the exercise training period rats were submitted to swimming
exercises until exhaustion and cardiac parameters were assessed.
Results: Exercising until exhaustion significantly increased serum cardiac biomarker
levels and cytokines, glycogen content and inhibited protein synthesis signaling also
led to cardiac electrical disturbances. When combined with exercise, leucine
supplementation led to further increases in the aforementioned parameters and also
significant increase in blood pressure and protein degradation signaling.
Conclusion: We report, for the first time, that leucine supplementation not only does not
prevent cardiac fatigue symptoms, but may also aggravate prolonged strenuous
exercise-induced cardiovascular disturbances in trained rats. Furthermore, we find that
exercising until exhaustion can cause cardiac electrical disturbances and cardiac
myocyte damage.
25
Abbreviations list Akt, Protein kinase B; AMPK, AMP-activated protein kinase α; CS, citrate synthase;
cTnI, Troponin I; cTnT, Troponin T; ECG, Electrocardiogram; IL-6, interleukin-6;
mTOR, mammalian target of rapamycin; p-AMPK, AMPK phosphorylation; TNF-α,
tumor necrosis factor-α;
Introduction
It is well established that exercise training induces a variety of cardiovascular
adaptations leading to enhanced sporting performances and health benefits. A meta-
analysis quantifying the dose-response relationship between physical activity and risk
of coronary heart disease stated that even low-to moderate-intensity leisure-time
exercise could induce cardioprotection (SATTELMAIR et al., 2011). Despite such
positive outcomes, numerous observational studies have raised concerns that
prolonged strenuous exercise training may be associated with increased risk of cardiac
arrhythmia and even primary cardiac arrest or sudden death (BENITO et al., 2011;
GEORGE et al., 2008; O'KEEFE et al., 2012).
Since prolonged exercising has been reported to result in skeletal muscle fatigue and
damage, as well as reduced performance, it seems reasonable to expect the same
response, namely cardiac fatigue, from cardiac muscle (DAWSON et al., 2003).
Concerns over the clinical consequences of individual acute bouts of prolonged
exercise are often dismissed because the changes reported are quite small and
transitory, however, there has been speculation in recent years that prolonged
exercising, whether in a single exposure or in a lifetime of activity, may actually have
some negative consequences for cardiovascular performance or health (GEORGE et
al., 2008). These prolonged strenuous exercise-induced cardiac disturbances can be
attributed to morphologic and metabolic factors. Morphologic factors range from
changes in cardiomyocytes membrane permeability and left ventricular dysfunction, to
elevations in cardiac-specific biomarkers, and metabolic factors from energy substrate
depletion to cardiac electrical abnormalities (BHELLA; LEVINE, 2010; SAHLEN et al.,
2009).
It has been demonstrated that branched chain amino acids, particularly leucine, can
reduce prolonged exercise-induced muscle damage and accelerate the recovery
process (GREER et al., 2007). Furthermore, leucine seems to be the most potent one
regarding the effects on protein synthesis and degradation and not only provides
substrates for gluconeogenesis, but also can supply tricarboxylic acid cycle with
different anaplerotic substrates (LI et al., 2011). Crowe et al. (CROWE;
26
WEATHERSON; BOWDEN, 2006) showed that six weeks’ dietary leucine
supplementation significantly improved endurance performance and upper body power
in outrigger canoeists. While leucine’s effects on skeletal muscle, i.e. preventing
muscle damage and fatigue are well established; it’s not known if the same occurs with
cardiac muscles.
The aim of this study was to investigate the effects of prolonged strenuous endurance
exercise with cardiovascular parameters and biomarkers of cardiac injury in trained
adult male rats and assess the use of leucine as an auxiliary substance to prevent the
likely cardiac adverse effects caused by strenuous exercise. Since the current
evidence is promising, we hypothesized that prolonged strenuous exercise would
induce cardiac disturbances (electrical, structural and biochemical) and that leucine
supplementation could prevent, or at least mitigate, those outcomes.
Material and Methods
Animals and diets
Twenty-four male wistar rats (12 weeks old, weighing 351 g ± 28.87) were obtained
from the animal facilities of the University of Campinas (São Paulo, Brazil). They were
housed in collective cages at 22-24°C on a 12-h light-and-dark cycle, with free access
to tap water and food. The semi-purified isocaloric diets were a normal protein (C),
containing 18% protein (REEVES; NIELSEN; FAHEY, 1993); or leucine (L), containing
15% protein plus 3% of L-leucine. Approximately 70% carbohydrate (sucrose, dextrin
and starch), 7% fat (soybean oil) and 5% fiber (purified micro-cellulose) were added to
the diets. Vitamin and mineral mix, as well as cystine and choline, supplemented the
diets. The control diet had 1.6% of L-leucine, and a leucine-rich diet contained 4.6% L-
leucine, according to a previous study from our group (CRUZ; GOMES-MARCONDES,
2014). Leucine-supplemented diet has led to a significant increase in plasma leucine
concentration in fetuses from tumor-bearing pregnant mice (VIANA; GOMES-
MARCONDES, 2013) and adults rats (data not shown). Two groups were fed the
control diet: sedentary control (C) and trained (T); and two other groups were fed the
leucine-rich diet: control-leucine supplemented (CL) and trained-leucine supplemented
(TL). All the experimental procedures employed were in accordance with the Ethics
Committee on Animal Experimentation of Unicamp (CEEA/IB/UNICAMP, protocol
2888-1).
Training Protocol
The T and TL groups were submitted to the swimming protocol adapted from Santos et
al. (BARBOSA DOS SANTOS et al., 2013), 5 d.wk−1 for 6 wk, in a water tank (90 x 70
27
x 70 cm and water temperature at 31 ± 1ºC). All of the rats were adapted to the water
during the first week of the experiment. The adaptation process consisted of keeping
the animals in shallow water, initially for 20 min and then progressively increasing 10
min/day and 10 cm water/day for 5 days. Exercise sessions began with 60 min/day at
the second experimental week, carrying constant loads (added to the tail) of 20 g
(approximately 6% of initial body weight). Initially for 10 minutes of these 60 minutes,
increased by 10 more minutes each week, until it reached 60 min of loaded swimming
training in the sixth and last week of experiment. Three days after the exercise-training
period, rats were submitted to swimming exercise carrying the same load until
exhaustion. Animals were sacrificed under anesthesia (ketamine and xylazine, 90
mg/kg/bw and 45 mg/kg/bw, respectively, i.p.) between 3-4 hours after exercise bout, in
order to reach cytokine peak after swimming exhaustion test (LOUIS et al., 2007;
SUZUKI et al., 2002).
Electrocardiogram (ECG)
ECG was performed before and after experiment periods. Anesthetized rats (ketamine
and xylazine, 90mg/kg/bw and 45mg/kg/bw, respectively, i.p.) were kept in the supine
position with spontaneous breathing for ECG recording. The electrodes were
connected to the computer channels (Heart Ware System), and six standard waves
were recorded (BARBOSA DOS SANTOS et al., 2013).
Blood Pressure
To determine arterial systolic and diastolic blood pressure, a cannula was inserted into
the left femoral artery and connected to BP-1-Analog single-channel transducer signal
conditioner (World Precision Instruments, USA) (KANG et al., 2006).
