Ursolic acid directly promotes protein accretion in myotubes but does not affect myoblast proliferation

Ursolic acid (UA) has been recently proposed as a potential candidate for the treatment of muscle wasting conditions because of its protein sparring/anabolic effects. Despite this finding, it is unknown whether this response is the consequence of a direct effect on the muscle fibre or if it is mediated by neural or other systemic factors. In the present study, we sought to determine if UA has direct effects in skeletal muscle cells, whether it can increase myoblast proliferation and whether UA can become myotoxic at higher doses. Our results demonstrate that UA directly promoted protein accretion in cultured myotubes but did not modulate myoblast proliferation. At higher doses, UA compromised cell viability in both myoblasts and myotubes. We conclude that the anabolic properties of UA seen in vivo and in vitro are likely a direct effect on the muscle cell, but at higher doses, the benefits decline in favour of a myotoxic outcome. Copyright (C) 2012 John Wiley & Sons, Ltd.

Myostatin: genetic variants, therapy and gene doping

Since its discovery, myostatin (MSTN) has been at the forefront of muscle therapy research because intrinsic mutations or inhibition of this protein, by either pharmacological or genetic means, result in muscle hypertrophy and hyperplasia. In addition to muscle growth, MSTN inhibition potentially disturbs connective tissue, leads to strength modulation, facilitates myoblast transplantation, promotes tissue regeneration, induces adipose tissue thermogenesis and increases muscle oxidative phenotype. It is also known that current advances in gene therapy have an impact on sports because of the illicit use of such methods. However, the adverse effects of these methods, their impact on athletic performance in humans and the means of detecting gene doping are as yet unknown. The aim of the present review is to discuss biosynthesis, genetic variants, pharmacological/genetic manipulation, doping and athletic performance in relation to the MSTN pathway. As will be concluded from the manuscript, MSTN emerges as a promising molecule for combating muscle wasting diseases and for triggering wide-ranging discussion in view of its possible use in gene doping.

Transcriptional dynamics of the human DMD locus

The human DMD locus encodes dystrophin protein. Absence or reduced levels of dystrophin (DMD or BMD phenotype, respectively) lead to progressive muscle wasting. Little is known about the complex coordination of dystrophin expression and its transcriptional regulation is a field of intense interest. In this work we found that DMD locus harbours multiple long non coding RNAs which orchestrate and control transcription of muscle dystrophin mRNA isoforms. These lncRNAs are tissue-specific and highly expressed during myogenesis, suggesting a possible role in tissue-specific expression of DMD gene isoforms. Their forced ectopic expression in human muscle and neuronal cells leads to a specific and negative regulation of endogenous dystrophin full lenght isoforms. An intriguing aspect regarding the transcription of the DMD locus is the gene size (2.4Mb). The mechanism that ensures the complete synthesis of the primary transcript and the coordinated splicing of 79 exons is still completely unknown. By ChIP-on-chip analyses, we discovered novel regions never been involved before in the transcription regulation of the DMD locus. Specifically, we observed enrichments for Pol II, P-Ser2, P-Ser5, Ac-H3 and 2Me-H3K4 in an intronic region of 3Kb (approximately 21Kb) downstream of the end of DMD exon 52 and in a region of 4Kb spanning the DMD exon 62. Interestingly, this latter region and the TSS of Dp71 are strongly marked by 3Me-H3K36, an histone modification associated with the regulation of splicing process. Furthermore, we also observed strong presence of open chromatin marks (Ac-H3 and 2Me-H3K4) around intron 34 and the exon 45 without presence of RNA pol II. We speculate that these two regions may exert an enhancer-like function on Dp427m promoter, although further investigations are necessary. Finally, we investigated the nuclear-cytoplasmic compartmentalization of the muscular dystrophin mRNA and, specifically, we verified whether the exon skipping therapy could influence its cellular distribution.

