Alzheimer’s disease (AD), the most common form of dementia in the elderly, is a global issue affecting about 24 million individuals. Because AD is a systemic pathology, dementia is not the only leading factor contributing to loss of independence in AD patients. AD may also impair skeletal muscle metabolism and function. Creatine (CR) supplementation may enhance skeletal muscle hypertrophy/mass and function in sarcopenia and muscular dystrophies, but has yet to be studied in AD. This study examined the effect of oral CR on muscle metabolism in a triple-transgenic (3xTg) AD mouse model. Twenty-four, 3×Tg AD mice (~8 month-old) were randomly assigned to control (CON) or CR (3% w/w) diet. Bodyweights and feed intakes were measured throughout the 8-week study. Lower limb (quadriceps muscle; QM and gastrocnemius; GM) and upper limb muscles (triceps; TM) were collected to analyze levels of CR, total protein, DNA, RNA, amino acids (AA), adenosine triphosphate (ATP), adenosine diphosphate (ADP), total and phosphorylated p70 ribosomal S6 kinase (p70S6K). Data (mean ± SEM) were assessed by analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) post hoc test. In comparison to the CON group, CR supplementation increased CR content in both GM (p=0.002) and QM (p=0.037), with higher (p=0.032) ATP/ADP ratio in CR in comparison with CON in QM. A higher protein concentration (p<0.0001) was notable in GM of CR supplemented group vs. CON. Total branched-chain AA levels in QM increased 2-fold (p< 0.0001) in CR groups. Additionally, CR resulted in a higher (p<0.05) protein/DNA ratio; an index of muscle cell size, in both QM and GM for CR groups. The index of cell capacity for protein synthesis (RNA/DNA ratio) in GM was also higher (p=0.001) in CR groups. However, phosphorylation (activation) level of p70S6K, an integral component in protein synthesis signalling pathway, did not show any significant differences in female (p=0.161) and male (p=0.292) CR supplemented groups compared with CON. To conclude, CR supplementation is capable of inducing muscle hypertrophy/growth parameters in the 3×Tg AD mouse model, thereby enhancing protein synthesis capacity in skeletal muscles, thus possibly promoting muscle function in AD. / May 2016

Acute effects of repeated sprinting upon serum creatine kinase (CK), lactic dehydrogenase (LDH), and isozymal activities were studied in five collegiate runners (R_s) and six non-runners (NR_s ). After an intermittent running treadmill test, blood sampling showed three-fold mean increases in CK with no change in LDH in both groups; group differences were insignificant (p>.05). Results suggest (1) intense anaerobic exercise produces moderate enzyme elevations; (2) relatively equivalent exercise intensities are critical to enzyme responses in exercising individuals of varying fitness levels; and (3) exercise-induced enzyme release may be consequential to muscle cell membrane permeability changes from decreased intracellular high-energy phosphates.

