Orientador: Maria Cristina Cintra Gomes Marcondes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T13:25:51Z (GMT). No. of bitstreams: 1 Cruz_BreadLeandroGomesda_D.pdf: 1846989 bytes, checksum: 30a328ba2d64a0e66c6bb74d4c81d492 (MD5) Previous issue date: 2014 / Resumo: O câncer é uma das principais causas de morte no mundo e o quadro de caquexia provocado por alguns tipos de tumor é um dos grandes responsáveis por isso. A caquexia instaurada em pacientes com câncer, sendo mais prevalente nos tumores gastrointestinal, pulmonar, pancreático e mamário, é caracterizada, dentre outros processos, pela perda involuntária de peso, devido a constante espoliação sobre a massa magra corporal. Estudos que tenham como objetivo a manutenção da massa magra em organismos portadores de tumor caquético são importantes para contribuir com a redução de óbitos e preservar a qualidade de vida das pessoas com câncer. Nos últimos anos, a leucina tem mostrado ser eficaz na manutenção da massa magra corpórea através do estímulo de síntese protéica muscular e inibição da degradação de proteína, principalmente da massa magra corpórea. Logo, entender como a presença da leucina estimula a síntese protéica e atua de forma protetora em organismo no estado caquético tem se mostrado uma necessidade crescente. Desse modo, a proposta deste trabalho foi avaliar, ao longo do tempo, os efeitos de dieta rica em leucina sobre a sinalização de síntese e degradação protéica, envolvendo o complexo mTOR em músculos de ratos portadores do carcinossarcoma de Walker 256. Os animais foram distribuídos em grupos de acordo com a inoculação tumoral e/ou esquema nutricional com dieta rica em leucina, sendo sacrificados em três momentos diferentes do desenvolvimento tumoral (7º, 14º e 21º dias após a implantação do tumor). No músculo gastrocnêmio foram analisadas as proteínas-chave da via mTOR, como RAG A GTPase, ERK/MAP4K3, PKB/Akt, mTOR, p70S6K1, Jnk, IRS-1, STAT3, e STAT6 e, tambem, foram avaliadas as proteínas de degradação protéica pertencentes ao sistema ubiquitina-proteossomo (11S, 19S e 20S) e citocinas IL-4, IL-6, IL-10, TNF? e INF?. Os resultados mostram que o desenvolvimento tumoral reduziu a ativação de RAG-A, associada com queda de IRS-1, aumento da PKB/Akt e Erk/MAP4K3 no 21º dia e manutenção de p70S6K1; também houve aumento dos níveis de STAT-3 e STAT-6 em ratos portadores de tumor. Entretanto, a presença de leucina na dieta modulou etapas chave da via mTOR pelo desencadeamento da ativação aumentada de RAG-A e mTOR junto com a manutenção dos níveis de JNK, STAT-3 e STAT-6 no músculo durante o desenvolvimento do tumor de Walker no organismo caquético. A análise da sinalização para degradação protéica mostrou que o crescimento tumoral promoveu, simultaneamente, diminuição de proteína muscular, acentuado aumento de citocinas pró-inflamatórias (TNF?, IL-6 e IFN?) e aumento progressivo das subunidades proteossômicas (19S e 20S), sendo que a suplementação com leucina atenuou essa ativação. Os resultados obtidos apoiam o efeito benéfico do uso de leucina e esclarece as vias metabólicas utilizadas por este aminoácido, contribuindo para melhor compreensão da ação in vivo desse aminoácido sobre a caquexia / Abstract: Cancer is one of the most important causes of death worldwide and the process of cachexia caused by some types of tumour is largely responsible for this. Cancer-cachexia state established in these patients is characterized, among other processes, by involuntary loss of weight due to constant spoliation of lean body mass. Many studies aim to focus in maintenance of lean body mass in cachectic tumour-bearing host, contributing to the reduction of deaths and improving the quality of life of cancer patients. In recent years, leucine has been shown to be effective in maintaining lean body mass by stimulating muscle protein synthesis and inhibiting the proteolysis. Therefore, there are increased needs in understanding how the presence of leucine stimulates protein synthesis and acts protectively in the cachectic state. The aim of this work is to evaluate the effects of leucine-rich diet in a time-course model on signalling protein synthesis and degradation involving mTOR complex in muscles of Walker 256 carcinoma-bearing rats. Animals were divided into experimental groups based on the tumour inoculation and/or fed a nutritional supplementation diet rich in leucine. Animals were sacrificed at three different times depending on the tumour development (7, 14 and 21 