Dr Andrew J Murton
email: andrew.murton@nottingham.ac.uk

D Constantin

P L Greenhaff

The involvement of the ubiquitin proteasome system in human skeletal muscle remodelling and atrophy

Keywords: Exercise, skeletal muscle, atrophy, inflammation, ubiquitin proteasome system, human

Skeletal muscle exhibits great plasticity in response to altered activity levels, ultimately resulting in tissue remodelling and substantial changes in mass. Animal research would suggest that the ubiquitin proteasome system, in particular the ubiquitin ligases MAFbx/atrogin-1 and MuRF1, are instrumental to the processes underlying these changes. This review article therefore examines the role of proteasomal-mediated protein degradation in human skeletal muscle in health and disease. Specifically, the effects of exercise, disuse and inflammatory disease states on the ubiquitin proteasome system in human skeletal muscle are examined. The article also identifies several inconsistencies between published human studies and data obtained from animal models of muscle atrophy, highlighting the need for a more comprehensive examination of the molecular events responsible for modulating muscle mass in humans.

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Introduction

The dynamic adaptation of skeletal muscle to changing requirements has been acknowledged for decades, but the molecular events underpinning these adaptations have only just started to become clearer. Intricately regulated anabolic and catabolic pathways are responsible for the maintenance of muscle protein content, which represents the bulk of skeletal muscle mass, and imbalance between these pathways results in a change in muscle mass that can sometimes be rapid.

Classically, increases in skeletal muscle mass have been associated with individuals partaking in resistance exercise programs [START_REF] Tesch | Skeletal muscle adaptations consequent to long-term heavy resistance exercise[END_REF] or after the administration of anabolic agents, such as testosterone [START_REF] Kadi | Effects of anabolic steroids on the muscle cells of strength-trained athletes[END_REF][START_REF] Sinha-Hikim | Testosterone-induced increase in muscle size in healthy young men is associated with muscle fiber hypertrophy[END_REF]. Conversely, a loss of muscle mass has been observed following disuse [START_REF] Jones | Disuse atrophy and exercise rehabilitation in humans profoundly affects the expression of genes associated with the regulation of skeletal muscle mass[END_REF][START_REF] Mulder | Knee extensor fatigability after bedrest for 8 weeks with and without countermeasure[END_REF][START_REF] De Boer | The temporal responses of protein synthesis, gene expression and cell signalling in human quadriceps muscle and patellar tendon to disuse[END_REF] and a number of disease states, including but not limited to; sepsis [START_REF] Hasselgren | Novel aspects on the regulation of muscle wasting in sepsis[END_REF], cancer cachexia [START_REF] Berger | Oxandrolone in AIDS-wasting myopathy[END_REF][START_REF] Severinsen | Atrophy of foot muscles in diabetic patients can be detected with ultrasonography[END_REF], AIDS [START_REF] Berger | Oxandrolone in AIDS-wasting myopathy[END_REF], diabetes mellitus [START_REF] Severinsen | Atrophy of foot muscles in diabetic patients can be detected with ultrasonography[END_REF][START_REF] Pupim | Increased muscle protein breakdown in chronic hemodialysis patients with type 2 diabetes mellitus[END_REF] and renal failure [START_REF] Rajan | Muscle wasting in chronic kidney disease: the role of the ubiquitin proteasome system and its clinical impact[END_REF][START_REF] Mcintyre | Patients receiving maintenance dialysis have more severe functionally significant skeletal muscle wasting than patients with dialysis-independent chronic kidney disease[END_REF], with sometimes rapid onset and profound consequences.

In eukaryotic cells, four main mechanisms are responsible for the majority of cellular protein degradation, mediated via the actions of either cysteine-dependent aspartate specific proteases (caspases) [START_REF] Du | Activation of caspase-3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions[END_REF][START_REF] Nunez | Caspases: the proteases of the apoptotic pathway[END_REF], cathepsins [START_REF] Bechet | Lysosomal proteolysis in skeletal muscle[END_REF], calcium-dependent calpains [START_REF] Bartoli | Calpains in muscle wasting[END_REF][START_REF] Costelli | Ca2+dependent proteolysis in muscle wasting[END_REF], or the ubiquitin proteasome system (UPS) [START_REF] Jackman | The molecular basis of skeletal muscle atrophy[END_REF][START_REF] Attaix | The ubiquitin-proteasome system and skeletal muscle wasting[END_REF]. A substantial body of evidence has accumulated implicating the UPS as the principal regulator of skeletal muscle atrophy [START_REF] Lecker | Muscle protein breakdown and the critical role of the ubiquitin-proteasome pathway in normal and disease states[END_REF]. This has led many researchers to focus on both the components and regulators of the UPS in skeletal muscle, with the hopes of providing a greater understanding of the mechanisms responsible for increased skeletal muscle protein degradation, and identifying clinically relevant therapeutic targets to ablate disease-induced muscle atrophy.

To date, the vast majority of the information about the mechanisms responsible for muscle atrophy has been obtained from cell line and small mammal (primarily rodents) based research, with limited information coming to light concerning the molecular mechanisms modulating skeletal muscle mass in humans.

Whilst the similarity in organ systems, systemic physiology and genes between the

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rodent and human species is useful, the validity of information obtained from nonhuman studies needs to be questioned when considering their direct relevance to human situations of muscle hypertrophy/atrophy. Indeed, several reported observations give rise to this stance. Firstly, most studies utilise animals that are immature and still growing, where high rates of muscle protein synthesis, low metabolic stability (i.e. a weak capacity to maintain homeostasis) and a comparatively high basal metabolic rate are commonly observed [START_REF] Demetrius | Of mice and men -When it comes to studying ageing and the means to slow it down, mice are not just small humans[END_REF]. In contrast, most human studies involve subjects of adult age where, in the absence of pathological events, display body weights that are effectively stable for months at a time, experience high metabolic stability, and have much lower specific metabolic rates that include lower rates of muscle protein turnover. Specific to skeletal muscle, the response of the processes governing muscle protein synthesis to insulin differs between rodents and humans, notably where insulin, even at supraphysiological doses, fails to augment muscle protein synthesis in humans without prior amino acid administration [START_REF] Gelfand | Effect of physiologic hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man[END_REF][START_REF] Bennet | Euglycemic hyperinsulinemia augments amino acid uptake by human leg tissues during hyperaminoacidemia[END_REF]. These observations, in conjunction with the discrepancies of both the stress response to experimental procedures and the general severity of the atrophy inducing conditions between animal models and studies conducted in informed, consenting, human adults, provides some cause for concern when translating cell-line/animal data to the human situation.

Nevertheless, findings from cell line, animal and human based research, has consistently pointed to the UPS as a pivotal component to the instigation and regulation of muscle protein breakdown. However, to date, a comprehensive examination of the literature pertaining to the regulation of the UPS in modulating human skeletal muscle mass, particularly in regard to the effects of exercise and disease, has not been undertaken. In this review article therefore, we will first outline the proposed involvement of the UPS in muscle atrophy, as discovered in cell lines and small animals. This will be followed by a detailed examination of published studies investigating the effects of exercise on the UPS pathways in human skeletal

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muscle. Subsequently, conditions that favour muscle atrophy will be examined and discussed, including the use of exercise to modulate the atrophic response of catabolic disease states.