Heart failure (HP) is a common, disabling and deadly condition that causes high rates of morbidity and mortality worldwide. It is widely recognised that the failing heart is energy-starved, and that restoring energy homeostasis is a promising approach towards improving cardiac output. This thesis aims to address the role of energetics in the failing heart, by focussing on modulation of the creatine transporter (CrT). Creatine (Cr), together with the phosphocreatine shuttle, plays a vital role in maintaining energy supplies via ATP in times of high energy demand. Key to the regulation of intracellular [Cr] is the CrT, a Na+ and Cl - dependent membrane transporter. Previous CrT genetic mouse models include a knockout model, found to still express cardiac CrT, and a cardiac-specific CrT overexpressing (OE) model with large variations in myocardial [Cr] between animals and Cr levels high enough to cause spontaneous hypertrophy. To overcome the shortfalls of this CrT-OE model, a novel in vivo model of temporal inducible expression of CrT is described, using a cardiac-specific tetracycline inducible (Tet-On) system . ..,. .A' Ten transgenic lines (RCT) were created with a construct containing . zhe CrT-HA (CrT cDNA with an haemagglutinin epitope tag), following successful doxycyline-inducibility in vitro. Eight lines showed germline transmission, with LV CrT OE achieved in an individual mouse that displayed double LV [Cr] compared to WT. Issues with the inducer line (rtTA) were ruled out by its use in the creation of a luciferase overexpressing mouse line; all mice tested demonstrated LV luciferase expression in response to doxycycline feeding. The failure to overexpress CrT could be attributed to position or copy number dependent suppression, or to position effect variegation in the case of the single OE mouse obtained. Subsequent work focus sed on regulatory pathways in vitro in a cell line of mouse fibroblasts stably overexpressing CrT·HA. Post-translational modifications (PTMs) had been previously suggested to regulate CrT activity. Two N-linked glycosylation sites exist, in addition to the putative phosphorylation sites. Inhibition of glycosylation by tunicamycin led to decreased CrT activity, reflected by decreased Cr uptake capacity. Strategies to confirm the presence of phosphorylation were employed, including isolation of CrT -HA by immunoprecipitation and subsequent LC-MS / MS analysis to identify PTMs. Although the presence of CrT was confirmed in 5 different sized species- one previously unreported- inadequate sequence coverage prevented identification of any PTM sites. Tyrosine phosphorylation was not detected using a phosphospecific antibody on immunopurified CrT -HA. Candidate signalling pathways in vitro were then investigated to elucidate CrT regulation, namely the IGF-IR signalling pathway. This study included a cardiomyocyte-like mouse cell line (HL-l) in addition to 3T3-CrT -HA. Exposure of cells to extracellular insulin, growth hormone and IGF-1 led to increased Cr uptake of 125% - 300% of normal. Pharmacological inhibition of the downstream kinases PKA and PKC reduced the effect of insulin and GH, while PMA, sapintoxin (STX) and Go 6976 induced CrT activity. The mammalian target of rapamycin (mTOR) is also a candidate regulator of CrT, as incubation with rapamycin decreased Cr uptake in 3T3-CrT -HA. Finally, a targeted approach on transcription factors in the 5'UTR region of mouse CrT identified HEYl as a highly conserved site. In siRNA experiments, HEYl was found to exert a mild effect on CrT activity, suggesting that regulation at the transcriptional level merits further investigation. Together, this work has provided novel insights into the modulation of CrT in vitro, identifying molecular and pharmacological targets in a known therapeutic signalling pathway. Further work could potentially develop these findings by identifying candidate compounds that would increase CrT activity, potentially in a tissue-specific manner. 3

Changes in myocardial energetics have been implicated in the pathophysiology of heart failure (HF). However, the precise contribution of creatine (Cr) / phosphocreatine (PCr) / creatine kinase (CK) energy buffer and transfer remains unclear. The aim of this thesis was to study the effects on murine cardiac function of both impairment and enhancement of creatine metabolism. In order to longitudinally follow the cause and effect relationship of myocardial creatine concentration, a non-invasive method of quantification was required. Cardiac Cr levels measured in vivo by 1H-MRS were therefore compared with gold-standard invasive HPLC and found to correlate over a wide-range (r2=0.91). 1H-MRS was reproducible for measuring Cr levels in the heart, brain, and skeletal muscle. The cardiac phenotype of a novel model of creatine depletion, the AGAT-/- mouse, was characterized using in vivo MRI, 1H-MRS and LV catheterisation, under conditions of gradually reducing Cr concentrations; zero Cr; and attempted phenotype rescue with dietary Cr. For the first time in the heart, the rate of Cr turnover was quantified (~3 % per day) and demonstrated that cardiac function was preserved even when creatine levels reduced by ~70-90%. Total absence of myocardial Cr induced impairment of inotropic and lusitropic cardiac function and reduced inotropic reserve. Cardiac dysfunction was only partially rescued by replenishment of the Cr pool, suggesting this to be a consequence of long-term adaptations to chronic low Cr. Finally, we tested the hypothesis that combined elevation of myocardial creatine and ribose would be beneficial in a mouse model of chronic HF by increasing cardiac energy availability. Despite an increase in myocardial ribose concentration, this did not prevent loss of total adenine nucleotides (TAN), and there was no improvement in post-infarct LV remodeling or function. Future studies are needed to explore alternative approaches for maintaining TAN in combination with total creatine.