days after tumour implantation), and the gastrocnemius muscles were analysed as mTOR pathway and ubiquitin-proteasome via. The results showed that the tumour development has reduced the activation of RAG-A, associated with a decrease of IRS-1, increased PKB / Akt and Erk / MAP4K3 at day 21 and maintaining p70S6K1, there has also been increasing levels of STAT-3 and STAT-6 in tumour-bearing rats. Meanwhile, the presence of leucine in the diet modulated the key steps of the mTOR pathway for triggering the increased RAG-A and mTOR activation along with the maintenance of levels of JNK, STAT-3 and STAT-6 in the muscle during tumour development in cachectic host. The gastrocnemius muscle signalling of protein degradation indicated by the ubiquitin-proteasome subunits (11S, 19S and 20S) and pro- and anti-inflammatory cytokines were marked increase of pro-inflammatory cytokines (TNF?, IL-6 and IFN?) and a progressive increase in the proteasome subunits (19S and 20S) associated with simultaneously decreased muscle protein. The supplementation with leucine attenuated these parameters, suggesting the beneficial effect of the use of leucine and clarifies the metabolic pathways used by this amino acid, contributing to better understanding of the in vivo action of this amino acid on cachexia / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Loss of muscle due to aging is often associated with significant detrimental effects. Therefore, it is crucial to understand signaling molecules that may trigger the muscle loss or prevent the process. The transcription factor, Nuclear Factor Kappa-B (NF-κB), is associated with both catabolic and anabolic pathways of muscle metabolism and may be involved in age-related muscle loss. Leucine is an essential amino acid that is required for both protein synthesis and intracellular signaling pathways that regulate protein synthesis and degradation. The current study examined muscle NF-kB levels in male Sprague-Dawley rats, aged 6 (adult) and 21 months (old) fed a diet containing suboptimal leucine levels for 10-17 days. We found that old rats consumed less grams of food per body weight (BW) each day than adult rats (1.45% g diet/g BW vs. 2.4% g diet/g BW). Weight loss during the study was not significantly different between age groups. However the average mass of gastrocnemius and soleus muscles (g muscle/g BW) was significantly lower in old rats. Reduction in gastrocnemius (g muscle/g BW*10²) was associated with 1.8 fold higher muscle cell NF-κB in old vs. adult rats (p = 0.0443). There was also a higher level of ubiquitinated proteins in old gastrocnemius muscle cells relative to the adult gastrocnemius, however differences did not reach statistical significance. For tibialis anterior muscle, the average mass (g muscle/g BW*10²), NF-κB levels and ubiquitinated proteins were not significantly different between adult and old rats. Our findings suggest that aging affects muscle loss and NF-kB in a tissue-specific manner in rats fed a diet with suboptimal leucine levels.

The effects of supplementation of the leucine metabolite β-hydroxy-β- methylbutyrate (HMB) were examined in a resistance training study. Thirty- nine men and 36 women between the ages of 20-40 y were randomized to either a placebo (P) supplemented or HMB supplemented (3.0 g HMB/d) group in two gender cohorts. All subjects trained three times per week for 4 wk. In the HMB group, plasma creatine phosphokinase levels tended to be suppressed compared to the placebo group following the 4 wk of resistance training (HMB:174.4 ± 26.8 to 173.5 ± 17.0 U/L; P:155.0 ± 20.8 to 195.2 ± 23.5 U/L). There were no significant differences in strength gains based on prior training status or gender with HMB supplementation. The HMB group had a greater increase in upper body strength than the placebo group (HMB:7.5 ± 0.6 kg; P:5.2 ± 0.6 kg; P = 0.008). The HMB groups increased fat-free weight by 1.4 ± 0.2 kg and decreased percent fat by 1.1% ± 0.2% while the placebo groups increased fat-free weight by 0.9 ± 0.2 kg and decreased percent fat by 0.5% ± 0.2% (fat-free weight P = 0.08, percent fat P = 0.08, HMB compared to placebo). In summary, this is the first short-term study to investigate the roles of gender and training status on the effects of HMB supplementation on strength and body composition. This study showed, regardless of gender or training status, HMB may increase upper body strength and minimize muscle damage when combined with an exercise program.