Glycogen content
Gastrocnemius muscles were quickly removed, frozen immediately in liquid nitrogen
and stored at -80°C until further analysis. Muscle glycogen content was estimated
colorimetrically based on the method described by Lo et al. (LO; RUSSELL; TAYLOR,
1970). The absorbance was read on a plate CHAMELEON V Multilabel Microplate
Reader (Hidex, Finland) at 620nm.
Citrate synthase activity
For citrate synthase analyses ≈30 mg of gastrocnemius muscle was homogenized
in ice cold extraction buffer (175 mM KCl, 2 mM EDTA, pH 7.4), centrifuged at 16.000 x
g, 20 min, at 4°C. An aliquot of supernatant was combined with reaction mixture
28
containing 0.1 M Tris, pH 8.3, 1 mM DTNB, 3 mM acetyl-CoA. Reaction was initiated
by adding 10 mM oxaloacetic acid to the extract. The absorbance was
spectrophotometrically measured at 412 nm in 30 sec interval for 5 min using a Dynex
MRX plate reader controlled through personal computer software (Revelation,
Dynatech Laboratories), as previously described (SRERE, 1969). All samples were
tested for linearity up to 5 min of reaction and values were normalized by protein
concentration (BRADFORD, 1976).
Western Blot
Heart samples (40μg) were homogenized and protein concentration was measured
using a colorimetric method (BRADFORD, 1976). The proteins were revealed using
primary antibodies against α-tubulin (1:20.000), phospho-mTORSer2448 (1:1.000).
phospho-AktThr308 (1:1.000), phospho-AMPK-αThr172 (1:1.000) (Cell Signaling, USA) and
proteasome subunits 20S, 19S (1: 1.000) (Enzo, USA), and secondary anti-mouse,
anti-rabbit and anti-goat antibodies (1:10.000, Cell Signaling, USA) after reaction with a
chemiluminescent reagent (Thermo Fisher Scientific, USA) was added and band
volume was captured using Alliance Captura 2.7 (UVItec, UK) and quantified using UVI
band -1D (UVI tec, UK).
Specific cardiac biomarkers
Blood samples were taken from the heart by ventricle puncture. Serum was separated
by centrifugation at 1,000 x g for 10 min at 4 °C and stored at −80 °C. The analysis of
serum cardiac-specific markers (Troponin I and T) and serum inflammatory markers
(TNF-α, IL-6) was determined using beads coupled with capture antibodies specific for
each protein of interest as specified by the manufacture Millipore® (Merck Millipore
Corporation, Darmstadt, Germany). The analysis was carried out on Xponent software
used with the Luminex® 200 (Luminex Corporation, Austin, TX, USA) equipment,
following the manufacturer’s technical procedures.
Statistical Analysis
The data is expressed as the mean ± SD. The data was analyzed statistically by
analysis of variance (ANOVA) followed by Tukey’s test to establish differences
between groups. We used Prism software (Graphpad Software Inc., San Diego, CA,
USA). The results were considered significant when P<0.05.
29
Results
Cardiac Functional Parameters
Relative heart weight increased in the T and TL groups when compared to the C and
CL group (figure 1). Although collectively there was no significant difference in
electrocardiographic parameters, individually there were some clinically relevant
changes (figure 2A and 2B), which are discussed later. Both arterial systolic and
diastolic blood pressure were determined approximately two hours after last exercise
bout. Systolic, diastolic and mean arterial pressure were significantly higher in TL group
when compared to all experimental groups (figure 3).
Cardiac Metabolic Parameters
The cardiac glycogen content was assessed in order to reveal the metabolic stress (i.e.
energetic demand) of exercising until exhaustion and was significantly elevated in
trained groups (T and TL) compared to sedentary groups (C and CL). This
enhancement was even higher in TL group when compared to T group (figure 1B).
AMP kinase α (AMPK) was also assessed to measure metabolic stress, since AMPK
reflects the energetic status of the cell. AMPK phosphorylation (p-AMPK) did not differ
significantly between groups (figure 4D)
Citrate synthase (CS) activity is the most important biomarker for mitochondrial density
in skeletal muscle and biochemical marker of the skeletal muscle oxidative adaptation
to a training intervention. CS activity did not differ significantly between groups (figure
1C).
Cardiac Structural Parameters - Protein Synthesis and Degradation Pathway
In order to evaluate treatment-induced cardiac protein synthesis, we assessed the
activation of two main key proteins in synthesis pathway, namely, Akt and mTOR.
Activation of Akt was inhibited in trained groups (T and TL) compared to control group.
Among trained groups, leucine supplementation increased Akt phosphorylation when
compared to exercise only (T group) (figure 4B). Compared to control group, mTOR
activation was significantly inhibited only in T group (figure 4C). The ubiquitin-
proteasome pathway is the most important pathway to protein degradation in cardiac
muscle during exercises. To analyze the effect of exercising until exhaustion in cardiac
protein degradation pathway and the modulatory effect of a leucine supplementation,
we evaluated some key proteins of this process as proteasome subunits 19S, and 20S
(Figures 5A, and 5B respectively). Both proteins were elevated only in TL group when
compared to all others groups.
30
Cardiac cell damage and systemic inflammation
To determine cardiomyocyte integrity we assess specific serum cardiac biomarker
levels, namely Troponin T and I (cTnT and cTnI, respectively). Both were significantly
elevated in trained groups (T and TL) compared to sedentary groups (C and CL). This
enhancement was even higher in TL group when compared to T group (figure 6A and
6B, respectively).
Then, we assessed interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels as
inflammation markers. Although IL-6 level was significantly elevated in trained groups
compared to control groups (figure 6C), TNF-α level did not differ significantly between
groups (figure 6D).
Discussion
It is generally accepted that leucine supplementation, can mitigate endurance exercise-
induced skeletal muscle damage and fatigue, accentuate muscle protein synthesis and
also improve recovery and muscle performance (ROWLANDS et al., 2014). But it is
unclear if leucine supplementation would lead to these outcomes in cardiac muscle and
if it could prevent cardiac fatigue. Here we report, for the first time, that leucine
supplementation not only does not prevent cardiac fatigue symptoms, but may also
aggravate prolonged strenuous exercise-induced cardiovascular disturbances in
trained rats. Furthermore, we find that exercising until exhaustion can cause cardiac
electrical disturbances and cardiac myocyte damage.
Prolonged strenuous exercise presents a unique hemodynamic and metabolic
challenge to cardiac muscle and may lead to transient impairment of cardiac function,
so called cardiac fatigue (SAHLEN et al., 2009). Therefore, the primary novel finding of
the present study was that leucine supplementation, when combined with prolonged
endurance exercise, can induce high blood pressure. This finding was highly
unexpected since endurance exercise has previously been reported to reduce blood
pressure in both hypertensive and normotensive subjects, in humans and rats
(HALLIWILL et al., 2013; WHELTON et al., 2002). However, leucine has recently been
related to hypertensive response through mTOR-induced hypothalamic sympathetic
stimulation pathway (HARLAN et al., 2013). Although a leucine dose-related rise in
arterial pressure has been found in this study, leucine was administered
intracerebroventricularly. Moreover, previous studies reported higher circulating leucine
in hypertensive subjects (NEWGARD et al., 2009) and correlated plasma leucine levels
with cardiovascular events (SHAH et al., 2010). It is not clear why this hemodynamic
response appeared in the present study only when leucine supplementation was
combined with prolonged exercise, nevertheless, it is worth mentioning that our
31
exercise protocol can be considered as high-intensity (imposed tail weight) and high-
volume, which could lead to overreaching syndrome and consequently to autonomic
cardiac dysfunction (BAUMERT et al., 2006).