Body composition and fuel metabolism after kidney grafting

Kidney transplant patients display decreased muscle mass and increased fat mass. Whether this altered body composition is due to glucocorticoid induced altered fuel metabolism is unclear. To answer this question, 16 kidney transplant patients were examined immediately after kidney transplantation (12 +/- 4 days, mean +/- SEM) and then during months 2, 5, 11 and 16, respectively, by whole body dual energy X-ray absorptiometry (Hologic QDR 1000W) and indirect calorimetry. Results were compared with those of 16 age, sex and body mass index matched healthy volunteers examined only once. All patients received dietary counselling with a step 1 diet of the American Heart Association and were advised to restrict their caloric intake to the resting energy expenditure plus 30%. Immediately after transplantation, lean mass of the trunk was higher by 7 +/- 1% (P < 0.05) and that of the limbs was lower by more than 10% (P < 0.01) in patients than in controls. In contrast, no difference in fat mass and resting energy expenditure could be detected between patients and controls. During the 16 months of observation, total fat mass increased in male (+4.9 +/- 1.5 kg), but not in female patients (0.1 +/- 0.8 kg). The change in fat mass observed in men was due to an increase in all subregions of the body analysed (trunk, arms+legs as well as head+neck), whereas in women only an increase in head+neck by 9 +/- 2% (P = 0.05) was detected. Body fat distribution remained unchanged in both sexes over the 16 months of observation. Lean mass of the trunk mainly decreased between days 11 and 42 (P < 0.01) and remained stable thereafter. After day 42, lean mass of arms and legs (mostly striated muscle) and head+neck progressively increased over the 14 months of observation by 1.6 +/- 0.6 kg (P < 0.05) and 0.4 +/- 0.1 kg (P < 0.01), respectively. Resting energy expenditure was similar in controls and patients at 42 days (30.0 +/- 0.7 vs. 31.0 +/- 0.9 kcal kg-1 lean mass) and did not change during the following 15 months of observation. However, composition of fuel used to sustain resting energy expenditure in the fasting state was altered in patients when compared with normal subjects, i.e. glucose oxidation was higher by more than 45% in patients (P < 0.01) during the second month after grafting, but gradually declined (P < 0.01) over the following 15 months to values similar to those observed in controls. Protein oxidation was elevated in renal transplant patients on prednisone at first measurement, a difference which tended to decline over the study period. In contrast to glucose and protein oxidation, fat oxidation was lower in patients 42 days after grafting (P < 0.01), but increased by more than 100% reaching values similar to those observed in controls after 16 months of study. Mean daily dose of prednisone per kg body weight correlated with the three components of fuel oxidation (r > 0.93, P < 0.01), i.e. protein, glucose and fat oxidation. These results indicate that in prednisone treated renal transplant patients fuel metabolism is regulated in a dose-dependent manner. Moreover, dietary measures, such as caloric and fat intake restriction as well as increase of protein intake, can prevent muscle wasting as well as part of the usually observed fat accumulation. Furthermore, the concept of preferential upper body fat accumulation as consequence of prednisone therapy in renal transplant patients has to be revised.

Analysis of the E1-ubiquitin activating enzyme, Uba1, in cell death and tissue growth in Drosophila

Ubiquitination is an essential process involved in basic biological processes such as the cell cycle and cell death. Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Subsequently, ubiquitin is transferred to target proteins via ubiquitin ligases (E3). Defects in ubiquitin conjugation have been implicated in several forms of malignancy, the pathogenesis of several genetic diseases, immune surveillance/viral pathogenesis, and the pathology of muscle wasting. However, the consequences of partial or complete loss of ubiquitin conjugation in multi-cellular organisms are not well understood. Here, we report the characterization of nba1, the sole E1 in Drosophila. We have determined that weak and strong nba1 alleluias behave genetically different and sometimes in opposing phenotypes. For example, weak uba1 alleluias protect cells from cell death whereas cells containing strong loss-of-function alleluias are highly apoptotic. These opposing phenotypes are due to differing sensitivities of cell death pathway components to ubiquitination level alterations. In addition, strong uba1 alleluias induce cell cycle arrest due to defects in the protein degradation of Cyclins. Surprisingly, clones of strong uba1 mutant alleluias stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner resulting in severe overgrowth phenotypes in the mosaic fly. I have determined that the observed overgrowth phenotypes were due to a failure to downregulate the Notch signaling pathway in nba1 mutant cells. Aberrant Notch signaling results in the secretion of a local cytokine and activation of JAK/STAT pathway in neighboring cells. In addition, we elucidated a model describing the regulation of the caspase Dronc in surviving cells. Binding of Dronc by its inhibitor Diap1 is necessary but not sufficient to inhibit Dronc function. Ubiquitin conjugation and Uba1 function is necessary for the negative regulation of Dronc. ^