The muscle atrophy 'program'

The increased expression of UPS constituents, including components of the 26S proteasome itself, and the prevention of increased proteolysis in atrophic conditions via the use of proteasome inhibitors [START_REF] Bailey | The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATP-dependent ubiquitinproteasome pathway[END_REF][START_REF] Price | Muscle wasting in insulinopenic rats results from activation of the ATPdependent, ubiquitin-proteasome proteolytic pathway by a mechanism including gene transcription[END_REF][START_REF] Tawa | Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles[END_REF], has led many to conclude that the UPS is intrinsically linked to the degradation of myofibril proteins in skeletal muscle [START_REF] Jagoe | What do we really know about the ubiquitinproteasome pathway in muscle atrophy?[END_REF]. Of note, it has been shown that the UPS is unable to degrade intact myofibrils [START_REF] Solomon | Importance of the ATP-ubiquitin-proteasome pathway in the degradation of soluble and myofibrillar proteins in rabbit muscle extracts[END_REF], suggesting that an alternative system is responsible for initial myofibril disruption, possibly the result of increased caspase-3 [START_REF] Du | Activation of caspase-3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions[END_REF] or calpain activity [START_REF] Williams | Sepsis stimulates release of myofilaments in skeletal muscle by a calcium-dependent mechanism[END_REF]. The UPS is an ATP-dependent proteolytic system that involves the degradation of target proteins, with substrates identified for degradation by the addition of ubiquitin (Ub) molecules, a process itself coordinated via the activity of a triplet of enzymes [START_REF] Passmore | Getting into position: the catalytic mechanisms of protein ubiquitylation[END_REF].

Ubiquitin transfer to targeted proteins represents a robust method for the specific targeting of protein families. In brief, Ub is first bound to the Ub-activating enzyme (E1) via a high-energy thioester bond in an ATP-dependent process (Fig. 1).

Ubiquitin is subsequently transferred from the Ub-activating enzyme to the Ubconjugating enzyme (E2) via the formation of a new thioester linkage between Ub and a cysteine residue of the E2 enzyme. Generally, albeit with a few notable exceptions [START_REF] Li | A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate[END_REF][START_REF] Hoeller | Dikic, E3independent monoubiquitination of ubiquitin-binding proteins[END_REF], the Ub monomer, catalysed by the action of an Ub-ligase enzyme (E3), is conjugated to the target protein via an isopeptide bond between the ε-amino group of a lysine residue in the target protein and the carboxy-terminal glycine residue 76 in Ub (for a review see [START_REF] Passmore | Getting into position: the catalytic mechanisms of protein ubiquitylation[END_REF]). The process is repeated until a minimum of four Ub monomers are covalently attached via lysine residue 48 of Ub to the target protein, the classical formation that is recognised by the 26S proteasome as a signal to degrade the target protein [START_REF] Thrower | Recognition of the polyubiquitin proteolytic signal[END_REF].

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In eukaryotic cells, only one Ub-activating enzyme has been characterised and, observed in relatively high abundance, it meets the somewhat-divergent demands placed upon it by the UPS [START_REF] Handley | Molecular cloning, sequence, and tissue distribution of the human ubiquitinactivating enzyme E1[END_REF][START_REF] Lecker | Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats[END_REF]. Indeed, even under normal physiological conditions, the UPS is constantly degrading damaged or malformed proteins [START_REF] Etlinger | Soluble ATP-dependent proteolytic system responsible for degradation of abnormal proteins in reticulocytes[END_REF], so as to maintain normal cell function. In humans, several dozens of Ub-conjugating enzymes are also present, in addition to hundreds of Ub-ligases [START_REF] Patton | Combinatorial control in ubiquitindependent proteolysis: don't Skp the F-box hypothesis[END_REF],

through which target specificity is established.

Following successful ubiquitination, and only once the criterion for recognition by the 26S proteasome has been met, proteins are unfolded and fed into the proteasome in an ATP-dependent process [START_REF] Attaix | The ubiquitin-proteasome system and skeletal muscle wasting[END_REF]. The 26S proteasome consist of a 20S catalytic core and 19S regulatory caps. Structurally, the 20S proteasome consists of four heptameric rings, formed from alpha subunits providing structural support [START_REF] Rivett | Regulation of proteasome structure and function[END_REF], and the beta subunits responsible for the chymotrypsin-like, trypsin-like and caspase-like activities (for a comprehensive review see [START_REF] Attaix | Mechanisms of ubiquitination and proteasome-dependent proteolysis in skeletal muscle[END_REF]). The proteasome cleaves tagged proteins into short oligonucleotides, after which the activity of tripeptidyl-peptidase II and exopeptidases result in almost complete degradation of the original protein [START_REF] Tomkinson | Tripeptidyl-peptidase II: A multi-purpose peptidase[END_REF].

In 2001, the mRNAs for two muscle-specific Ub-ligases were found to be elevated in the atrophied muscles of food-deprived mice [START_REF] Gomes | Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy[END_REF] and limb immobilised rats [START_REF] Bodine | Identification of ubiquitin ligases required for skeletal muscle atrophy[END_REF]. These two Ub-ligases, MAFbx/atrogin-1 and MuRF1, were collectively termed 'atrogins' and increased mRNA levels for both were subsequently observed in various animal models of muscle atrophy, including but not limited to; burn injury [START_REF] Lang | Burn-induced increase in atrogin-1 and MuRF-1 in skeletal muscle is glucocorticoid independent but downregulated by IGF-I[END_REF],

uremia [START_REF] Lecker | Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression[END_REF], diabetes mellitus [START_REF] Lecker | Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression[END_REF], denervation [START_REF] Bodine | Identification of ubiquitin ligases required for skeletal muscle atrophy[END_REF], unweighting [START_REF] Jones | Disuse atrophy and exercise rehabilitation in humans profoundly affects the expression of genes associated with the regulation of skeletal muscle mass[END_REF][START_REF] Lecker | Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression[END_REF],

dexamethasone administration [START_REF] Clarke | The E3 ligase MuRF1 degrades myosin heavy chain protein in dexamethasone-treated skeletal muscle[END_REF][START_REF] Frost | Hormone, cytokine, and nutritional regulation of sepsis-induced increases in atrogin-1 and MuRF1 in skeletal muscle[END_REF] and sepsis [START_REF] Li | TNFalpha acts via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/MAFbx in skeletal muscle[END_REF][START_REF] Cai | IKK beta/NF-kappa B activation causes severe muscle wasting in mice[END_REF]. Moreover, when knocked out, MAFbx/atrogin-1 -/-and MuRF1 -/-mice appeared resistant to the effects of denervation-induced muscle atrophy, with a 56% and 36% respective sparing of muscle loss, compared to littermate controls [START_REF] Bodine | Identification of ubiquitin ligases required for skeletal muscle atrophy[END_REF]. Consistent with these findings, it was suggested that MAFbx/atrogin-1 and MuRF1 may, in part, be responsible for the

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UPS mediated muscle protein degradation observed during muscle atrophy conditions [START_REF] Glass | Signalling pathways that mediate skeletal muscle hypertrophy and atrophy[END_REF].