O exercício de enduro é caracterizado por um esforço aeróbico prolongado, de intensidade variável em que o cavalo é submetido a um trabalho permanente o qual exige dos sistemas orgânicos a manutenção da homeostasia. A habilidade dos músculos em gerar energia rapidamente via produção de lactato, é essencial para o desempenho em exercícios de elevada intensidade. Entretanto, a produção de lactato pode também suprimir muitos dos processos vitais necessários pra sustentar a atividade muscular. A associação de lactato com a fadiga muscular tem levado à busca de suplementos alimentares que reduzem o acúmulo de lactato. N,N-Dimetilglicina (DMG), um intermediário do metabolismo da colina, é um suplemento atualmente comercializado, porém não há dados consistentes na literatura sobre sua eficácia para equinos atletas. Objetivando avaliar o efeito da DMG, utilizou-se 12 animais em treinamento para provas de enduro, dos quais seis receberam suplementação oral; utilizou-se delineamento inteiramente casualizado, com medidas repetidas no tempo. Foram coletadas amostras de sangue em sete tempos diferentes, a cada 15 dias; dessas amostras obteve-se valores das enzimas creatina quinase e aspartato aminotransferase e também valores de glicose e lactato, além de medições de freqüência cardíaca e respiratória. Após 30 dias da última amostragem, realizaram-se coletas de sangue em intervalos curtos de tempo, após exercício, a fim de se realizar uma curva de lactato, e também comparar dados analisados em laboratório com analisados via lactímetro. Dos dados analisados, houve interação entre tempo e aumento da enzima creatina quinase, o que pode ser justificado pelo aumento da demanda muscular durante exercício físico constante. A enzima aspartato aminotransferase diminuiu com o passar do tempo, em ambos os grupos, porém oscilou dentro da normalidade, o que pode caracterizar baixa permeabilidade da membrana celular, comum em animais condicionados. A alteração da glicose foi a mesma ao longo do tempo para ambos os grupos. As médias de lactato não diferiram no grupo suplementado, mas sua variação dentro do grupo não suplementado sugere que a suplementação com DMG pode influenciar na manutenção da integridade muscular. Em comparação entre as formas de dosagem do lactato plasmático, pode-se concluir que o lactímetro é uma ferramenta eficaz na obtenção de dados a campo, pois suas médias não diferiram das médias de lactato obtidas através de análise laboratorial. A suplementação oral com dimetilglicina não influenciou o desempenho atlético de cavalos em treinamento para enduro equestre. / The exercise endurance is characterized by a prolonged aerobic work, of varying intensity in which the horse is subjected to a permanent job which requires organ systems maintain homeostasis. The ability of muscles to generate energy quickly via production of lactate is essential for the performance of high intensity exercise. However, the production of lactate may also suppress many of the vital processes necessary to sustain muscle activity. The combination of lactate in muscle fatigue has led to the search for dietary supplements that reduce the accumulation of lactate. N, N-dimethylglycine (DMG), an intermediate in the metabolism of choline, is a supplement currently marketed, but there is no consistent data in the literature on its effectiveness for equine athletes. In order to evaluate the effect of DMG, we used 12 animals in training for endurance events, of which six received oral supplementation, we used a completely randomized design with repeated measures on time. Blood samples were collected in seven different times, every 15 days, these samples gave values of the enzymes creatine kinase and aspartate aminotransferase values as well as glucose and lactate, and easurements of heart rate and breathing. After 30 days the last sampling, there were blood samples at short intervals of time after exercise in order to perform a lactate curve, and also compare data analyzed in laboratory and analyzed via lactimeter. Of the data analyzed, there was interaction between time and increased the enzyme creatine kinase, which can be explained by increased demand constant muscle during exercise. The enzyme aspartate aminotransferase decreased over time in both groups, but varied within the normal range, which can characterize low permeability of the cell membrane, which is common in animals conditioned. The change in glucose was the same over time for both groups. The mean lactate did not differ in the supplemented group, but the variation in the unsupplemented group suggests that supplementation with DMG can influence the maintenance of muscle integrity. In comparison dosage forms of lactate, it can be concluded that the lactimeter is an effective tool for getting data into the field, because their means did not differ from average lactate obtained through laboratory analysis. Oral supplementation with dimethylglicine did not influence athletic performance of horses in training for endurance riding.