A second promoter provides an alternative target for therapeutic up-regulation of utrophin in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an inherited muscle-wasting disease caused by the absence of a muscle cytoskeletal protein, dystrophin. We have previously shown that utrophin, the autosomal homologue of dystrophin, is able to compensate for the absence of dystrophin in a mouse model of DMD; we have therefore undertaken a detailed study of the transcriptional regulation of utrophin to identify means of effecting its up-regulation in DMD muscle. We have previously isolated a promoter element lying within the CpG island at the 5′ end of the gene and have shown it to be synaptically regulated in vivo. In this paper, we show that there is an alternative promoter lying within the large second intron of the utrophin gene, 50 kb 3′ to exon 2. The promoter is highly regulated and drives transcription of a widely expressed unique first exon that splices into a common full-length mRNA at exon 3. The two utrophin promoters are independently regulated, and we predict that they respond to discrete sets of cellular signals. These findings significantly contribute to understanding the molecular physiology of utrophin expression and are important because the promoter reported here provides an alternative target for transcriptional activation of utrophin in DMD muscle. This promoter does not contain synaptic regulatory elements and might, therefore, be a more suitable target for pharmacological manipulation than the previously described promoter.

Efeitos do treinamento de força no músculo esquelético em ratos com caquexia induzida pelo câncer

A ausência de terapias eficazes para a caquexia permanece como um problema central para o tratamento do câncer no mundo. Em contrapartida, o treinamento de força (i.e. também conhecido como treinamento resistido) tem sido amplamente utilizado como uma estratégia não farmacológica anticatabólica, prevenindo a perda da massa e da função da musculatura esquelética. Entretanto, o papel terapêutico do treinamento de força na caquexia do câncer permanece apenas especulativo. Portanto, nesse estudo avaliamos se o treinamento de força poderia atenuar a perda da massa e da função da musculatura esquelética em um severo modelo de caquexia do câncer em ratos. Para isso, ratos machos da linhagem Wistar foram randomizados em quatro grupos experimentais: 1) ratos sedentários injetados com solução salina na medula óssea (Controle); 2) ratos injetados com solução salina na medula óssea e submetidos ao treinamento de força (Controle + T); 3) ratos sedentários injetados com células do tumor Walker 256 na medula óssea (Tumor); e 4) ratos injetados com células do tumor Walker 256 na medula óssea e submetidos ao treinamento de força (Tumor + T). Foram avaliados a massa e a área de secção transversa da musculatura esquelética, marcadores de disfunção metabólica e do turnover proteico, a função da musculatura esquelética in vivo e ex vivo, o consumo alimentar, o crescimento tumoral e a sobrevida dos grupos experimentais com tumor. O grupo Tumor apresentou atrofia muscular após quinze dias da injeção das células tumorais como pode ser observado pela redução na massa dos músculos Plantaris (- 20,5%) e EDL (-20%). A atrofia no músculo EDL foi confirmada por análises histológicas, demonstrando uma redução de 43,8% na área de secção transversa. Embora o treinamento de força tenha aumentado o conteúdo proteico da lactato desidrogenase e revertido totalmente o conteúdo da forma fosforilada de 4EBP-1 (i.e. repressor da transcrição de mRNA), ele não atuou na morfologia da musculatura esquelética nos animais com tumor. Além disso, o treinamento de força não atenuou a perda de função da musculatura esquelética, a anorexia, o crescimento tumoral ou a taxa de mortalidade. Contudo, a força muscular, avaliada pelo teste de 1RM, apresentou uma correlação negativa com a sobrevida dos animais (p = 0,02), sugerindo que a perda de força prediz a mortalidade nesse modelo experimental de caquexia do câncer. Em suma, a injeção de células do tumor Walker 256 na medula óssea induz caquexia do câncer em ratos. O treinamento de força não foi eficaz em atenuar a perda de massa e função da musculatura esquelética nesse modelo. Entretanto, a força muscular prediz a sobrevida dos animais, sugerindo que novos estudos são necessários para elucidar o possível efeito terapêutico do treinamento de força para atenuar a caquexia do câncer e a progressão tumoral

Dysfunction of respiratory muscles in critically ill patients on the intensive care unit.