Recent evidence has suggested that the transcriptional regulation of MAFbx/atrogin-1 and MuRF1 Ub-ligases are intrinsically linked to both cellular metabolic status and inflammatory state via a coordinated pathway of signalling events. Li and colleagues discovered that both H 2 O 2 and the catabolic cytokine tumor necrosis factor-alpha (TNFα), were competent inducers of MAFbx/atrogin-1 mRNA expression in skeletal muscle [START_REF] Li | Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes[END_REF]. Moreover, they observed that TNFα induced expression of MAFbx/atrogin-1 mRNA was blunted by the p38 MAPK inhibitors SB203580 and curcumin, suggesting that TNFα works via a p38 MAPK dependent mechanism to stimulate MAFbx/atrogin-1 expression [START_REF] Li | TNFalpha acts via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/MAFbx in skeletal muscle[END_REF]. At about the same time,

Cai and colleagues, with the use of constitutively active IκK mice (termed MIKK mice), showed an activation of NF-κB leads to an elevation in MuRF1, but not MAFbx/atrogin-1, mRNA [START_REF] Cai | IKK beta/NF-kappa B activation causes severe muscle wasting in mice[END_REF]. Additionally, in the tibialis anterior of MIKK mice, both fibre diameter and fibre cross sectional area were reduced to 56% and 32% respectively, compared to control mice [START_REF] Cai | IKK beta/NF-kappa B activation causes severe muscle wasting in mice[END_REF]. Moreover, MIKK mice crossed with MuRF1 -/-mice experienced a 50% rescue of protein degradation [START_REF] Cai | IKK beta/NF-kappa B activation causes severe muscle wasting in mice[END_REF], thus demonstrating that NF-κB mediated protein degradation is heavily dependent on the upregulation of the MuRF1 Ub-ligase. Collectively, these findings highlight that the transcriptional regulation of MAFbx/atrogin-1 and MuRF1 are sensitive to local inflammatory mediators (TNFα) and the NF-κB inflammatory pathway itself.

Furthermore, they show MAFbx/atrogin-1 and MuRF1 to be able to function independently of each other.

Transcription of the MAFbx/atrogin-1 and MuRF1 genes also appear under the control of the forkhead Foxo family of transcription factors. It had been known for some time that transgenic overexpression of Foxo1 resulted in an atrophic phenotype [START_REF] Kamei | Skeletal muscle FOXO1 (FKHR) transgenic mice have less skeletal muscle mass, downregulated type I (slow twitch/red muscle) fiber genes, and impaired glycemic control[END_REF]. Likewise, elevations in the forkhead transcription factors have been observed in muscle of animals subjected to starvation, glucocorticoid administration, diabetes,

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uremia and cancer [START_REF] Lecker | Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression[END_REF][START_REF] Furuyama | Forkhead transcription factor FOX01 (FKHR)-dependent induction of PDK4 gene expression in skeletal muscle during energy deprivation[END_REF]. The Foxo family of transcription factors are rendered inactive as a result of phosphorylation by AKT (also known as protein kinaseB) which results in the removal of the transcription factors from the nucleus where they are subsequently sequestered in the cytoplasm [START_REF] Brunet | Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor[END_REF]. AKT represents an important mediator of the protein synthesis initiation pathway and is responsible for regulating the formation of the 43S preinitiation complex via GSK3β inhibition, synthesis of ribosomal proteins, and binding of the terminal 7-methylguanosine mRNA cap to the eIF4F complex via mTOR activation (Fig. 2; for a comprehensive review see [START_REF] Nader | Translational control: Implications for skeletal muscle hypertrophy[END_REF]).

Consequently, as the Foxo1 transcription factor is also known to increase the transcription of pyruvate dehydrogenase kinase isoform 4 [START_REF] Furuyama | Forkhead transcription factor FOX01 (FKHR)-dependent induction of PDK4 gene expression in skeletal muscle during energy deprivation[END_REF][START_REF] Kwon | Protein kinase B alpha/Akt1 (PKB(alpha/Akt1) inhibits human pyruvate dehydrogenase kinase-4 gene induction by dexamethasone through inactivation of forkhead transcription factor, FKHR (Foxo1)[END_REF], a reversible inhibitor of the pyruvate dehydrogenase complex [START_REF] Holness | Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation[END_REF], and thus a regulator of the decarboxylation of pyruvate to acetyl-CoA in the TCA cycle, the regulation of MAFbx/atrogin-1 and MuRF1 mRNA expression by Foxo forkhead transcription factors appears intrinsically linked to the rate of muscle protein synthesis and cellular oxidative metabolism. In support, our group has recently shown lipopolysaccharideinduced endotoxaemia to result in the coordinated reductions of AKT protein levels and cytosolic phosphorylation of Foxo1 and Foxo3. These observations were accompanied by significant increases in MAFbx/atrogin-1 and PDK4 protein levels and significant reductions in muscle protein content and pyruvate dehydrogenase complex activity in rodent fast-twitch muscle leading to an impairment of carbohydrate oxidation [START_REF] Crossland | A potential role for Akt/FOXO signalling in both protein loss and the impairment of muscle carbohydrate oxidation during sepsis in rodent skeletal muscle[END_REF]. To clarify, during times of increased muscle protein synthesis, it would be hypothesised that MAFbx/atrogin-1 and MuRF1 mRNA expression would be suppressed, and vice versa. However, as discussed in detail later, in humans this relationship is not consistently observed.

Exercise and the UPS in human skeletal muscle

Eccentric contraction induced muscle "damage" and remodelling

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Exercise, an essential component of a healthy and balanced lifestyle, elicits substantial metabolic and functional demands upon skeletal muscle in humans. The nature of these demands depends greatly upon the mode, intensity and duration of the exercise [START_REF] Pollock | The recommended quantity and quality of exercise for developing and maintaining cardiorespiratory and muscular fitness, and flexibility in healthy adults[END_REF]. A number of human studies have examined the role of the UPS in skeletal muscle hypertrophy/remodelling following eccentric and concentric contractions and, to a lesser degree, endurance exercise. While either unaccustomed eccentric or concentric exercise elevates levels of both fractional protein synthesis rates and fractional protein breakdown rates [START_REF] Phillips | Mixed muscle protein synthesis and breakdown after resistance exercise in humans[END_REF], the cellular and subsequent morphological changes in response to the two exercise stimuli appear somewhat divergent. Unaccustomed or intense eccentric exercise has been associated with significant muscle damage, characterised by localised muscular pain, inflammation [START_REF] Fielding | Acute phase response in exercise. III. Neutrophil and IL-1 beta accumulation in skeletal muscle[END_REF], extensive sarcomere disruption, z-disk streaming [START_REF] Fridén | A morphological study of delayed muscle soreness[END_REF][START_REF] Fridén | Myofibrillar damage following intense eccentric exercise in man[END_REF] and increased serum creatine kinase activity [START_REF] Nosaka | Relationship between post-exercise plasma CK elevation and muscle mass involved in the exercise[END_REF] and troponin I levels [START_REF] Willoughby | Glucocorticoid receptor and ubiquitin expression after repeated eccentric exercise[END_REF][START_REF] Sorichter | Skeletal troponin I as a marker of exercise-induced muscle damage[END_REF].