Chick feeding tests were conducted to study the effects of dietary imbalances among the three branched chain amino acids on growth rate. All diets fed in these tests contained about 18% protein. The indispensable amino acids (IAA) were found in two of the diets fed in the proportions found by Dobson et al. (1964) to be well balanced. One of the diets had all IAA at 85% of these balanced levels while the other had them all at 125% of these levels. The tests involved reducing the isoleucine, leucine and valine levels in the high IAA diet to the low levels in all combinations. Growth rates were similar with the diets containing all ten IAA at the low levels, all ten at the high levels, or seven at the high levels and isoleucine, leucine and valine at the low levels. Generally, when only one or two of these three were reduced to the low levels, growth rates were lower. Thus there appeared to be a three-way interaction among these amino acids with the reduction in weight gain being the result of an imbalance and not of a deficiency. The changes in growth rate noted when the leucine level was reduced indicated that the leucine level in Dobson's balanced diet was relatively high; the isoleucine level appeared to be relatively low. The interaction between leucine and valine appeared to be more significant than the other two two-way interactions. The effect of dietary level of these amino acids on the branched chain amino acid transaminase (BAT) activity in the liver and kidneys was determined. The differences noted were inconsistent. There tended to be a slightly higher activity in chicks fed the high levels of these amino acids, but the differences certainly were not as great as the changes in arginase activity reported by others when imbalanced diets were fed. Chicks fed the diet low in valine and high in the other nine IAA were selected for fast or slow growth on this diet. They were raised to maturity and produced eggs that were hatched for feeding tests with the diets containing different levels of the branched chain amino acids. Only a limited number of chicks from the two strains were hatched. Performance of the chicks was similar to that of their parents when fed the low valine diet.

No description available.

Endurance exercise has been shown to both raise and lower leucine oxidation in studies in rodents. We studied the effects of a 38 d endurance exercise training program upon leucine turnover during a 90 min exercise bout at 60 % VO_2peak in 6 males and 6 females. Subjects were studied at both the same absolute (ABS) and relative (REL) exercise intensities post-training. Pre (PRE)- and post-training measurements were taken for analysis of: L-[1-^13C]leucine turnover, muscle branched-chain oxoacid dehydrogenase activity (BCOAD), muscle glycogen, phosphocreatine and ATP utilization, and resting enzyme activity of citrate synthase (CS) and NADH-cytochrome c-oxidoreductase (complex I-III). We also determined total substrate oxidation by indirect calorimetry, and plasma lactate, glucose, and insulin concentrations. The exercise training resulted in a significant increase in both CS (P < 0.001) and complex I- III (P < 0.05) activities. Leucine oxidation increased during exercise for the pre-training trial (P < 0.001), however, there was no increase for either the post-training ABS or REL trial. Leucine oxidation was significantly lower for females at all time points (P < 0.01). Total BCOAD activity was also significantly increased when comparing the PRE to both ABS and REL trials (P < 0.001). The % activation of BCOAD was significantly increased from t=0 to t=90 in both the PRE and REL exercise trials with the increase in PRE being greater (P < 0.001 (PRE), and P < 0.05 (REL)). Exercise RER was lower for females vs. males (P< 0.05). In addition, the ABS trial was significantly lower than PRE and REL (P < 0.01). Plasma lactate was significantly lower at all time points for ABS vs. PRE (P < 0.001) and REL vs. PRE at t=30 min of exercise (P < 0.001). Resting muscle glycogen was higher for both ABS and REL vs. PRE (P < 0.001). In conclusion, we found that 38 d of endurance exercise training significantly attenuated both leucine oxidation and BCOAD activation during 90 min of endurance exercise at 60 % VO_2peak for both absolute and relative exercise intensities. In addition, females were also shown to oxidize a greater proportion of energy from lipid and a lesser amount from carbohydrates and proteins during exercise. / Thesis / Master of Science (MS)