Muscular weakness and muscle wasting may often be observed in critically ill patients on intensive care units (ICUs) and may present as failure to wean from mechanical ventilation. Importantly, mounting data demonstrate that mechanical ventilation itself may induce progressive dysfunction of the main respiratory muscle, i.e. the diaphragm. The respective condition was termed 'ventilator-induced diaphragmatic dysfunction' (VIDD) and should be distinguished from peripheral muscular weakness as observed in 'ICU-acquired weakness (ICU-AW)'. Interestingly, VIDD and ICU-AW may often be observed in critically ill patients with, e.g. severe sepsis or septic shock, and recent data demonstrate that the pathophysiology of these conditions may overlap. VIDD may mainly be characterized on a histopathological level as disuse muscular atrophy, and data demonstrate increased proteolysis and decreased protein synthesis as important underlying pathomechanisms. However, atrophy alone does not explain the observed loss of muscular force. When, e.g. isolated muscle strips are examined and force is normalized for cross-sectional fibre area, the loss is disproportionally larger than would be expected by atrophy alone. Nevertheless, although the exact molecular pathways for the induction of proteolytic systems remain incompletely understood, data now suggest that VIDD may also be triggered by mechanisms including decreased diaphragmatic blood flow or increased oxidative stress. Here we provide a concise review on the available literature on respiratory muscle weakness and VIDD in the critically ill. Potential underlying pathomechanisms will be discussed before the background of current diagnostic options. Furthermore, we will elucidate and speculate on potential novel future therapeutic avenues.

Cleavage of caspases-1, -3, -6, -8 and -9 substrates by proteases in skeletal muscles from mice undergoing cancer cachexia

A prominent feature of several type of cancer is cachexia. This syndrome causes a marked loss of lean body mass and muscle wasting, and appears to be mediated by cytokines and tumour products. There are several proteases and proteolytic pathways that could be responsible for the protein breakdown. In the present study, we investigated whether caspases are involved in the proteolytic process of skeletal muscle catabolism observed in a murine model of cancer cachexia (MAC16), in comparison with a related tumour (MAC13), which does not induce cachexia. Using specific peptide substrates, there was an increase of 54% in the proteolytic activity of caspase-1, 84% of caspase-8, 98% of caspase-3 151% to caspase-6 and 177% of caspase-9, in the gastrocnemius muscle of animals bearing the MAC16 tumour (up to 25% weight loss), in relation to muscle from animals bearing the MAC13 tumour (1-5% weight loss). The dual pattern of 89 kDa and 25 kDa fragmentation of poly (ADP-ribose) polymerase (PARP) occurred in the muscle samples from animals bearing the MAC16 tumour and with a high amount of caspase-like activity. Cytochrome c was present in the cytosolic fractions of gastrocnemius muscles from both groups of animals, suggesting that cytochrome c release from mitochondria may be involved in caspase activation. There was no evidence for DNA fragmentation into a nucleosomal ladder typical of apoptosis in the muscles of either group of mice. This data supports a role for caspases in the catabolic events in muscle involved in the cancer cachexia syndrome. © 2001 Cancer Research Campaign.

The 'cancer cachectic factor'

The object of this study was to summarize information on catabolic factors produced by tumours which lead to tissue catabolism in cancer cachexia and to use this information for the development of effective therapy. The study population was made up of patients with cancer cachexia and weight loss greater than 1 kg month-1. They had a varied range of carcinomas, particularly pancreatic, but also of the breast, ovary, lung, colon and rectum. Cachectic factors were isolated by standard biochemical methods, and the mechanism of tissue catabolism was evaluated in vitro and in vivo. We isolated a 24-kDa sulphated glycoprotein produced by cachexia-inducing murine and human tumours, which induces catabolism of myofibrillar proteins in skeletal muscle and for this reason has been named proteolysis-inducing factor (PIF). PIF was shown to be present in a diverse range of carcinomas in patients whose rate of weight loss exceeded 1.0 kg month-1. Administration of PIF to normal mice produced a rapid decrease in body weight, which arose primarily from a loss of skeletal muscle, accompanied by increased mRNA levels for ubiquitin, the ubiquitin-carrier protein (E214k), and proteasome subunits. This suggests that PIF induces protein catabolism through an increased expression of the key components of the ATP-ubiquitin-dependent proteolytic pathway. The action of PIF was attenuated both in vitro and in vivo by eicosapentaenoic acid (EPA). Oral EPA has been found to stabilize the body weight of patients with advanced pancreatic cancer and, when combined with an energy- and protein-rich nutritional supplement, to produce weight gain arising solely from an increase in lean body mass. Nutritional supplementation alone is unable to reverse the process of muscle wasting in cancer patients, since this arises from activation of the ubiquitin proteasome pathway by PIF, which is independent of nutrient intake. EPA is able to down-regulate the increased expression of this pathway and prevents muscle wasting in cancer patients.