Concomitant with a rise in myofibrillar damage and inflammation/pain, muscle strength can be reduced by 50%, with a gradual recovery over the 5-10 day period post-exercise [START_REF] Sargeant | Human muscle function following prolonged eccentric exercise[END_REF][START_REF] Gibala | Changes in human skeletal muscle ultrastructure and force production after acute resistance exercise[END_REF]. Conversely, whilst a single bout of concentric resistance exercise in untrained subjects can also induce myofibrillar disruption, the degree of damage is far more limited than that of eccentric exercise [START_REF] Gibala | Changes in human skeletal muscle ultrastructure and force production after acute resistance exercise[END_REF]. However, it should be noted that mild eccentric and concentric exercise displays only limited damage and, moreover, evidence suggests that eccentric loading is important for maximising the hypertrophic response to resistance training [START_REF] Bamman | Mechanical load increases muscle IGF-I and androgen receptor mRNA concentrations in humans[END_REF][START_REF] Hather | Influence of eccentric actions on skeletal muscle adaptations to resistance training[END_REF].

Biopsies obtained form the vastus lateralis of healthy male subjects 24 h after completing an intense eccentric exercise protocol show increased proportions of myofibrillar disruption compared to samples taken immediately post exercise [START_REF] Féasson | Molecular adaptations of neuromuscular diseaseassociated proteins in response to eccentric exercise in human skeletal muscle[END_REF].

Thus, a progression of the exercise induced muscle damage is dependent not only on the initial mechanical stress, but also on post-exercise events [START_REF] Féasson | Molecular adaptations of neuromuscular diseaseassociated proteins in response to eccentric exercise in human skeletal muscle[END_REF]. Consistent with this suggestion, an elevation in the mRNA and protein expression of free Ub and components of the 20S proteasome in skeletal muscles of subjects following a single

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bout of eccentric exercise, in addition to increased levels of Ub conjugated proteins 48 h post exercise (Table 1), strongly suggest that the UPS may play a part in eccentric contraction induced muscle damage. A sole report demonstrating no change in the amount of Ub-conjugated proteins following eccentric exercise was potentially due to an insufficient period of time between the exercise intervention and the period of measurement (24 h; [START_REF] Stupka | Cellular adaptation to repeated eccentric exercise-induced muscle damage[END_REF]).

The induction of the UPS during eccentric contraction induced muscle damage appears instrumental in the remodelling of the skeletal muscle myofilaments;

tentatively instigated to limit skeletal muscle damage from future eccentric bouts.

Indeed, the degree of muscle damage associated with the original bout of eccentric exercise is attenuated following a second bout performed several weeks after the first [START_REF] Clarkson | Muscle function after exercise-induced muscle damage and rapid adaptation[END_REF][START_REF] Mair | Rapid adaptation to eccentric exercise-induced muscle damage[END_REF]. Thus, adaptations must have occurred to limit the muscle fibre susceptibility to mechanical stress [START_REF] Gibala | Changes in human skeletal muscle ultrastructure and force production after acute resistance exercise[END_REF]. Suggested remodelling events include the addition of sarcomeres in series, resulting in shorter average sarcomere length, altered motor until recruitment patterns [START_REF] Morgan | Early events in stretch-induced muscle damage[END_REF], and an increase in desmin protein levels resulting in a putative enhancement of z-disk stability [START_REF] Féasson | Molecular adaptations of neuromuscular diseaseassociated proteins in response to eccentric exercise in human skeletal muscle[END_REF]. Moreover, elevated levels of skeletal muscle heat shock protein's HSP27 and HSP72 have been observed following eccentric exercise [START_REF] Féasson | Molecular adaptations of neuromuscular diseaseassociated proteins in response to eccentric exercise in human skeletal muscle[END_REF][START_REF] Thompson | Exerciseinduced HSP27, HSP70 and MAPK responses in human skeletal muscle[END_REF][START_REF] Willoughby | HSP-72 and ubiquitin expression and caspase-3 activity after a single bout of eccentric exercise[END_REF]. The heat shock proteins represent a family of molecular chaperones; being expressed at times of cellular stress, they are responsible for both the maintenance of protein structure and orchestrating the repair of malformed/damaged proteins. As a consequence, heat shock proteins are considered to have anti-proteolytic and anti-apoptotic effects [START_REF] Morimoto | Cells in stress: transcriptional activation of heat shock genes[END_REF]. Collectively, this appears tantamount to the skeletal muscle cell trying to preserve function during a period of increased damage/remodelling elicited by exercise, which is dependent upon intensity [START_REF] Liu | Human skeletal muscle HSP70 response to physical training depends on exercise intensity[END_REF].

The role of the UPS in the initiation of the 'protective repeated bout effect' is less clear. While it had been hypothesised that a reduction in UPS mediated

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eccentric exercise performed several weeks apart, presumably because sarcomeric remodelling had already occurred, contrasting observations have thus far been reported. For example, Willoughby and colleagues [START_REF] Willoughby | Glucocorticoid receptor and ubiquitin expression after repeated eccentric exercise[END_REF] showed greater increases in muscle Ub protein content in healthy volunteers following the first, rather than a second eccentric exercise bout conducted 3 weeks apart. Conversely, increased Ubconjugation in skeletal muscle samples of the vastus lateralis have been observed on the second of two exercise sessions (involving eccentric leg press and knee extension exercises) separated by >5 weeks [START_REF] Stupka | Cellular adaptation to repeated eccentric exercise-induced muscle damage[END_REF]. Thus, further work is required to rationalise these two observations and determine the involvement of the UPS in this adaptive response. It also seems that to provide meaningful information these experiments need to be performed in conjunction with measurements of muscle protein turnover.

The signal(s) responsible for instigating increased skeletal muscle UPSmediated proteolysis following eccentric exercise are currently unknown, but the cytokines have garnered much attention. Exercise induced muscle damage is known to stimulate an acute phase inflammatory response resulting in the infiltration of neutrophils and macrophages into skeletal muscle [START_REF] Fielding | Acute phase response in exercise. III. Neutrophil and IL-1 beta accumulation in skeletal muscle[END_REF]. Indeed, a single bout of eccentric exercise is known to increase macrophage counts per surface unit of tissue in skeletal muscle compared to controls [START_REF] Stupka | Cellular adaptation to repeated eccentric exercise-induced muscle damage[END_REF]. Moreover, downhill running, which requires extensive eccentric contractions of the vastus lateralis muscle, results in localised elevations of TNFα and interleukin-6 (IL-6) mRNA and induces substantial muscle damage [START_REF] Hamada | Senescence of human skeletal muscle impairs the local inflammatory cytokine response to acute eccentric exercise[END_REF]. Interestingly, contracting skeletal muscle is one of the major sites of IL-6 production [START_REF] Féasson | Molecular adaptations of neuromuscular diseaseassociated proteins in response to eccentric exercise in human skeletal muscle[END_REF], however, the source of the elevated TNFα is less clear but is most likely to be macrophage-derived. Given that both TNFα and IL-6 are capable of inducing muscle atrophy [START_REF] Li | TNFalpha acts via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/MAFbx in skeletal muscle[END_REF][START_REF] Coletti | Tumor necrosis factor-alpha gene transfer induces cachexia and inhibits muscle regeneration[END_REF][START_REF] Li | Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NFkappa B activation in response to tumor necrosis factor alpha[END_REF][START_REF] Janssen | Interleukin-6 causes myocardial failure and skeletal muscle atrophy in rats[END_REF][START_REF] Haddad | IL-6-induced skeletal muscle atrophy[END_REF] and of activating the UPS [START_REF] Li | TNFalpha acts via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/MAFbx in skeletal muscle[END_REF][START_REF] Li | TNFalpha increases ubiquitin-conjugating activity in skeletal muscle by upregulating UbcH2/E2(20k)[END_REF][START_REF] Tsujinaka | Interleukin 6 receptor antibody inhibits muscle atrophy and modulates proteolytic systems in interleukin 6 transgenic mice[END_REF], it is tempting to suggest that they may be involved in the instigation of the remodelling process following eccentric damage. However, no evidence in humans yet exists to confirm these postulations. Moreover, given that eccentric exercise