Duchene muscular dystrophy (DMD) is a lethal X-linked disease that afflicts approximately 1 in 3500 newborn males. Boys with DMD will become progressively weaker causing wheelchair dependence by their early teens and death by their mid to late twenties. Currently there is no cure for DMD, the exact mechanism of disease action remains elusive, and treatments to improve quality of life are limited. Two areas of DMD research that could begin to fill this void and provide simple, cost effective therapy aimed to improve quality of life are neutriceutical and exercise therapies. We hypothesized that leucine, a branched chain amino acid (BCAA) with anabolic properties, given to sedentary and exercised x-linked dystrophic mice (mdx) over 4 weeks would improve skeletal muscle function and decrease markers of skeletal muscle degradation. In sedentary mdx mice, leucine improved tetanic extensor digitorum longus (EDL) stress (p < 0.05), gastrocnemius mammalian target or rapamycin (mTOR) phosphorylation (p < 0.05), while decreasing the rate of real-time calpain activity in flexor digitorum brevis (FDB) fibers (p < 0.05) compared to sedentary mice given no leucine. In exercised mdx mice, leucine improved total running distance over the 4 week testing period by 40% (p < 0.02) and increased EDL stress at every frequency recorded (p < 0.05). Our data lead us to the conclusion that the BCAA leucine can increase EDL muscle stress in dystrophic animals, and that the effects of leucine treatment are enhanced when leucine supplementation is combined with exercise. Leucine supplementation should be explored further and in higher order species of muscular dystrophy to determine if its use could provide clinical improvements in DMD patients. / Ph. D.

Résumé : La protéolyse par les proprotéines convertases (PC) représente une étape cruciale de maturation pour de nombreux peptides et protéines destinés aux voies de sécrétions cellulaires. Parmi ces substrats des PC, de nombreuses molécules participent aux étapes clés de progression tumorale. L’enzyme PACE4, une des PC, est d’ailleurs surexprimée dans le cancer de la prostate et des études antérieures réalisées dans notre laboratoire démontrent que cette protéase occupe un rôle essentiel et non redondant dans la progression du cancer de la prostate. Puisque PACE4 représente une cible thérapeutique potentielle pour le traitement du cancer de la prostate, l’objectif principal des travaux présentés dans cette thèse vise à développer un inhibiteur pharmacologique de PACE4 et d’évaluer son potentiel thérapeutique. Le développement d’inhibiteur spécifique à la PACE4 représente un défi de taille, puisque le site actif des PC démontre un fort niveau d’homologie. Bien que les sous-sites S1 à S4 des PC soient hautement conservés, la littérature suggère qu’il existe des déterminants moléculaires exploitables au sein des sous-sites S5 à S8. Ainsi, ces observations suggèrent qu’une sélectivité d’inhibition pourrait provenir de courts peptides. En analysant les profils d’inhibition obtenus pour divers peptides inhibiteurs des PC, la séquence Ac-LLLLRVKR-NH[indice inférieur 2], nommée Multi-Leu, a été identifiée comme inhibiteur permettant d’obtenir une préférence d’inhibition 20 fois plus importante pour PACE4 que pour Furine. Dans le but d’améliorer les propriétés pharmacocinétiques du composé et permettre son utilisation comme inhibiteur pharmacologique, des études de relation structure-activité ont été conduites. Ces études ont permis de déterminer que le peptide Multi-Leu est sujet à une dégradation par des exopeptidases, et par conséquent l’addition d’acides aminés non naturels aux extrémités N et C-terminales permet d’augmenter la stabilité du composé. Suivant cette étude, la modification amidinobenzylamide (Amba) a été introduite en position P1, permettant d’augmenter la puissance d’inhibition du composé, ainsi qu’un stéréoisomère D-Leucine en position P8. La caractérisation du potentiel inhibiteur du peptide Ac-[DLeu]LLLRVK-Amba démontre l’efficacité du peptide in vivo alors qu’une administration par voie intra veineuse du composé parvient à freiner la progression tumorale dans un