In our study, both trained groups presented significant increase in IL-6 level. Even
though an association between serum IL-6 concentration and mortality (SU et al.,
2013) and myocardial remodeling (MELENDEZ et al., 2010) has recently been shown,
these outcomes occurred invariably when there was a preexisting disease. Thus, it is
important to keep in mind that although mostly regarded as a pro-inflammatory
cytokine, IL-6 also has many regenerative or anti-inflammatory activities (SCHELLER
et al., 2011). Furthermore, IL-6 has an important metabolic function during exercise
and may represent a link between skeletal muscle and organs such as liver and
adipose tissue, since studies have clearly demonstrated that contracting muscles
without any muscle damage can induce a marked elevation in plasma IL-6. In fact, it
has been suggested that an exercise-induced increase in plasma IL-6 level can exert
an important role in mediating the beneficial health effects of exercise in inactivity and
obesity-related disorders such as diabetes and cardiovascular disease (PEDERSEN et
al., 2004; SCHELLER et al., 2011).
As previously reported and as might be expected, exercising until exhaustion, when
alone, induced an increase in troponin levels, even in trained rats (CHEN et al., 2000).
Serum cTnT and cTnI measurements are specific in the assessment of cardiac injury
even in the presence of skeletal muscle damage. Another novel finding of our study
was that leucine supplementation associated to chronic high-intensity endurance
training may significantly increase myocardial injury when compared to training alone.
The arterial hypertension response found only in trained leucine supplementation group
may explain the significant increase in cTnI and cTnT found in this group, since it is
well established that hypertension can induce cardiac cell damage, endothelial
dysfunction and ultimately lead to strokes and cardiovascular events (VASAN et al.,
2001). Thus an arterial hypertensive condition combined with exercising until
exhaustion can have an additive effect on cardiac cell damage. Although someone
might argue that increases in cardiac troponins are only mild and transitory, reflecting a
physiological troponin release from the free cytosolic pool rather than damaged
contractile elements (SCHARHAG et al., 2008) this was definitely not the case in TL
group. Since the cTnT level was three times higher than the T group and clinically
relevant cardiac electrical disturbance was found in some ECG parameters.
Taken collectively, there was no significant difference in electrocardiographic
parameters between experimental groups. It is important to understand, however, that
there is a broad normal range in electrocardiogram parameters. Hence, some of them
32
should not be taken collectively; otherwise, clinically relevant cardiac electrical
disturbance may not be noted. Moreover, the ECG provides indirect evidence of
structural cardiac changes and remodeling that affects automaticity, impulse
propagation and other mechanisms of arrhythmia. Thus, individual pre and post-
experimental period analysis should be advised in these studies. Although the ECG is
not a direct measurement of cardiac function, it may be used to infer the anatomical
orientation of the heart, disturbances of rhythm and conduction, the presence of
ischemic injury, and the influence of altered electrolyte concentrations, drugs, disease,
or nutritional deficiencies (FARRAJ; HAZARI; CASCIO, 2011). A number of studies
(MIDDLETON et al., 2007; SHAVE et al., 2004) have shown that these prolonged
exercises inducing diastolic and systolic changes are physiological, mainly in trained
subjects (PELLICCIA et al., 2000) and our results shows that it seems to be true in
most of the cases. However, despite no statistical relevance, we presented some
important individual changes in both trained and trained leucine supplemented groups.
In our study, one out of five rats of the T group presented significant QTc interval
prolongation (103ms to 207ms, pre and post-experimental period, respectively), which
is suggestive of increased life-threatening ventricular arrhythmias and risk of sudden
death (SAHLEN et al., 2009; STRAUS et al., 2006). Moreover, two out of five rats of
the TL group also presented QTc interval prolongation (90ms to 150ms and 50ms to
90ms) and one of them presented prolonged Tpeak-end (4ms to 18ms) and inverted T
wave (figure 2B), which taken together with troponin level, strongly suggested cardiac
injury and myocardial ischemia (SAHLEN et al., 2009). The transient characteristic of
ECG parameter changes reported in previous studies suggests that the impact of
prolonged endurance training upon cardiac electrical disturbances is not harmful. Our
individual data, however, suggests otherwise. It is likely that some subjects are more
susceptible to these prolonged strenuous exercise-induced cardiac disturbances than
others, as previously proposed (ARO et al., 2012; SAHLEN et al., 2009). Thus in
susceptible individuals (such as those with underlying cardiac disease or with a
particular genetic predisposition) these modest and mostly transient cardiac electrical
instability could, in fact, lead to a significant risk of cardiac events. It has already been
demonstrated that individuals can respond very differently to exercise training, and part
of these results have been related to genetics (ASTORINO; SCHUBERT, 2014). Our
results demonstrated that prolonged endurance exercise combined with leucine
supplementation may induce a state of electrical instability, which has been associated
with increased propensity for ventricular arrhythmias and cardiac sudden death.
As expected, exercise protocols lead to increased relative heart weight. It can be
explained by increased glycogen content and glycogen molecule bonded-water
33
(PHILP; HARGREAVES; BAAR, 2012), furthermore, although not assessed, it is
reasonable to expect that exercise-induced miofibrilar hypertrophy may have occurred
(ROWLANDS et al., 2014). Endurance exercise led to significant glycogen content
enhancement and when combined with leucine supplementation, this enhancement
was even higher. It is well established that individuals who exercise on a regular basis
generally have higher muscle glycogen levels than their sedentary counterparts, due to
glycogen supercompensation after glycogen-depleting exercise. Part of this adaptation
comes from an endurance exercise-induced increase in GLUT-4 content in the skeletal
muscle. It seems that transportation of glucose is the rate-limiting step in muscle
glycogen accumulation under physiological conditions (GREIWE et al., 1999).
Although, there is still quite a bit of uncertainty on the precise role of leucine in
regulating glucose metabolism, it is known that leucine interacts positively with insulin
signaling pathway. It was recently demonstrated, in vitro, that leucine increases the
insulin-mediated glycogen synthesis by 50% (DI CAMILLO et al., 2014), which could
explain the significant increase in cardiac glycogen in the trained leucine supplemented
group when compared with the trained group only.
The endurance exercise training did not increase cardiac citrate synthase activity.
Although it is generally recognized that skeletal muscle citrate synthase activity is
elevated by endurance exercise training, it seems not to be the case in cardiac muscle.
Siu et al (SIU et al., 2003) showed that 8 weeks of endurance treadmill training did not
increase cardiac citrate synthase activity. It was suggested that myocardium has a
sufficient preexisting oxidative capacity to supply the energy requirement during
exercise. Interestingly, prolonged endurance exercise had no effect on p-AMPK level.