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contractions often result in only a small percentage of severely damaged fibres and yet global reduced rates of glucose uptake to the order of 20-30% are observed, a systemic factor may be responsible for the events that follow eccentric-induced muscle damage [START_REF] Asp | Eccentric exercise decreases maximal insulin action in humans: muscle and systemic effects[END_REF][START_REF] Kirwan | Eccentric exercise induces transient insulin resistance in healthy individuals[END_REF][START_REF] Newham | Ultrastructural changes after concentric and eccentric contractions of human muscle[END_REF]. [START_REF] Deldicque | Effects of resistance exercise with and without creatine supplementation on gene expression and cell signaling in human skeletal muscle[END_REF] which, in conjunction with localised muscle inflammation and NF-κB activation [START_REF] Ji | Role of nuclear factor kappaB and mitogenactivated protein kinase signaling in exercise-induced antioxidant enzyme adaptation[END_REF], could represent the primary elements responsible for exercise-induced expression of the atrogenes and thus, the early protein breakdown component of exercise.

Resistance training induced muscle mass gains

However, skeletal muscle contraction also induces a marked elevation in plasma IL-6 [START_REF] Pedersen | Muscle-derived interleukin-6: possible biological effects[END_REF]. Moreover, the degree of IL-6 expression is dependent on the intensity, duration and mode of the exercise [START_REF] Pedersen | The metabolic role of IL-6 produced during exercise: is IL-6 an exercise factor?[END_REF] and appears muscle derived [START_REF] Ostrowski | Evidence that interleukin-6 is produced in human skeletal muscle during prolonged running[END_REF].

Interestingly, exercise also results in increased circulating levels of anti-inflammatory cytokines and the inflammatory cytokine inhibitors, IL-1 receptor antagonist and soluble TNFα receptors [START_REF] Pedersen | Muscle-derived interleukin-6: possible biological effects[END_REF][START_REF] Pedersen | Searching for the exercise factor: is IL-6 a candidate?[END_REF][START_REF] Febbraio | Muscle-derived interleukin-6: mechanisms for activation and possible biological roles[END_REF]; only very intense exercise stimulates TNFα expression [START_REF] Pedersen | Role of myokines in exercise and metabolism[END_REF]. In addition, IL-6 is typically the first cytokine to appear in the circulation during exercise and shows the most marked expression amongst the elevated cytokines. Intriguingly, treatment of cachectic murine colon adenocarcinoma with anti-mouse IL-6 antibody was successful at ablating the skeletal muscle atrophy commonly observed [START_REF] Strassmann | Suramin interferes with interleukin-6 receptor binding in vitro and inhibits colon-26-mediated experimental cancer cachexia in vivo[END_REF]. Furthermore, in C2C12 myotubes, IL-6 shortens the halflife of long-lived proteins [START_REF] Ebisui | Interleukin-6 induces proteolysis by activating intracellular proteases (cathepsins B and L, proteasome) in C2C12 myotubes[END_REF]. Concordantly, transgenic mice overexpressing IL-6 experience profound muscle atrophy and elevated Ub gene expression, which is preventable by the administration of an IL-6 receptor antagonist [START_REF] Tsujinaka | Interleukin 6 receptor antibody inhibits muscle atrophy and modulates proteolytic systems in interleukin 6 transgenic mice[END_REF]. Therefore, the exercise-induced expression of IL-6 could, tentatively, represent an alternative stimulus following resistance exercise for the early elevation in the UPS.

UPS in muscle atrophy conditions in humans

Skeletal muscle comprises ~40% of the total body mass of an average adult human and has energy requirements even at rest. Moreover, the inherent structure of skeletal muscle represents a substantial reservoir of protein that can be utilised by hepatic tissue during times of stress [START_REF] Hasselgren | Current concepts of protein turnover and amino acid transport in liver and skeletal muscle during sepsis[END_REF]. During periods of prolonged inactivity or disease, a substantial reduction in muscle mass is commonly observed, reducing total muscular energy requirements and, during disease, providing substrates for hepatic gluconeogenesis and acute phase protein production [START_REF] Hasselgren | Current concepts of protein turnover and amino acid transport in liver and skeletal muscle during sepsis[END_REF]. Despite these beneficial features, a dramatic loss of skeletal muscle mass can be debilitating, resulting in prolonged times to recovery, increased risk of subsequent injury and a severe burden on health care provisions. Pathological and environmental states that can induce skeletal muscle atrophy can be broadly divided into those with and without an inflammatory stimulus. Examples of the former include bed rest/immobilisation, spinal cord injury and fasting; whilst states with an inflammatory element include cancer, AIDS, sepsis and perhaps sarcopenia in the frail elderly.

Despite both cohorts experiencing skeletal muscle atrophy, the mechanistic events between groups most likely differ and are examined in detail below.

Non-inflammatory skeletal muscle atrophy

Muscle disuse through limb immobilisation, bed rest or spinal cord injury represent the majority of non-inflammatory related atrophy seen in the clinical setting, and the degree of muscle loss can be appreciably large. Indeed, two weeks of legimmobilisation has been shown to result in an almost 5% reduction in quadriceps lean mass and a 27% fall in isometric strength [START_REF] Jones | Disuse atrophy and exercise rehabilitation in humans profoundly affects the expression of genes associated with the regulation of skeletal muscle mass[END_REF]. Likewise, a 20% reduction in type I muscle fibre cross sectional area and a 30% reduction in type II fibre area, has been