modèle de xénogreffes de cancer de la prostate. Dans ce modèle, l’analogue du peptide Multi-Leu parvient à bloquer la néovascularisation tumorale en plus d’induire la quiescence et l’apoptose dans les tumeurs traitées. // Abstract : Numerous secreted peptides or proteins require a proteolytic activation by the proprotein convertases (PC) to fully gain their biologic activities. Among substrates of the PC family, there exist various cancer-related molecules. The enzyme PACE4, one of the seven kexinlike PC has been demonstrated to be overexpressed in prostate cancer and to have a nonredundant role in prostate cancer progression. Since PACE4 is a validated therapeutic target for prostate cancer, the main aim of this thesis was to develop a pharmacologic PACE4 inhibitor and to evaluate its therapeutic potential. The development of PACE4 specific inhibitors represents a challenge since PC share an important homology level within their active site. Whereas S1 to S4 subsites appear to be highly homologous within the PC family, litterature suggests that significant differences exist in subsites S5 and beyond, suggesting that peptide compound could result in specific inhibitors. In this thesis, the Multi-Leu peptide (Ac-LLLLRVKR-NH[subscript 2]) was identified as a selective PACE4 inhibitor, which displays a 20-fold inhibitory preference toward PACE4 over furin. In order to improve Multi-Leu peptide pharmacokinetic profile, structureactivities relationship studies were performed and allowed for the identification of two modifications that increase both inhibitory properties and stability of this molecule. Peptide resulting from introduction of an arginine mimetic residue amidinobenzylamide (Amba) in P1 and a stereoisomer D-Leucine in position P8 displayed an improved pharmacokinetic profile. Futhermore, intravenous administration of the compound Ac-[DLeu]LLLRVKAmba significantly inhibited prostate cancer progression in a LNCaP xenograft model of prostate cancer. This Multi-Leu peptide analog also inhibited tumor neovascularisation along with inducing cell quiescence and apoptosis in tumors of treated animals.

Expression studies have implicated Fibronectin Leucine Rich Transmembrane protein 2 (FLRT2) in cranial neural crest cell migration and pre-chondrogenic cell condensation during craniofacial skeletogenesis. This aim of this study was to characterize the expression of FLRT2 and its relationship to the extracellular matrix (ECM) in ATDC5 chondroprogenitor cells. Immunofluorescence studies localized FLRT2 to the cell membrane as well as exracellularly, where it colocalized with fibronectin. FLRT2 was identified in the ATDC5-derived ECM after cell extraction. Further to its colocalization with fibronectin, FLRT2 associated with fibronectin-coated beads in cell cultures. Co-immunoprecipitation confirmed that FLRT2 and fibronectin interact, either directly or indirectly. Blocking fibronectin fibril formation in ATDC5 cell cultures demonstrated a concomitant decrease in extracellular FLRT2 accumulation. It appears that FLRT2 may exist in both a membrane-bound and a shed form. Either or both of these forms may participate in cell-ECM interactions in cooperation with fibronectin or other ECM proteins.

Expression studies have implicated Fibronectin Leucine Rich Transmembrane protein 2 (FLRT2) in cranial neural crest cell migration and pre-chondrogenic cell condensation during craniofacial skeletogenesis. This aim of this study was to characterize the expression of FLRT2 and its relationship to the extracellular matrix (ECM) in ATDC5 chondroprogenitor cells. Immunofluorescence studies localized FLRT2 to the cell membrane as well as exracellularly, where it colocalized with fibronectin. FLRT2 was identified in the ATDC5-derived ECM after cell extraction. Further to its colocalization with fibronectin, FLRT2 associated with fibronectin-coated beads in cell cultures. Co-immunoprecipitation confirmed that FLRT2 and fibronectin interact, either directly or indirectly. Blocking fibronectin fibril formation in ATDC5 cell cultures demonstrated a concomitant decrease in extracellular FLRT2 accumulation. It appears that FLRT2 may exist in both a membrane-bound and a shed form. Either or both of these forms may participate in cell-ECM interactions in cooperation with fibronectin or other ECM proteins.