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Orientador: Jaime Amaya-Farfán Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos Resumo: As proteínas do soro do leite (PSL) têm mostrado características de alimento funcional e suas propriedades podem ser ampliadas durante a digestão. Um dos efeitos do consumo da PSL hidrolisada (PSLH) é a indução da produção das heat shock proteins (HSPs), que são proteínas endógenas integrantes de um sistema protetor que promove a resistência e tolerância celular diante de condições adversas ao organismo. Evidências apontam que a indução das HSPs promove a citoproteção de fibras musculares, sobrevida celular, preservação da função muscular, reparação do dano muscular e prevenção da injúria térmica e oxidativa. Em estudo de nossa autoria, mostramos que a PSLH é capaz de aumentar a expressão a HSP70, hipotetizando-se que peptídeos bioativos presentes na PSLH atuem como sinalizadores metabólicos e expliquem várias das funções positivas atribuídas ao hidrolisado. Ainda, é sabido que a suplementação com glutamina estimula a expressão do sistema HSP, inclusive durante o exercício, porém pouco se sabe sobre a possibilidade de que outros aminoácidos possam exercer efeitos similares ao da glutamina. O objetivo do presente estudo foi investigar o efeito das proteínas, alguns peptídeos e aminoácidos, presentes nas PSL, nas HSPs (HSP25, HSP60, HSP70, HSP90) e parâmetros metabólicos relacionados, em ratos submetidos ao exercício como fonte de alteração da homeostase. O estudo foi dividido em três experimentos: o primeiro, usando a PSLH como fonte proteica da dieta, o segundo, com cinco peptídeos bioativos (Isoleucil-leucina (lle-Leu), Leucil-isoleucina (Leu-lle), Valil-leucina (Val-Leu), Leucil-valina (Leu-Val)) e, o terceiro, com os aminoácidos (L-Leucina, L-isoleucina, L-valina, L-arginina, L-glutamina). Todos os animais foram submetidos ao exercício agudo em esteira rolante com a intenção de induzir o estresse e a alteração na homeostase, exceto os grupos controle, que permaneceram em repouso. Os resultados mostraram que o consumo da PSLH aumentou a expressão da HSP90 em sedentários e exercitados; entretanto nenhuma alteração foi provocada pelo exercício ou pela dieta na HSP60 ou HSP25. O peptídeo Leu-Val aumentou a expressão das HSP90, HSP70 e HSP25 no músculo, enquanto que o lle-Leu elevou a HSP70 muscular e sérica, e HSP60 no músculo. A suplementação com arginina induziu a HSP70 e HSP90 na mesma intensidade que a glutamina. Todos os aminoácidos elevaram a HSP60, em associação com o exercício, exceto a valina. Nenhum dos aminoácidos afetou a expressão da HSP25. Com base nos dados conclui-se que, enquanto a PSLH estimula as HSPs e vários fatores relacionados à preservação celular em músculo de ratos, os dipeptídeos Leu-Val e lle-Leu se destacaram por estimular a expressão de HSPs e diversos outros parâmetros, e que a arginina pode ter função protetora, semelhante à da glutamina. Sugere-se ainda que os peptídeos e aminoácidos selecionados estejam envolvidos ou sejam os responsáveis pelas várias funções positivas atribuídas ao consumo da proteína do soro do leite hidrolisada e, especialmente, como alternativa para elevar as HSPs e atenuar injúrias celulares Abstract: The whey proteins (WPC) are known to have functional food characteristics, which may be magnified during digestion. One of the advantages hydrolyzed WPC (WPH) is to induce the production of heat shock proteins (HSPs), known to be endogenous proteins, members of a cell-shielding system that promotes cell resistance to adverse conditions to the body. Evidence suggests that the induction of HSPs promotes cytoprotection of muscle fibers, cell survival, preservation of muscle function, muscle damage repair and prevention of thermal and oxidative injury. In a study of our own we proved that PSLH is able to increase the expression of HSP70, hypothesizing that bioactive peptides present in PSLH act as metabolic flags thus explaining many of the positive functions assigned to the hydrolyzate. In addition, it is known that glutamine supplementation stimulates the expression of the HSP system, including during exercise, but the possibility that other amino acids could exert similar effects to those of glutamine has not been explored. The aim of this study was, therefore, to investigate the effect that the protein, some strategic peptides and amino acids, present in the whey