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observed following 60-days bed rest [START_REF] Salanova | Molecular biomarkers monitoring human skeletal muscle fibres and microvasculature following long-term bed rest with and without countermeasures[END_REF]. While elements of the UPS, notably levels of ubiquitinated proteins [START_REF] Ogawa | Ubiquitin ligase gene expression in healthy volunteers with 20-day bedrest[END_REF] and HC6 mRNA [START_REF] Jones | Disuse atrophy and exercise rehabilitation in humans profoundly affects the expression of genes associated with the regulation of skeletal muscle mass[END_REF], appear elevated during disuse, a consistent elevation of MAFbx/atrogin-1 and MuRF1 mRNA has not been observed in human skeletal muscle in the studies performed to date (Table 2). While some reports have detailed an increase in MAFbx/atrogin-1 [START_REF] Jones | Disuse atrophy and exercise rehabilitation in humans profoundly affects the expression of genes associated with the regulation of skeletal muscle mass[END_REF][START_REF] Ogawa | Ubiquitin ligase gene expression in healthy volunteers with 20-day bedrest[END_REF][START_REF] Chen | Transcriptional pathways associated with skeletal muscle disuse atrophy in humans[END_REF] mRNA to disuse stimuli in humans, a number have reported no change or a reduction in the mRNA levels of MAFbx/atrogin-1 [START_REF] De Boer | The temporal responses of protein synthesis, gene expression and cell signalling in human quadriceps muscle and patellar tendon to disuse[END_REF] or MuRF1 [START_REF] Jones | Disuse atrophy and exercise rehabilitation in humans profoundly affects the expression of genes associated with the regulation of skeletal muscle mass[END_REF][START_REF] De Boer | The temporal responses of protein synthesis, gene expression and cell signalling in human quadriceps muscle and patellar tendon to disuse[END_REF][START_REF] Salanova | Molecular biomarkers monitoring human skeletal muscle fibres and microvasculature following long-term bed rest with and without countermeasures[END_REF][START_REF] Ogawa | Ubiquitin ligase gene expression in healthy volunteers with 20-day bedrest[END_REF]. In comparison, a classical elevation of the atrogenes has been observed in animals following denervation [START_REF] Bodine | Identification of ubiquitin ligases required for skeletal muscle atrophy[END_REF], hind limb suspension [START_REF] Bodine | Identification of ubiquitin ligases required for skeletal muscle atrophy[END_REF], or space-flight [START_REF] Nikawa | Skeletal muscle gene expression in space-flown rats[END_REF]. Numerous explanations exist for the disparity between animal models of disuse, where large and consistent elevations in expression of the atrogenes have been observed, and humans, where variable findings in regard to MAFbx/atrogin-1 and MuRF1 levels have thus far been reported. Most notable is the generally greater severity of the procedures performed on animals to induce muscle disuse compared to that employed in human volunteers/patients. Furthermore, with the desire to limit invasive procedures in humans, the number of muscle biopsies performed to obtain tissue is usually minimised; as a consequence, a comprehensive examination of the temporal expression of the atrogins in response to disuse in the human, particularly in the first few days after onset, is currently lacking. A lone report examining temporal levels of MAFbx/atrogin-1 and MuRF1 mRNA reinforces the need for such work to be undertaken as a matter of course. de Boer and colleagues [START_REF] De Boer | The temporal responses of protein synthesis, gene expression and cell signalling in human quadriceps muscle and patellar tendon to disuse[END_REF] showed 10 days limb immobilisation to result in elevated mRNA levels for MuRF1, but not MAFbx/atrogin-1, in the vastus lateralis of healthy human volunteers; however, 11 further days of limb immobilisation saw a subsequent fall in mRNA levels for both atrogenes. Given these shortcomings which, in conjunction with a general absence of data on the protein expression of the two atrogins (with the exception of [START_REF] Salanova | Molecular biomarkers monitoring human skeletal muscle fibres and microvasculature following long-term bed rest with and without countermeasures[END_REF]), it is not currently possible to discern the functional relevance of MAFbx/atrogin-1 and MuRF1 to the human condition of muscle disuse atrophy. The lack of protein levels of the atrogins The Ub-ligases Cbl-b, Siah-1A and Nedd4, originally identified in animals following unloading [START_REF] Nikawa | Skeletal muscle gene expression in space-flown rats[END_REF][START_REF] Koncarevic | The ubiquitin-protein ligase Nedd4 targets Notch1 in skeletal muscle and distinguishes the subset of atrophies caused by reduced muscle tension[END_REF], have been tentatively linked to disuse atrophy.

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Preliminary evidence from humans has shown a statistically significant, albeit small (~2.5-fold increase compared to controls) rise in Cbl-b, but not Siah-1 A, mRNA levels following disuse atrophy [START_REF] Ogawa | Ubiquitin ligase gene expression in healthy volunteers with 20-day bedrest[END_REF]; whilst the involvement of Nedd4 in muscle atrophy remains to be examined in human skeletal muscle. Thus, an examination of the involvement of Cbl-b and Nedd4 in the induction of disuse atrophy is warranted.

Interestingly, a target of Cbl-b Ub-ligase activity includes growth factor receptors, possibly including IRS-1 [START_REF] Ogawa | Ubiquitin ligase gene expression in healthy volunteers with 20-day bedrest[END_REF]. Furthermore, with a bona-fide target of Nedd4 being Notch1, a protein associated with satellite cell proliferation and differentiation [START_REF] Koncarevic | The ubiquitin-protein ligase Nedd4 targets Notch1 in skeletal muscle and distinguishes the subset of atrophies caused by reduced muscle tension[END_REF],

the further study of both Ub-ligases appears judicious

The advent and adoption of microarray technology has allowed the rapid identification of global changes in gene expression following an applied intervention and represents a valuable approach, particularly when integrated with functional measurements in a time-dependent experimental paradigm. However, to date, only a handful of human studies investigating muscle atrophy events have adopted the microarray approach. Despite this, several novel observations related to proteins

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associated with the UPS have been observed in three differing models of human muscle disuse atrophy. Firstly, in the immediate days following spinal cord injury (2 and 5 days from injury) increased expression changes in Ub E3-ligase C and the 26S proteasome non-ATP-ase regulatory subunit 11 has been revealed using the Affymetrix GeneChip approach [START_REF] Urso | Alterations in mRNA expression and protein products following spinal cord injury in humans[END_REF]. Furthermore, increases in the Ub-conjugating enzyme, UBE2E, as well as the Ub-ligase, FBXO9, have been identified in muscle biopsies following limb immobilisation [START_REF] Urso | Analysis of human skeletal muscle after 48 h immobilization reveals alterations in mRNA and protein for extracellular matrix components[END_REF]. These immobilisation-induced changes were in addition to elevations in USP-6, a deubiquitinating-enzyme responsible for cleaving Ub from Ub tagged proteins, thereby purportedly allowing efficient recycling of Ub monomers during catabolic states, and SUMO-1, a small Ub modifier, previously postulated to prevent UPS-mediated protein degradation of conjugated proteins [START_REF] Urso | Analysis of human skeletal muscle after 48 h immobilization reveals alterations in mRNA and protein for extracellular matrix components[END_REF][START_REF] Desterro | SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation[END_REF]. Lastly, 4-11 days of cast mediated immobilisation following ankle fracture resulted in 227 genes being differentially expressed compared to muscle from healthy control subjects prior to cast mediated immobilisation, with all bar 26 genes downregulated [START_REF] Chen | Transcriptional pathways associated with skeletal muscle disuse atrophy in humans[END_REF]. Interestingly, 96% of the transcriptional changes that occurred following limb immobilisation in ankle fracture patients were also observed in the contralateral leg of the patients, but these changes were not observed in the non-immobilised limb from cast-immobilised healthy volunteers [START_REF] Chen | Transcriptional pathways associated with skeletal muscle disuse atrophy in humans[END_REF]. Although perplexing, it is possible that the observed transcriptional changes in the contralateral leg of ankle fracture patients may be due to increased bed rest/decreased mobility following ankle fracture, highlighting the additional complexity of studying patient populations where a component of the observed muscle atrophy could be due to decreased activity levels [START_REF] Chen | Transcriptional pathways associated with skeletal muscle disuse atrophy in humans[END_REF]. However, elevated levels of MAFbx/atrogin-1 was only observed in samples isolated from patients with ankle fractures, an observation that is supportive of its role as a regulator of muscle atrophy; although in contrast, other members of the UPS such as E2D3 and E2N were downregulated in the muscle of ankle fracture patients when compared to healthy control subjects prior to cast mediated immobilisation [START_REF] Chen | Transcriptional pathways associated with skeletal muscle disuse atrophy in humans[END_REF].