A realização de uma sessão com ações excêntricas provoca dano na estrutura muscular. Durante o processo de recuperação, essa estrutura sofre adaptações que a protegem da ocorrência de dano nas sessões subseqüentes. Essas adaptações são chamadas de Efeito da Carga Repetida (ECR). Esse efeito foi estudado com a realização de apenas duas sessões de exercícios. A velocidade da ação excêntrica também pode contribuir para a variabilidade do dano induzido. O objetivo desse estudo foi investigar através da análise dos indicadores indiretos, creatina quinase (CK), força, dor, circunferência e amplitude de movimento (ADM), o dano induzido por diferentes velocidades da ação excêntrica e o efeito da carga repetida com a realização das diferentes velocidades (60ºs-1 (Exc60) e 180ºs-1 (Exc180)); e verificar se o efeito da carga repetida seria maior com a realização de três sessões de exercícios. Os resultados dos indicadores analisados tiveram alterações semelhantes nos grupos Exc60 e Exc180, sugerindo que as diferentes velocidades parecem não interferir na magnitude do dano induzido. O ECR não foi diferente entre as velocidades, pois o comportamento das variáveis analisadas foi semelhante entre os dois grupos nas duas sessões iniciais. A realização da terceira sessão de exercícios excêntricos não promove o aumento do efeito protetor, visto que não houve diferenças significantes entre a segunda e a terceira sessão. Indicando que o ECR advém principalmente da realização da primeira sessão / Performing a bout of eccentric exercise causes muscle damage. During recovery, some adaptations occur that can protect muscle structure. These adaptations are known as Repeated Bout Effect. However, this phenomenon has been studied with two bouts. Velocity of eccentric action has been referred as one possible factor which can affect the extension of muscle damage. The aim of this study was to investigate muscle damage induced by different velocities, the repeated bout effect with different velocities and to verify if the repeated bout effect could be larger if three bouts of eccentric exercise were performed. Results of indirect markers of muscle damage (CK, DOR, upper-arm circumference, maximal isometric force) showed similar alterations in groups Exc60 and Exc180, suggesting that different velocities do not affect the extension of muscle damage. Repeated bout effect is not different between velocities, because changes in markers were comparable in both groups after the first two bouts. After performing a third bout of eccentric exercise, there was not any significant differences between second and third bouts. It indicates that the first bout is responsible for the adaptations of the repeated bout effect

Hypertrophy of the heart is tightly bound to the metabolic adaptations and a cellular remodeling. An important and dynamic system contributing to the maintenance of energy homeostasis is the creatine kinase system (CK). The microcompartmentalization of CK isoforms maintains the flux of ATP between energy production and consumption sites and ensures the effectiveness of the CK system. Changes in expression and activity of CK isoforms during hypertrophy are already well described - to extend this knowledge, this thesis quantified changes in association of cytosolic CK isoforms and sarcomeres. Another essential system, maintaining homeostasis in overloaded heart is composed of the hexokinase (HK) isoforms, located also in cytosol and in mitochondrial compartment. HK1 is associated with mitochondria under physiological conditions, maintaining mitochondrial membrane potential, while HK2 is located mainly in the cytosol. Under stress conditions translocation of HK2 into mitochondrial membrane occurs, which increases the direct supply of ADP to complex V of the respiratory chain and decreases the probability of apoptosis activation. We analyzed association of individual HK isoforms with mitochondria within the second aim of this thesis. Third aim of the thesis was to characterize changes in the CK and M...

Purpose: To examine the effects of deer antler velvet on muscular strength, performance, and markers of delayed onset muscle soreness following a 10-week resistance training period. Participants: 16 resistance-trained males (18-35) volunteered. Measures: DEXA, 1-RM, a power test, and a 70% performance trial were measured. Creatine kinase and self-reported soreness levels were measured following an eccentric trial. Results: No pre-experimental significant differences existed between the groups for any of the variables measured. There were no significant differences between the groups regarding body composition, strength, muscular performance, or improvements in creatine kinase and soreness levels from pre to post-intervention. Both groups demonstrated significant (p<0.05) increases in creatine kinase and soreness levels immediately post-exercise and 48 hours following the eccentric trial at the 0 and 10-week measurement periods. Conclusions: Deer antler velvet does not improve muscle size, strength, or performance. Nor does it reduce markers of DOMS following a 10-week supplementation period.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "May 2003." Typescript. Vita. Includes bibliographical references (leaves 102-113).

Alzheimer Disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and dementia. Both energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain. With synchrotron FTIR microscopy, extensive deposits of crystalline creatine (Cr) had been discovered in TgCRND8 mouse brain tissue by previous students in our lab. In this thesis, regions of hippocampus and caudate of 5 pairs of transgenic mice and their non-transgenic littermate controls were mapped using Raman and IR microspectroscopy to find clues to Cr origin in transgenic mouse brain. Raman spectra obtained at higher spatial resolution (1-2 µm) were used for better delineation of the Cr crystalline deposits and their environs. These results indicate that Cr crystals were formed after snap-freezing and desiccation of brain tissue. Therefore, it can be speculated that Cr might be exist in solution form in vivo.