Induction of protein catabolism and the ubiquitin-proteasome pathway by mild oxidative stress

Muscle wasting in cancer cachexia is associated with increased levels of malondialdehyde (MDA) in gastrocnemius muscles, suggesting an increased oxidative stress. To determine whether oxidative stress contributes to muscle protein catabolism, an in vitro model system, consisting of C2C12 myotubes, was treated with either 0.2 mM FeSO4, 0.1 mM H2O2, or both, to replicate the rise in MDA content in cachexia. All treatments caused an increased protein catabolism and a decreased myosin expression. There was an increase in the proteasome chymotrypsin-like enzyme activity, while immunoblotting showed an increased expression of the 20S proteasome α-subunits, p42, and the ubiquitin-conjugating enzyme, E214k. These results show that mild oxidative stress increases protein degradation in skeletal muscle by causing an increased expression of the major components of the ubiquitin-proteasome pathway. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

In vitro assessment of the combined effect of eicosapentaenoic acid, green tea extract and curcumin C3 on protein loss in C2C12 myotubes

EPA has been clinically shown to reduce muscle wasting during cancer cachexia. This study investigates whether curcumin or green tea extract (GTE) enhances the ability of low doses of eicosapentaenoic acid (EPA) to reduce loss of muscle protein in an in vitro model. A low dose of EPA with minimal anti-cachectic activity was chosen to evaluate any potential synergistic effect with curcumin or GTE. Depression of protein synthesis and increase in degradation was determined in C2C12 myotubes in response to tumour necrosis factor-α (TNF-α) and proteolysis-inducing factor (PIF). EPA (50 μM) or curcumin (10 μg ml−1) alone had little effect on protein degradation caused by PIF but the combination produced complete inhibition, as did the combination with GTE (10 μg ml−1). In response to TNF-α (25 ng ml−1)-induced protein degradation, EPA had a small, but not significant effect on protein degradation; however, when curcumin and GTE were combined with EPA, the effect was enhanced. EPA completely attenuated the depression of protein synthesis caused by TNF-α, but not that caused by PIF. The combination of EPA with curcumin produced a significant increase in protein synthesis to both agents. GTE alone or in combination with EPA had no effect on the depression of protein synthesis by TNF-α, but did significantly increase protein synthesis in PIF-treated cells. Both TNF-α and PIF significantly reduced myotube diameter from 17 to 13 μm for TNF-α (23.5%) and 15 μm (11.8%) for PIF However the triple combination of EPA, curcumin and GTE returned diameters to values not significantly different from the control. These results suggest that either curcumin or GTE or the combination could enhance the anti-catabolic effect of EPA on lean body mass.

Cancer cachexia

Purpose of review: Although cachexia has a major effect on both the morbidity and mortality of cancer patients, information on the mechanisms responsible for this condition is limited. This review summarizes recent data in this area. Recent findings: Cachexia is defined as loss of muscle, with or without fat, frequently associated with anorexia, inflammation and insulin resistance. Loss of adipose mass is due to an increased lipolysis through an increased expression of hormone-sensitive lipase. Adipose tissue does not contribute to the inflammatory response. There is an increased phosphorylation of both protein kinase R (PKR) and eukaryotic initiation factor 2 on the α-subunit in skeletal muscle of cachectic cancer patients, which would lead to muscle atrophy through a depression in protein synthesis and an increase in degradation. Mice lacking the ubiquitin ligase MuRF1 are less susceptible to muscle wasting under amino acid deprivation. Expression of MuRF1 and atrogin-1 is increased by oxidative stress, whereas nitric oxide may protect against muscle atrophy. Levels of interleukin (IL)-6 correlate with cachexia and death due to an increase in tumour burden. Ghrelin analogues and melanocortin receptor antagonists increase food intake and may have a role in the treatment of cachexia. Summary: These findings provide impetus for the development of new therapeutic agents. © 2010 Wolters Kluwer Health

Exercise rehabilitation following intensive care unit discharge for recovery from critical illness: Executive summary of a Cochrane Collaboration systematic review