Our hypothesis was that great energetic stress induced by prolonged endurance bout
would increase p-AMPK level, and leucine supplementation would attenuate this
metabolic stress and AMPK activation. However, these results, as well as all cell
signaling pathways, are highly time point-dependent. As far as we know, very few
studies have examined cardiac p-AMPK level after endurance exercise, and one of
these (OGURA et al., 2011) showed that p-AMPK peaked immediately after 30 minutes
of endurance exercise and returned to basal level after only 30 minutes. It is likely that
we have failed to coincide with peak exercise-induced phosphorylation of cardiac
AMPK-mediated signaling. Furthermore, since the glycogen level did not decrease
significantly (when compared to control groups) after exercising until exhaustion, it is
expected that AMPK activation remains unaltered. Taken together these results
suggest that even high systemic metabolic stress induced by prolonged endurance
exercise does not exceed heart capacity and does not represent an energetic threat for
this organ, at least in trained subjects.
34
Protein synthesis signaling was significantly inhibited after the endurance exercise and
leucine supplementation prevented it. Both Akt and mTOR phosphorylation was
significantly reduced in T group but not in TL. It has been previously demonstrated that
cardiac p-mTOR was also diminished during one hour after 30 minutes of endurance
training (OGURA et al., 2011). On the other hand, Mascher et al (MASCHER et al.,
2007) demonstrated increased skeletal muscle Akt and mTOR phosphorylation after
one hour of endurance training. The type of exercise, the intensity and duration of the
exercise, besides exercise training history are critical factors in the regulation of protein
synthesis (CAMERA et al., 2010; COFFEY et al., 2005), and could explain the different
Akt and mTOR responses to the endurance exercise previously reported. These results
are also highly time point-dependent, and it may explain why, in our study, both Akt
and mTOR activation were inhibited after exercise, since the previous study
(MASCHER et al., 2007) showed that both had already returned to basal level 3 hours
after endurance exercise. Furthermore, in the present study, exercise was done until
exhaustion, with a mean exercise duration of 3 hours and 22 minutes (± 10 minutes),
which could have led to this different protein synthesis signaling response. A very
recent study has also demonstrated increased mTOR phosphorylation after endurance
exercise and leucine supplementation (ROWLANDS et al., 2014). Although it has been
viewed only in skeletal muscle, it’s likely that cardiac mTOR response to endurance
exercise is similar.
Surprisingly, protein degradation signaling was significantly increased only after
endurance exercise and leucine supplementation (TL group). Autophagy has
physiological functions in maintaining cellular homeostasis especially during energy-
deficit situations. During exercise, mechanical and metabolic homeostasis is disturbed,
leading to muscle cell damage, thus, under such a stressful condition, muscle tissue
can remove damaged proteins and organelles by autophagy to release useful materials
to the nutrient pool inside the muscle cells (TAM; SIU, 2014). The reason for this result
is not entirely clear, but it seems reasonable to assume that it is a response to
hypertension-induced pressure overload (mechanical stress), since it has been
demonstrated that the cardiac proteasome system is activated during pressure
overload (CACCIAPUOTI, 2014).
In conclusion, and in contrast to our original hypothesis, leucine supplementation failed
to prevent cardiac fatigue symptoms, and may also aggravate prolonged strenuous
exercise-induced cardiovascular disturbances in trained rats. The major exercise-
induced cardiac disturbances do not appear to be metabolic/energetic but electrical
and structural. Prolonged endurance exercise does not seem to exceed cardiac
energetic capacity, hence does not represent an energy threat to this organ, at least in
35
trained subjects. However, prolonged endurance exercise may induce, in susceptible
individuals, a state of cardiac electrical instability, which has been associated with
ventricular arrhythmias and cardiac sudden death. This situation may be worsened
when combined with leucine supplementation, which led to increased blood pressure
and cardiac injury. Additional studies are needed to fully elucidate which factors
(genetic and/or metabolic) lead to this electrical susceptibility.
Acknowledgments We are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) for a fellowship granted to Santos, G.B., and to Ajinomoto Interamericana
Indústria e Comércio Ltda (Brazil) for supplying the leucine.
Conflict of Interest The authors declare no conflict of interest.
Author Contribution All authors approved the final version for publication.
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39
Figure 1. Effects of a leucine supplementation and endurance training on: A, relative
heart weight (g/100g). B, cardiac glycogen content (g/100g). C, citrate synthase activity
(µmol/ml/min). Experimental groups were divided as follows: sedentary control (C),
control-supplemented (CL), trained (T) and trained-supplemented (TL). Data are
expressed as means ± SD (n=6). Different letters indicate significant differences
between groups (p<0.05).
Figure 1
40
Figure 2. Effects of a leucine supplementation and endurance training on cardiac
electrical activity. A, QTc interval (ms). B, T wave amplitude (mV). Data are expressed
as means ± SD (bars) and individually (lines), before and after experimental period.
Figure 2
41
Figure 3. Effects of a leucine supplementation and endurance training on
hemodynamics: A, systolic arterial pressure (mmHg). B, diastolic arterial pressure
(mmHg). C, mean arterial pressure (mmHg). Experimental groups were divided as
follows: sedentary control (C), control-supplemented (CL), trained (T) and trained-
supplemented (TL). Data are expressed as means ± SD (n=6). Different letters indicate
significant differences between groups (p<0.05).
Figure 3
42
Figure 4. Effects of a leucine supplementation and endurance training on protein
synthesis signaling. A, representative blot for Akt, mTOR and AMPK phosphorylation.
B, Akt activation. C, mTOR activation. D, AMPK activation. Experimental groups were
divided as follows: sedentary control (C), control-supplemented (CL), trained (T) and
trained-supplemented (TL). Data are expressed as means ± SD (n=6). Different letters
indicate significant differences between groups (p<0.05).
Figure 4
43
Figure 5. Effects of a leucine supplementation and endurance training on protein
degradation signaling. A, representative blot for 20S proteasome and 19S regulatory
units, B, 19S content. C, 20S content. Experimental groups were divided as follows:
sedentary control (C), control-supplemented (CL), trained (T) and trained-
supplemented (TL). Data are expressed as means ± SD (n=6). Different letters indicate
significant differences between groups (p<0.05).
Figure 5
44
Figure 6. Effects of a leucine supplementation and endurance training on specific
cardiac biomarkers and inflammatory response: A, serum cardiac TnT (pg/ml). B,
serum cardiac TnI (pg/ml). C, serum IL-6 (pg/ml). D, serum TNF-α (pg/ml).
Experimental groups were divided as follows: sedentary control (C), control-
supplemented (CL), trained (T) and trained-supplemented (TL). Data are expressed as
means ± SD (n=6). Different letters indicate significant differences between groups
(p<0.05).
Figure 6
45
3.2. Artigo 2 foi submetido ao periódico “Nutrition”
_____________________________________________________________________
EFFECTS OF LONG-TERM LEUCINE SUPPLEMENTATION ON METABOLIC AND
MOLECULAR RESPONSES IN THE SKELETAL MUSCLE OF TRAINED RATS
SUBMITTED TO EXHAUSTIVE EXERCISE
Authors:
Gustavo Barbosa dos Santos1, MSc
André Gustavo de Oliveira1, MSc
Maria Cristina Cintra Gomes Marcondes1, PhD
Miguel Arcanjo Areas1, PhD
Affiliation:
1Department of Structural and Functional Biology, Institute of Biology, University of
Campinas (UNICAMP), Campinas, São Paulo, Brazil.