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With two notable exceptions [START_REF] Salanova | Molecular biomarkers monitoring human skeletal muscle fibres and microvasculature following long-term bed rest with and without countermeasures[END_REF][START_REF] Léger | Human skeletal muscle atrophy in amyotrophic lateral sclerosis reveals a reduction in Akt and an increase in atrogin-1[END_REF], the general absence of protein measurements for the two atrogins is concerning and introduces compounding issues in the interpretation of current mRNA data for the role of the UPS in muscle atrophy. Interestingly, it has been suggested that the loss of lean body mass observed following disuse induced muscle atrophy in the human is primarily the result of a decrease in muscle protein synthesis [START_REF] Rennie | Letter to the Editor on the Journal Club article by Barker and Traber[END_REF]. Reductions in muscle protein synthesis following disuse atrophy have been reported by several groups, despite the maintenance of adequate nutritional intake [START_REF] Ferrando | Prolonged bed rest decreases skeletal muscle and whole body protein synthesis[END_REF][START_REF] Paddon-Jones | Atrophy and impaired muscle protein synthesis during prolonged inactivity and stress[END_REF]. de Boer and colleagues [START_REF] De Boer | The temporal responses of protein synthesis, gene expression and cell signalling in human quadriceps muscle and patellar tendon to disuse[END_REF] detected a 50% reduction in myofibrillar protein synthesis after 10 days of limb suspension, which was similarly suppressed 11 days later. They approximated that if daily mixed muscle protein synthesis fell by 50% to 1.0-1.25% per day, protein breakdown rates would not need to be elevated above the basal rate of 2.0-2.5% per day to account for the 0.5% fall per day in cross sectional area. However, given the existence of the purported AKT/Foxo/atrogene axis [103,[START_REF] Sandri | Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy[END_REF], this observation of a marked reduction in muscle protein synthesis during human limb immobilisation cannot be easily reconciled with the proposal of unchanged muscle protein breakdown and moreover, is not in concordance with experimental observations of increased muscle protein breakdown in animals following either denervation [START_REF] Goldspink | The effects of denervation on protein turnover of rat skeletal muscle[END_REF],

hindlimb suspension [START_REF] Taillandier | Coordinate activation of lysosomal, Ca 2+-activated and ATP-ubiquitin-dependent proteinases in the unweighted rat soleus muscle[END_REF] or limb immobilisation [START_REF] Krawiec | Hindlimb casting decreases muscle mass in part by proteasome-dependent proteolysis but independent of protein synthesis[END_REF]. In reality, evidence of changes in muscle protein degradation in humans over the time-course of muscle disuse remains to be demonstrated and represents an essential component in the determination of the true significance of UPS-mediated degradation during immobilisation induced atrophy.

Despite the aforementioned scepticism held by some in regard to the role of muscle protein breakdown in human muscle disuse, confocal microscopy of vastus lateralis muscle samples taken after 60-days bed rest has revealed MuRF1 immunoreactivity in the cytosolic/myofibrillar compartment of myofibres, with strong cytosolic immunofluorescence in subpopulations of atrophic myofibres [START_REF] Salanova | Molecular biomarkers monitoring human skeletal muscle fibres and microvasculature following long-term bed rest with and without countermeasures[END_REF].

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Furthermore, work emanating from our group has shown the suppression of limb protein breakdown that occurs following intravenous amino acid infusion during periods of elevated serum insulin (30 mU•l -1 ), occur in conjunction with a fall in MAFbx/atrogin-1 and proteasome subunit HC2 protein levels, suggesting that MAFbx/atrogin-1 may in part be responsible for increased muscle protein breakdown in human muscle [START_REF] Greenhaff | Disassociation between the effects of amino acids and insulin on signalling, ubiquitin-ligases and protein turnover in human muscle[END_REF]. Notwithstanding where measurements of muscle protein breakdown remain outstanding, such observations would be in agreement with suggestions that the atrogins play a role in the loss of muscle mass during disuse, possibly by regulating cellular signalling mechanisms. Ubiquitin tagging and/or UPSmediated degradation has long been considered a viable mechanism in the regulation of distinct pathways, as observed in the regulation of NF-κB activity by IκB (for a review see [START_REF] Perkins | Post-translational modifications regulating the activity and function of the nuclear factor kappa B pathway[END_REF]). Interestingly, reduced muscle protein synthesis following amino acid deprivation has been observed in wild-type but not MuRF1 -/-mice implicating MuRF1 as a negative regulator of muscle protein synthesis [START_REF] Koyama | Muscle RING-finger protein-1 (MuRF1) as a connector of muscle energy metabolism and protein synthesis[END_REF]. This is further reinforced by reports of MuRF1 -/-MuRF2 -/-mice displaying significant muscle hypertrophy and elevated levels of p70S6K in its active form, but notably, without changes in total levels of multiubiquitinated proteins and thus, presumably, degradation [START_REF] Witt | MURF-1 and MURF-2 target a specific subset of myofibrillar proteins redundantly: towards understanding MURF-dependent muscle ubiquitination[END_REF]. These findings, in conjunction with the currently known targets of the atrogins, which in skeletal muscle include MyoD [START_REF] Tintignac | Degradation of MyoD mediated by the SCF (MAFbx) ubiquitin ligase[END_REF] and eIF3f [START_REF] Lagirand-Cantaloube | The initiation factor eIF3-f is a major target for Atrogin1/MAFbx function in skeletal muscle atrophy[END_REF] for MAFbx/atrogin-1, and myosin heavy chain [START_REF] Glass | Signalling pathways that mediate skeletal muscle hypertrophy and atrophy[END_REF], creatine kinase [START_REF] Koyama | Muscle RING-finger protein-1 (MuRF1) as a connector of muscle energy metabolism and protein synthesis[END_REF] and possibly the myofibrillar proteins titin, nebulin and myosin light chain-2 [START_REF] Witt | MURF-1 and MURF-2 target a specific subset of myofibrillar proteins redundantly: towards understanding MURF-dependent muscle ubiquitination[END_REF] for MuRF1, suggest that in addition to proteolysis of myofibrillar proteins, the atrogins may also be responsible for suppressing anabolic and hyperplastic signals, including muscle protein synthesis. While the extent to which these findings are applicable to human skeletal muscle is unknown, it is intriguing to note that such observations are compatible with current reports in humans, although they do not explain why elevations in MAFbx/atrogin-1 and MuRF1 are not consistently observed in all models of disuse, though this could simply be due to the timing of muscle sampling