hydrolyzate could have on the HSPs (HSP25, HAP60, HSP70, HSP90) and related parameters in rats that were exercised to offset homeostasis. The study was divided in three experiments: in the first, the PSLH was the dietary protein source, the second was a supplementation study with five bioactive peptides (isoleucyl-leucine (Ile-Leu) Leucyl-isoleucine (Leu-Ile), valyl-leucine (Val-Leu) Leucyl-valine (Leu-Val)), and the third with free amino acids: L-Leucine, L-isoleucine, L-valine, L-arginine and L-glutamine. All animals were submitted to acute exercise on a treadmill with the intention of inducing stress and offset homeostasis, except the control group, which remained at rest. The results showed that consumption of WPH increased the expression of HSP90 in both sedentary and exercised animals. However, no changes were caused by exercise or diet in the HSP60 or HSP25. Leu-Val increased HSP90, HSP70 and HSP25 expression in muscle, while Ile-Leu increased muscle and serum HSP70 and HSP60 in muscle. Supplementation with arginine induced HSP70 and HSP90 at the same intensity as glutamine. All amino acids increased HSP60 associated with the exercise, except for valine, but none affected the expression of HSP25. Based on the above data we conclude that while WPH stimulates HSPs and stimulates various factors related to cell preservation in rat muscle, dipeptides Leu-Val and Ile-Leu stood out for stimulating the expression of HSPs and various other parameters, and that arginine may have a protective function, similar to that of glutamine. The data also suggest that the selected peptides and amino acids are responsible for several positive functions attributed to the consumption of hydrolyzed whey protein and especially as an alternative to raise HSPs and mitigate cellular injuries Doutorado Nutrição Experimental e Aplicada à Tecnologia de Alimentos Doutora em Alimentos e Nutrição FAPESP 2013/02862-0

Made available in DSpace on 2015-10-21T13:13:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-03-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Aim: To examine the influence of L-arginine supplementation in combination with physical training on mitochondrial biomarkers from gastrocnemius muscle and its relationship with physical performance.Main methods: Male Wistar rats were divided into four groups: control sedentary (SD), sedentary supplemented with L-arginine (SDLA), trained (TR) and trained supplemented with L-arginine (TRLA). Supplementation of L-arginine was administered by gavage (62.5 mg/ml/day/rat). Physical training consisted of 60 min/day, 5 days/week, 0% grade, speed of 1.2 km/h. The study lasted 8 weeks. Skeletal muscle mitochondrial enriched fraction as well as cytoplasmic fractions were obtained for Western blotting and biochemical analyses. Protein expressions of transcriptor coactivator (PGC-l alpha.), transcriptor factors (mtTFA), ATP synthase subunit c, cytochrome oxidase (COXIV), constitutive nitric oxide synthases (eNOS and nNOS), Cu/Zn-superoxide dismutase (SOD) and manganese-SOD (Mn-SOD) were evaluated. We also assessed in plasma: lipid profile, glycemia and malondialdehyde (MDA) levels. The nitrite/nitrate (NOx-) levels were measured in both plasma and cytosol fraction of the gastrocnemius muscle.Key findings: 8-week L-arginine supplementation associated with physical training was effective in promoting greater tolerance to exercise that was accompanied by up-regulation of the protein expressions of mtTFA, PGC-1 alpha, ATP synthase subunit c, COXIV, Cu/Zn-SOD and Mn-SOD. The upstream pathway was associated with improvement of NO bioavailability, but not in NO production since no changes in nNOS or eNOS protein expressions were observed.Significance: This combination would be an alternative approach for preventing cardiometabolic diseases given that in overt diseases a profound impairment in the physical performance of the patients is observed. (C) 2015 Elsevier Inc. All rights reserved. Univ Estadual Paulista, UNESP, Inst Biosci, Lab Cardiovasc Physiol &Phys Act, Rio Claro, SP, Brazil Univ Estadual Campinas, Inst Biol, Dept Struct &Funct Biol, Campinas, SP, Brazil Univ Estadual Campinas, UNICAMP, FCM, Clin Pathol, Campinas, SP, Brazil Univ Estadual Campinas, Fac Food Engn, Dept Food &Nutr, Campinas, SP, Brazil Univ Estadual Paulista, UNESP, Inst Biosci, Lab Cardiovasc Physiol &Phys Act, Departamento de Educação Física, Rio Claro, SP, Brazil