Corresponding author:
G.B. Santos, +55 (019) 3521-6196, [email protected]
Departamento de Biologia Funcional e Molecular
Instituto de Biologia - Caixa Postal 6109
Av. Bertrand Russell - Bloco O
Universidade Estadual de Campinas - UNICAMP
CEP-13083-865
Campinas - SP
Keywords for review: Leucine; endurance performance; exercise; exhaustion;
skeletal muscle; metabolism;
46
Abstract Background: Protein ingestion, during period of endurance training, may positively
affect performance and muscle glycogen resynthesis. Emerging evidence indicates a
role for dietary leucine in promoting adaptive remodeling. Although there is some
evidence of an ergogenic effect of leucine supplementation on acute response to
exercise, there is a paucity of information on whether long-term leucine
supplementation influences the adaptive response to chronic endurance training and
performance.
Aim: The main aim of our study was to assess the role of long-term leucine
supplementation on molecular and metabolic response in skeletal muscle of trained
rats after an exhaustion test.
Methods: Twenty-four male wistar rats were randomly allocated into four groups. Two
of them (control and trained groups) received a balanced control diet (18% protein) and
the other two (control leucine and trained leucine groups) leucine-rich diet (15% protein
with 3% leucine) for 6 weeks. The trained groups were submitted to 1 hour of
swimming exercise, 5 d.wk−1 for 6 wk. Three days after the exercise training period,
trained groups were submitted to swimming exercise until exhaustion and muscle
metabolic and molecular parameters were assessed.
Results: Endurance training increased citrate synthase activity significantly, while
exercise until exhaustion increased cytokine levels, and inhibited protein synthesis
signaling. Long-term leucine supplementation has enhanced muscle glycogen level in
trained rats (0.37±0.16 vs. 0.20±0.01 µmol/ml/min, in TL and C groups, respectively)
and citrate synthase activity in sedentary ones (0.27±0.04 vs. 0.17±0.03 g/100g, in CL
and C groups, respectively). However, it failed to enhance endurance performance of
trained rats submitted to an exhaustion test, and did not prevent exercise-induced
reduction in Akt (significant decrease of 49 and 62% in T and TL groups, respectively,
when compared to C group) and mTOR activation (significant decrease of 47 and 44%
in T and TL groups, respectively, when compared to C group).
Conclusion: Long-term leucine supplementation can improve metabolic parameters of
skeletal muscle (i.e. citrate synthase activity and glycogen content when combined with
exercise), however it does not improve endurance performance nor prevent Akt and
mTOR exercise-induced reduction.
Abbreviations list Akt, Protein kinase B; AMPK, AMP-activated protein kinase α; CS, citrate synthase; IL-
6, interleukin-6; mTOR (mammalian target of rapamycin); p-AMPK, AMPK
phosphorylation; PGC-1α, peroxisome proliferator-activated receptor-gamma
coactivator; TNF-α, tumor necrosis factor-α;
47
Introduction Chronic adaptation of skeletal muscle to high-intensity endurance exercise is strongly
dependent on nutrition (HAWLEY et al., 2011). Protein ingestion, during period of
endurance training, may positively affect performance, muscle glycogen resynthesis.
Emerging evidence indicates a role for dietary protein and amino acids in mitigating
skeletal muscle damage and increasing muscle protein turnover to promote adaptive
remodeling (HAWLEY et al., 2007). It has been demonstrated that branched chain
amino acids, particularly leucine, can reduce prolonged exercise-induced muscle
damage and accelerate the recovery process (GREER et al., 2007). Leucine seems to
be the most potent one regarding the effects on protein synthesis and degradation. It
not only provides substrates for gluconeogenesis, but also can supply tricarboxylic acid
cycle with different anaplerotic substrates (LI et al., 2011). These effects are especially
important for competitive endurance athletes, since they often train or compete
intensely on a daily basis, sometimes until physical exhaustion, generating a constant
state of tissue catabolism and muscle wasting.
Crowe et al. (CROWE et al., 2006) showed that dietary leucine supplementation
significantly improved endurance performance and upper body power in outrigger
canoeists. Furthermore, Rowlands et al (ROWLANDS et al., 2008) assessed the effect
of a high protein recovery diet on endurance performance at different times. Under
these conditions, the high-protein diet had no impact on the 15-h subsequent
performance when compared to isocaloric low protein control group. However, it
substantially enhanced the 60-h subsequent performance, suggesting that the period
when performance is assessed is important, and that a protein supplementation could
have a role in chronic adaptive response to endurance training. Although some
evidence for an ergogenic effect of leucine supplementation on acute response to
exercise, mainly regarding protein synthesis (ROWLANDS et al., 2014) and muscle
recovery (NELSON et al., 2012; THOMSON; ALI; ROWLANDS, 2011), has been
reported, there is a paucity of information on whether long-term leucine
supplementation influences the adaptive response to chronic endurance training and
performance.
The aim of this study was to investigate the role of long-term leucine supplementation
on metabolic and molecular responses in skeletal muscle of trained adult rats
submitted to exercise until exhaustion. We hypothesized that exercise until exhaustion
would induce some muscle disturbances (energetic, structural and biochemical),
leading to fatigue. Also, leucine supplementation could prevent or mitigate these
outcomes, enhancing endurance training-induced muscle adaptation and contributing
to improve endurance exercise performance.
48
Material and Methods
Animals and diets
Twenty-four male wistar rats (12 weeks old, weighing 351 g ± 28.87) were obtained
from the animal facilities of the University of Campinas (São Paulo, Brazil). They were
housed in collective cages at 22-24°C on a 12-h light-and-dark cycle, with free access
to tap water and food. The semi-purified isocaloric diets were a normal protein (C),
containing 18% protein (REEVES et al., 1993); or leucine (L), containing 15% protein
plus 3% of L-leucine. Approximately 70% carbohydrate (sucrose, dextrin and starch),
7% fat (soybean oil) and 5% fiber (purified micro-cellulose) were added to the diets.
Vitamin and mineral mix, as well as cystine and choline, supplemented the diets. The
control diet had 1.6% of L-leucine, and a leucine-rich diet contained 4.6% L-leucine,
according to a previous study from our group (CRUZ; GOMES-MARCONDES, 2014).
Leucine-supplemented diet has led to a significant increase in plasma leucine
concentration in fetuses from tumor-bearing pregnant mice (VIANA; GOMES-
MARCONDES, 2013) and adults rats (data not shown). Two groups were fed the
control diet: sedentary control (C) and trained (T); and two other groups were fed the
leucine-rich diet: control-leucine supplemented (CL) and trained-leucine supplemented
(TL). All the experimental procedures employed were in accordance with the Ethics
Committee on Animal Experimentation of Unicamp (CEEA/IB/UNICAMP, protocol
2888-1).
Training Protocol
The T and TL groups were submitted to the swimming protocol adapted from Santos et
al. (BARBOSA DOS SANTOS et al., 2013), 5 d.wk−1 for 6 wk, in a water tank (90 x 70
x 70 cm and water temperature at 31 ± 1ºC). All of the rats were adapted to the water
during the first week of the experiment. The adaptation process consisted of keeping
the animals in shallow water, initially for 20 min and then progressively increasing 10
min/day and 10 cm water/day for 5 days. Exercise sessions began with 60 min/day at
the second experimental week, carrying constant loads (added to the tail) of 20 g
(approximately 6% of initial body weight). Initially for 10 minutes of these 60 minutes,
increased by 10 more minutes each week, until it reached 60 min of loaded swimming
training in the sixth and last week of experiment. Three days after the exercise training
period, rats were submitted to swimming exercise carrying the same load until
exhaustion. Animals were sacrificed under anesthesia (ketamine and xylazine, 90
mg/kg/bw and 45 mg/kg/bw, respectively, i.p.) between 3-4 hours after exercise bout, in
order to reach cytokine peak after swimming exhaustion test (LOUIS et al., 2007;
SUZUKI et al., 2002).