Skeletal muscle wasting and weakness are major complications of critical illness and underlie the profound physical and functional impairments experienced by survivors after discharge from the intensive care unit (ICU). Exercise-based rehabilitation has been shown to be beneficial when delivered during ICU admission. This review aimed to determine the effectiveness of exercise rehabilitation initiated after ICU discharge on primary outcomes of functional exercise capacity and health-related quality of life. We sought randomized controlled trials, quasi-randomized controlled trials, and controlled clinical trials comparing an exercise intervention commenced after ICU discharge vs. any other intervention or a control or ‘usual care’ programme in adult survivors of critical illness. Cochrane Central Register of Controlled Trials, Medical Literature Analysis and Retrieval System Online (MEDLINE), Excerpta Medica Database, and Cumulative Index to Nursing and Allied Health Literature databases were searched up to February 2015. Dual, independent screening of results, data extraction, and quality appraisal were performed. We included six trials involving 483 patients. Overall quality of evidence for both outcomes was very low. All studies evaluated functional exercise capacity, with three reporting positive effects in favour of the intervention. Only two studies evaluated health-related quality of life and neither reported differences between intervention and control groups. Meta-analyses of data were precluded due to variation in study design, types of interventions, and selection and reporting of outcome measurements. We were unable to determine an overall effect on functional exercise capacity or health-related quality of life of interventions initiated after ICU discharge for survivors of critical illness. Findings from ongoing studies are awaited. Future studies need to address methodological aspects of study design and conduct to enhance rigour, quality, and synthesis.

Protein quality and distribution determines anabolic signaling, cellular energetics, and body composition

Building and maintaining muscle is critical to the quality of life for adults and elderly. Physical activity and nutrition are important factors for long-term muscle health. In particular, dietary protein – including protein distribution and quality – are under-appreciated determinants of muscle health for adults. The most unequivocal evidence for the benefit of optimal dietary protein at individual meals is derived from studies of weight management. During the catabolic condition of weight loss, higher protein diets attenuate loss of lean tissue and partition weight loss to body fat when compared with commonly recommended high carbohydrate, low protein diets. Muscle protein turnover is a continuous process in which proteins are degraded, and replaced by newly synthesized proteins. Muscle growth occurs when protein synthesis exceeds protein degradation. Regulation of protein synthesis is complex, with multiple signals influencing this process. The mammalian target of rapamycin (mTORC1) pathway has been identified as a particularly important regulator of protein synthesis, via stimulation of translation initiation. Key regulatory points of translation initiation effected by mTORC1 include assembly of the eukaryotic initiation factor 4F (eIF4F) complex and phosphorylation of the 70 kilodalton ribosomal protein S6 kinase (S6K1). Assembly of the eIF4F initiation complex involves phosphorylation of the inhibitory eIF4E binding protein-1 (4E-BP1), which releases the initiation factor eIF4E and allows it to bind with eIF4G. Binding of eIF4E with eIF4G promotes preparation of the mRNA for binding to the 43S pre-initiation complex. Consumption of the amino acid leucine (Leu) is a key factor determining the anabolic response of muscle protein synthesis (MPS) and mTORC1 signaling to a meal. Research from this dissertation demonstrates that the peak activation of MPS following a complete meal is proportional to the Leu content of a meal and its ability to elevate plasma Leu. Leu has also been implicated as an inhibitor of muscle protein degradation (MPD). In particular, there is evidence suggesting that in muscle wasting conditions Leu supplementation attenuates expression of the ubiquitin-proteosome pathway, which is the primary mode of intracellular protein degradation. However, this is untested in healthy, physiological feeding models. Therefore, an experiment was performed to see if feeding isonitrogenous protein sources with different Leu contents to healthy adult rats would differentially impact ubiquitin-proteosome (protein degradation) outcomes; and if these outcomes are related to the meal responses of plasma Leu. Results showed that higher Leu diets were able to attenuate total proteasome content but had no effect on ubiquitin proteins. This research shows that dietary Leu determines postprandial muscle anabolism. In a parallel line of research, the effects of dietary Leu on changes in muscle mass overtime were investigated. Animals consuming higher Leu diets had larger gastrocnemius muscle weights; furthermore, gastrocnemius muscle weights were correlated with postprandial changes in MPS (r=0.471, P<0.01) and plasma Leu (r=0.400, P=0.01). These results show that the effect of Leu on ubiquitin-proteosome pathways is minimal for healthy adult rats consuming adequate diets. Thus, long-term changes in muscle mass observed in adult rats are likely due to the differences in MPS, rather than MPD. Factors determining the duration of Leu-stimulated MPS were further investigated. Despite continued elevations in plasma Leu and associated translation initiation factors (e.g., S6K1 and 4E-BP1), MPS returned to basal levels ~3 hours after a meal. However, administration of additional nutrients in the form of carbohydrate, Leu, or both ~2 hours after a meal was able to extend the elevation of MPS, in a time and dose dependent manner. This effect led to a novel discovery that decreases in translation elongation activity was associated with increases in activity of AMP kinase, a key cellular energy sensor. This research shows that the Leu density of dietary protein determines anabolic signaling, thereby affecting cellular energetics and body composition.