49
Glycogen content
Gastrocnemius muscles were quickly removed, frozen immediately in liquid nitrogen
and stored at -80°C until further analysis. Muscle glycogen content was estimated
colorimetrically based on the method described by Lo et al. (LO et al., 1970). The
absorbance was read on a plate CHAMELEON V Multilabel Microplate Reader (Hidex,
Finland) at 620nm.
Citrate synthase activity
For citrate synthase analyses ≈30 mg of gastrocnemius muscle was homogenized
in ice cold extraction buffer (175 mM KCl, 2 mM EDTA, pH 7.4), centrifuged at 16.000 x
g, 20 min, at 4°C. An aliquot of supernatant was combined with reaction mixture
containing 0.1 M Tris, pH 8.3, 1 mM DTNB, 3 mM actetyl-CoA. Reaction was initiated
by adding 10 mM oxaloacetic acid to the extract. The absorbance was
spectrophotometrically measured at 412 nm in 30 sec interval for 5 min using a Dynex
MRX plate reader controlled through personal computer software (Revelation,
Dynatech Laboratories), as previously described (SRERE, 1969). All samples were
tested for linearity up to 5 min of reaction and values were normalized by protein
concentration (BRADFORD, 1976).
Western Blot
Muscle samples (40 μg) were homogenized and protein concentration was measured
using a colorimetric method (BRADFORD, 1976). The proteins were revealed using
primary antibodies against α-tubulin (1:20.000), phospho-mTORSer2448 (1:1.000).
Phospho-AktThr308 (1:1.000), phospho-AMPK-αThr172 (1:1.000) (Cell Signaling, Danvers,
MA, USA), proteasome subunits 20S, 19S (1: 1.000) (Enzo, USA), and secondary anti-
mouse, anti-rabbit and anti-goat antibodies (1:10.000, Cell Signaling, Danvers, MA,
USA) after reaction with a chemiluminescent reagent (Thermo Fisher Scientific,
Waltham, MA, USA) were added and band volume was captured using Alliance
Captura 2.7 (UVItec, Cambridge, UK) and quantified using UVI band -1D (UVI tec,
Cambridge, UK).
Inflammatory biomarkers
Blood samples were taken from the heart by ventricle puncture. Serum was separated
by centrifugation at 1,000 x g for 10 min at 4 °C and stored at −80 °C. The analysis of
serum inflammatory markers (TNF-α, IL-6) was determined using beads coupled with
capture antibodies specific for each protein of interest as specified by the manufacture
50
Millipore® (Merck Millipore Corporation, Darmstadt, Germany). The analysis was
carried out on Xponent software used with the Luminex® 200 (Luminex Corporation,
Austin, TX, USA) equipment, following the manufacturer’s technical procedures.
Statistical Analysis
The data is expressed as the mean ± SD. The data was analyzed statistically by
analysis of variance (ANOVA) followed by Tukey’s test to establish differences
between groups. We used Prism software (Graphpad Software Inc., San Diego, CA,
USA). The results were considered significant when P<0.05.
Results Functional Parameters Relative heart weight significantly increased in the T and TL groups when compared to
the C and CL group, although there were no differences in relative gastrocnemius and
soleus weight between groups. Leucine supplementation, regardless of whether they
were submitted to endurance training, significantly increased weight gain even when
compared to their respective control groups. Leucine supplementation did not enhance
exercise performance when compared with exercise only (table 1).
Muscle Metabolic Parameters Citrate synthase (CS) activity is the most important biomarker for mitochondrial density
in skeletal muscle. It is also a biochemical marker of the skeletal muscle oxidative
adaptation to a training intervention. Leucine supplementation significantly increased
CS activity when compared to sedentary groups, but this outcome was overcome when
leucine supplementation was combined with the exercise protocol (figure 1A). The
gastrocnemius glycogen content was assessed in order to reveal the metabolic stress
(i.e. energy demand) of exercise until exhaustion. Glycogen content was significantly
elevated only in trained leucine-supplemented group (TL), when compared to all
experimental groups (figure 1B). AMP kinase α (AMPK) was also assessed to measure
metabolic stress, since AMPK reflects the energy status of the cell. AMPK
phosphorylation (p-AMPK) was significantly enhanced in trained groups compared to
sedentary groups (figure 1C).
Muscle Structural Parameters - Protein Synthesis In order to evaluate treatment-induced muscle protein synthesis, we assessed the
activation of two main key proteins in synthesis pathway, namely, Akt and mTOR.
Activation of Akt was inhibited in trained groups (T and TL) compared to sedentary
groups (C and CL) after exercise until exhaustion. Surprisingly, among trained groups,
leucine supplementation did not prevent Akt inhibition, when compared with exercise
51
only (T group) (figure 2B). A very similar pattern was found in mTOR activation, since
exercise until exhaustion significantly decreased mTOR phosphorylation when
compared to sedentary groups and leucine was not able to prevent this reduction
(figure 2C).
Exercise-induced systemic inflammation We assessed interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels as
inflammatory markers. Although IL-6 level was significantly elevated in trained groups
compared to control groups (figure 3A), TNF-α level did not differ significantly between
groups (figure 3B).
Discussion Although it is generally accepted that leucine supplementation can mitigate endurance
exercise-induced skeletal muscle damage and fatigue, while also improving recovery
and muscle performance (ROWLANDS et al., 2014), recent studies have challenged
this belief. They show no beneficial role of leucine supplementation on performance, or
demonstrate even worsened metabolic response (COSTA JUNIOR et al., 2015;
NELSON et al., 2012). Here we report that leucine supplementation, combined with
endurance training, failed to improve time to exhaustion when compared to endurance
training only. Furthermore, leucine supplementation in endurance-trained rats was not
better than only exercising in improving oxidative capacity and molecular response and
failed to mitigate exercise-induced inflammatory. On the other hand, endurance
exercise training combined with leucine supplementation significantly enhanced
glycogen content when compared to leucine supplementation only or exercise only.
During exercising skeletal muscle is damaged mechanically and metabolic
homeostasis is disturbed (TAM; SIU, 2014). It has been recently demonstrated that
leucine can significantly enhance protein synthesis (ROWLANDS et al., 2014),
glycogen resynthesis (BERARDI; NOREEN; LEMON, 2008) and subsequent high-
intensity endurance performance and may also mitigate muscle damage (GREER et
al., 2007; THOMSON et al., 2011) after endurance exercise. These enhanced
endurance training-induced adaptation, may eventually contribute to improve
endurance exercise performance (HAWLEY et al., 2007). Despite promising current
evidence that using leucine supplementation chronically boosts endurance
performance, our data suggests that it does not seem to be the case in an exercise
until exhaustion scenario. Therefore, the primary novel finding of the present study was
that leucine supplementation, combined with endurance training, does not enhance
exercise until exhaustion performance, nor does it prevent exercise-induced increased
inflammatory response or decreased molecular signaling of protein synthesis.
52
Interestingly, leucine supplementation did not mitigate p-AMPK response until
exhaustive exercise. Since AMPK reflects the energy status of the cell, the great
metabolic stress induced by a bout of exercise until exhaustion was expected to
increase p-AMPK level, and leucine supplementation could mitigate this metabolic
stress and, therefore, AMPK activation. Our hypothesis was partly correct, since
exercise bout did enhance p-AMPK levels and glycogen content was significantly
higher (approximately 95%) in TL group when compared to T group, suggesting a
lower metabolic stress, or at least a higher energy availability. Thus, exercise-induced
fatigue did not occur due to energy substrate depletion in this case. However, the
reason why this lower metabolic stress (i.e. normal muscle glycogen) in TL group did
not lead to a lower p-AMPK concentration remains to be clarified, considering that
muscle glycogen seems to be an important factor in the regulation of muscle AMPK
activity. Also, studies have shown that glycogen can be a powerful negative controller
of AMPK (JORGENSEN; RICHTER; WOJTASZEWSKI, 2006). That calcium signaling
may also have a role in activating AMPK in muscle during exercise has been proposed
(JORGENSEN et al., 2006). It would be plausible to speculate that some other factors
(e.g. calcium signaling) can potentially overrule muscle glycogen depletion and
stimulate AMPK phosphorylation during exercise until exhaustion, even without the
occurrence of significant metabolic stress.
Besides its role as a constituent of protein, leucine also functions as regulator of
translation initiation of protein synthesis, a modulator of the insulin signal cascade, and
a nitrogen donor for muscle production of alanine and glutamine (NORTON; LAYMAN,
2006). Since the potential for leucine to impact on these metabolic processes depends
on its intracellular concentration, and leucine reaches peripheral tissues in direct
proportion to its dietary intake (NORTON; LAYMAN, 2006), we hypothesized that long-
term leucine supplementation could at least mitigate exhaustive endurance exercise-
induced reduction Akt and mTOR phosphorylation. However, protein synthesis
signaling was significantly inhibited after exercise until exhaustion and leucine
supplementation failed to prevent it. Both Akt and mTOR phosphorylation was
significantly reduced in exercise groups (T and TL). Contrary to our findings, Mascher
et al (MASCHER et al., 2007) demonstrated increased skeletal muscle Akt and mTOR
phosphorylation after one hour of endurance training. The type of exercise, the
intensity and duration of the exercise, as well as the exercise training history are critical
factors in the regulation of protein synthesis (CAMERA et al., 2010; COFFEY et al.,
2005) and could explain the different Akt and mTOR responses to the endurance
exercise previously reported. These results are also highly time-point dependent. In our
study they may explain why both Akt and mTOR activation was inhibited after exercise,
53
since a previous study (MASCHER et al., 2007) showed that both have already
returned to basal level 3 hours after endurance exercise. Furthermore, in the present
study, exercise was done until exhaustion, with mean exercise duration of 3 hours and
22 minutes (±10 minutes), which could have led to the different protein synthesis
signaling response. Exhaustive endurance exercise seems to inhibit muscle protein
synthesis with the magnitude of the depression related to the intensity and duration of
the activity. It has been established that skeletal muscle protein synthesis decreases by
about 30% during contractile activity (ROSE; RICHTER, 2009) and protein turnover
remains negative after endurance exercise until adequate dietary protein and energy
are available for recovery (NORTON; LAYMAN, 2006).
In our study, after exercise until exhaustion rats remained in a fasted state, because we
wanted to assess the long-term effect of leucine supplementation. It has been
proposed that ingestion of leucine directly after endurance exercise stimulated protein
synthesis in the recovery period (BLOMSTRAND et al., 2006) which could contribute to
improve endurance exercise performance. Anthony et al (ANTHONY; ANTHONY;
LAYMAN, 1999), have previously demonstrated that leucine ingestion, along with
carbohydrates, after prolonged endurance exercise, enhances muscle glycogen
content, insulin level and muscle protein synthesis. A very recent study has also
demonstrated increased mTOR phosphorylation after endurance exercise and leucine
supplementation (ROWLANDS et al., 2014). Differently from our study, those three
aforementioned studies assessed protein synthesis acutely, that is, right after leucine
ingestion. Nonetheless, while it would be tempting to conclude that acute responses
within specific markers may provide insight into chronic adaptations, the extrapolation
of acute response to potential adaptive responses after a single bout of unfamiliar
exercise should be cautiously interpreted (ATHERTON; SMITH, 2012). Thereby, our
data suggests that leucine-induced enhancement on protein synthesis is rather acute,
and chronic supplementation has no effect on protein synthesis after a bout of
exercise until exhaustion.
Concerning endurance performance, it is hard to explain why long-term leucine
supplementation did not enhance exercise performance, since many metabolic
parameters were improved by leucine supplementation. Surprisingly, leucine
supplementation significantly enhanced CS activity. It was recently demonstrated
(COSTA JUNIOR et al., 2015) that leucine can stimulate PGC-1α activation which
would explain enhanced CS activity. This study also showed enhanced CS activity,
after 12 weeks of leucine supplementation. However, differently from our study, this
outcome was still present in trained leucine-supplemented group. It may have occurred
due to the different exercise intensities between these studies. While we used high
54
intensity endurance exercise followed by a bout of exercise until exhaustion, they used
low intensity exercise. Thus, leucine seems to enhance CS activity but this outcome
can be overcome by exercising, if the intensity is high enough. Oddly, in this prior
study, increased CS activity in the sedentary leucine-supplemented group did not lead
to improvement in exercise performance (VO2max increase). Similarly, in our study, time
to exhaustion was not significantly different between trained groups (table 1),
demonstrating that long-term leucine supplementation did not enhance endurance
performance of the animals submitted to this exercise protocol. This result corroborates
with Araujo Jr. et al. (DE ARAUJO et al., 2006) , who also did not find significant
differences in the time to exhaustion in adult male wistar rats submitted to a swimming
exhaustion test, after six weeks of branched chain amino acids supplementation.
These authors pointed out that a significant decrease in glycaemia during exercise until
exhaustion could explain fatigue and, therefore, performance. Our results suggest that
this seems to be unlikely. Even though we have not assessed glycaemia in our study,
both trained groups presented normal levels of muscle glycogen after exercise until
exhaustion when compared to control groups. Also, TL group presented significant
enhancement of muscle glycogen compared to all experimental groups. Thus, when
combined with endurance exercise, long-term leucine supplementation has beneficial
effects on substrate availability. Furthermore, it has been demonstrated that higher pre-
exercise muscle glycogen concentration can delay fatigue (BERARDI et al., 2006).
Therefore, these results suggest that fatigue may have occurred due to any cause
other than substrate availability in our experimental conditions. Hence, the role of long-
term leucine supplementation on oxidative metabolism and endurance performance
should be further investigated. It has been proposed that exercise-induced ammonia
accumulation could generate fatigue due to excess cerebral ammonia and the inability
of peripheral organs to detoxify it (DE ARAUJO et al., 2006) should be considered.
In order to evaluate inflammation response due to exercise until exhaustion, we
assessed IL-6 and TNF-α levels. Both trained groups presented significant increase in
IL-6 level. It is important to keep in mind that, although mostly regarded as a pro-
inflammatory cytokine, IL-6 also has anti-inflammatory properties (REIHMANE; DELA,
2014; SCHELLER et al., 2011). The exercise-induced increase in plasma IL-6 leads to
increased circulating levels of well-known anti-inflammatory cytokines. Moreover, both
exercise and IL-6 infusion suppress TNF-α production in humans (REIHMANE; DELA,
2014). In our case, there was no difference of TNF-α level between the groups. It may
have occurred because TNF-α is only stimulated by very intense exercise (REIHMANE;
DELA, 2014). Despite its long duration, exercise until exhaustion does not impose
great intensity. Besides, there were no eccentric muscle contractions since it was
55
performed in water. Moreover, IL-6 has an important metabolic function during exercise
and may represent a link between skeletal muscle and organs such as the liver and the
adipose tissue. Studies have clearly demonstrated that contracting muscles, without
any muscle damage, can induce a marked elevation in plasma IL-6 (PEDERSEN et al.,
2004; SCHELLER et al., 2011). IL-6 production is modulated by the carbohydrate
availability in skeletal muscles, suggesting that IL-6 acts as an “energy sensor”
(REIHMANE; DELA, 2014). IL-6 was shown to enhance AMPK activity in both skeletal
muscle and adipose tissue (PEDERSEN et al., 2007), which has occurred in the
present study.
In conclusion, although long-term leucine supplementation has enhanced the muscle
glycogen level in trained rats and citrate synthase activity in sedentary ones, it failed to
enhance endurance performance of trained rats submitted to an exhaustion test.
Furthermore, it did not prevent exercise-induced reduction in protein synthesis
signaling. These results suggest that nutrient-mediated acute molecular and metabolic
effects that may support homeostatic restoration and adaptive remodeling of skeletal
muscle to endurance exercise does not necessarily occur in long-term training.
Acknowledgments We are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) for a fellowship granted to Santos, G.B., and to Ajinomoto Interamericana
Indústria e Comércio Ltda (Brazil) for supplying the leucine.
Conflict of Interest The authors declare no conflict of interest.
Author Contribution All authors approved the final version for publication.
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59
FIGURE CAPTIONS Figure 1. Effects of leucine supplementation and endurance training on: A, citrate
synthase activity (µmol/ml/min). B, gastrocnemius glycogen content (g/100g). C,
representative blot for AMPK phosphorylation. D, AMPK activation. Experimental
groups were divided as follows: sedentary control (C), control-supplemented (CL),
trained (T) and trained-supplemented (TL). Data are expressed as means ± SD (n=6).
Different letters indicate significant differences between groups (p<0.05).
Figure 2. Effects of leucine supplementation and endurance training on protein
synthesis signaling. A, representative blot for Akt and mTOR phosphorylation. B, Akt
activation. C, mTOR activation. Experimental groups were divided as follows:
sedentary control (C), control-supplemented (CL), trained (T) and trained-
supplemented (TL). Data are expressed as means ± SD (n=6). Different letters indicate
significant differences between groups (p<0.05).
Figure 3. Effects of leucine supplementation and endurance training on inflammatory
response biomarkers. A, IL-6 content. B, TNF-α content. Experimental groups were
divided as follows: sedentary control (C), control-supplemented (CL), trained (T) and
trained-supplemented (TL). Data are expressed as means ± SD (n=6). Different letters
indicate significant differences between groups (p<0.05).
60
Table 1
Data are expressed as means±SD (n=6). Different letters indicate significant differences between groups (p<0.05).
C CL T TL
Body weight gain (g) 76.40±11.52a 102.2±10.57b 64.17±13.89a 92.17±11.99b
Heart (g/100g) 3.0±0.26a 2.8±0.19a 3.6±0.44b 3.5±0.19b
Gastrocnemius (g/100g)
6.9±0.43 6.2±1.6 6.8±0.84 5.6±0.44
Soleus (g/100g) 0.36±0.04 0.32±0.03 0.38±0.03 0.33±0.05
Time to exhaustion (min)
- - 208.9±19.5 195.3±9.8
61
Figure 1
D
62
Figure 2
63
Figure 3
64
4. Conclusão
Após a análise molecular e funcional dos músculos cardíaco e esquelético em ratos
suplementados com leucina e submetidos ao exercício prolongado e extenuante de
natação, concluímos que:
1. O exercício extenuante de endurance, nas condições experimentais deste
estudo, não excedeu a capacidade energética cardíaca, não representando,
portanto, uma ameaça energética a este órgão, pelo menos, em indivíduos
treinados.
2. O exercício extenuante de longa duração pode induzir, em indivíduos
suscetíveis, um estado de instabilidade elétrica cardíaca, associado a arritmias
e morte súbita. Esta situação pode ser potencializada quando combinada à
suplementação de leucina, que induziu ao aumento da pressão arterial e lesão
cardíaca.
3. A suplementação de leucina, além de não prevenir os sintomas da fadiga
cardíaca, pode ainda, agravar as alterações cardiovasculares induzidas pelo
exercício extenuante de longa duração.
4. Embora a suplementação de leucina possa aumentar a concentração de
glicogênio muscular (gastrocnêmio), em ratos treinados, e a atividade da
enzima citrato sintase, em ratos sedentários, ela falhou em melhorar o
desempenho de endurance em ratos treinados submetidos a um teste de
exaustão e não preveniu a diminuição na sinalização de síntese proteica no
musculo esquelético.
Assim, estes resultados sugerem que os efeitos metabólicos e moleculares agudos,
mediados pela leucina, que, em teoria, poderiam de forma crônica, potencializar a
resposta adaptativa do músculo cardíaco e esquelético ao exercício de endurance não
ocorrem. Concluímos, também, que os músculos cardíaco e esquelético, respondem
de forma diferente ao estresse mecânico e metabólico induzido pelo exercício
extenuante de longa duração. E por último, que a leucina não é uma substância capaz
de auxiliar na prevenção dos efeitos cardíacos adversos induzidos pelo exercício
extenuante de longa duração, podendo, inclusive, potencializar estes efeitos.
65
5. Atividade concomitante à tese
Artigo publicado: “Melatonin reduces oxidative stress and cardiovascular
changes induced by stanozolol in rats exposed to swimming exercise” Barbosa
Dos Santos G, Machado Rodrigues MJ, Gonçalves EM, Cintra Gomes
Marcondes MC, Areas MA. Eurasian J Med. 2013 Oct;45(3):155-62. doi:
10.5152/eajm.2013.33.
66
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7. Anexos
Profa. Dra. Rachel Meneguello
Presidente
Comissão Central de Pós-Graduação
Declaração
As cópias de artigos de minha autoria ou de minha co-autoria, já publicados ou
submetidos para publicação em revistas científicas ou anais de congressos sujeitos a
arbitragem, que constam da minha Dissertação/Tese de Mestrado/Doutorado,
intitulada "Alterações cardiovasculares induzidas pelo exercício extenuante de
longa duração em ratos suplementados com leucina", não infringem os
dispositivos da Lei n.° 9.610/98, nem o direito autoral de qualquer editora.
Campinas, 20 / 05 / 2015
__________________________________
Autor Gustavo Barbosa dos Santos
RG n.° 25.282.527-5
________________________________
Orientador Prof. Dr. Miguel Arcanjo Areas
RG n.° 6.874.289-7
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