Inflammatory-mediated atrophy

Many disease states that are characterised by systemic inflammation are also associated with a muscle mass deficit, which can have a rapid onset. Moreover, a number but albeit not all, inflammatory disease states also display increased levels of both UPS components and the two atrogenes (Table 2). Interestingly, mRNA levels for proteasomal subunits and Ub, in addition to proteasomal chymotrypsin-like activity measured in muscle samples of patients suffering from cancer, appear greatest in individuals experiencing significant weight loss [START_REF] Khal | Increased expression of proteasome subunits in skeletal muscle of cancer patients with weight loss[END_REF][START_REF] Bossola | Increased muscle ubiquitin mRNA levels in gastric cancer patients[END_REF][START_REF] Bossola | Increased muscle proteasome activity correlates with disease severity in gastric cancer patients[END_REF]. Moreover, mRNA levels of proteasomal subunits HC2 and HC5 in cancer cachectic patients appeared dependent on the degree of weight loss [START_REF] Khal | Increased expression of proteasome subunits in skeletal muscle of cancer patients with weight loss[END_REF]. Patients experiencing a weight loss greater than 10%, where muscle protein content starts to become significantly depressed, show a parabolic relationship between the two subunits and weight loss; with HC2 and HC5 mRNA increasing maximally for patients with 12-19% weight loss and declining levels thereafter [START_REF] Khal | Increased expression of proteasome subunits in skeletal muscle of cancer patients with weight loss[END_REF]. Likewise, the Ub-conjugating enzyme E2 14K also showed a similar parabolic relationship with weight loss as the two proteasomal subunits [START_REF] Khal | Increased expression of proteasome subunits in skeletal muscle of cancer patients with weight loss[END_REF]. Importantly, significantly higher circulating levels of both IL-6 and C-reactive protein (CRP), an acute phase protein commonly employed as a marker for the magnitude of systemic inflammation, have been observed in weight-losing cancer patients compared to cancer patients without weight loss [START_REF] Scott | The relationship between weight loss and interleukin 6 in non-small-cell lung cancer[END_REF].

Furthermore, plasma levels of CRP and IL-6 appeared to correlate with skeletal muscle Ub mRNA levels in weight losing cancer patients [START_REF] Dejong | Systemic inflammation correlates with increased expression of skeletal muscle ubiquitin but not uncoupling proteins in cancer cachexia[END_REF]; while gastrointestinal cancer patients receiving the anti-inflammatory compound, ibuprofen, in conjunction

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with an appetite stimulator (megestrol acetate), demonstrated significant improvements in weight, mid-upper arm circumference and quality of life scores compared to patients receiving megestrol acetate alone, independent of changes in appetite [START_REF] Mcmillan | A prospective randomized study of megestrol acetate and ibuprofen in gastrointestinal cancer patients with weight loss[END_REF]. Collectively, these findings would suggest that in cancer cachexia, the UPS is actively involved in the initial stages of muscle protein loss and furthermore, implicate the associated inflammatory response as a potential regulator of the catabolic processes underlying cancer cachexia.

Inflammatory states result in the systemic production of a multitude of inflammatory and anti-inflammatory cytokines. Moreover, the cytokine response observed differs from that elicited by exercise, where increases in TNFα and IL-1β are generally not observed [START_REF] Petersen | The anti-inflammatory effect of exercise[END_REF]. Interestingly, numerous reports have demonstrated that the cytokines are capable of eliciting UPS-mediated protein degradation in skeletal muscle (for a review see [START_REF] Späte | Proinflammatory cytokines and skeletal muscle[END_REF]). Thus, a purported cytokine induction of the UPS could be responsible for the observed differences between disuse and inflammatory mediated muscle loss. However, determining causality for the cytokines in inflammatory states, especially in humans, remains particularly problematic.

Induction of the UPS has also been documented in the skeletal muscles of patients admitted to Intensive Care Units (ICU), a subpopulation of patients that often present with pronounced inflammatory responses [START_REF] Winkelman | Inactivity and inflammation in the critically ill patient[END_REF]. In muscle samples from septic patients obtained during abdominal surgery, an elevation in mRNA levels for Ub and proteasomal subunit HC3 was detected [START_REF] Tiao | Sepsis is associated with increased mRNAs of the ubiquitinproteasome proteolytic pathway in human skeletal muscle[END_REF]. In a subsequent study which utilised patients representing a larger spectrum of admissions to the ICU, including individuals suffering from sepsis, vascular disease or road traffic accident trauma, a mean daily decrease in cross sectional area of 4% for type II and 3% for type I fibres was observed in addition to an overall increase in Ub protein levels [START_REF] Helliwell | Muscle fibre atrophy in critically ill patients is associated with the loss of myosin filaments and the presence of lysosomal enzymes and ubiquitin[END_REF].

Furthermore, a separate study of ICU patients detailed a significant positive correlation between the levels of UbB polyubiquitin mRNA and the rate of protein degradation 8-12 days after admission, as determined by the rate of intracellular

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phenylalanine appearance by the isotope dilution technique [START_REF] Biolo | Contribution of the ubiquitin-proteasome pathway to overall muscle proteolysis in hypercatabolic patients[END_REF], with the authors concluding that the rate of proteolysis after trauma is mainly regulated at the level of transcription. Additionally, these findings are in concordance with the work by Mansoor and colleagues [START_REF] Mansoor | Increased mRNA levels for components of the lysosomal, Ca2+-activated, and ATP-ubiquitin-dependent proteolytic pathways in skeletal muscle from head trauma patients[END_REF], who demonstrated significantly increased mRNA levels of Ub, E2 14K and HC2 in head trauma patients presenting with negative nitrogen balance and indices of increased protein breakdown (endogenous leucine production). Lastly, increased levels of MAFbx/atrogin-1 mRNA has been observed following acute quadriplegic myopathy, a subacute muscle disorder characterised by generalised progressive muscle weakness and atrophy that is predominantly associated with a patient history of sepsis, multiple organ failure, surgery and intensive care admission [START_REF] Di Giovanni | Constitutive activation of MAPK cascade in acute quadriplegic myopathy[END_REF]. Collectively, these findings highlight the catabolic consequences of trauma in its various forms and demonstrate evidence that the reductions in muscle protein content are, at least in part, mediated via the actions of the UPS. However, a spectrum of critically ill patients will likely contain a host of different clinical presentations, each able to modify the catabolic response. Such possible influences include massive systemic inflammation, starvation, immobilisation, insulin resistance and denervation. This represents a limitation in the ability to determine the primary initiator of the catabolic response when utilising patient populations, if indeed such a thing exists.

Unfortunately, the number of studies that have examined the transcriptional changes of the two atrogenes in human inflammatory states is severely limited. While elevations in MAFbx/atrogin-1 mRNA have been observed in amyotrophic lateral sclerosis patients [START_REF] Léger | Human skeletal muscle atrophy in amyotrophic lateral sclerosis reveals a reduction in Akt and an increase in atrogin-1[END_REF], conflicting results have been observed in chronic obstructive pulmonary disease (COPD) sufferers, where reports of both an elevation [START_REF] Doucet | Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease[END_REF] and no significant change [START_REF] Ottenheijm | Activation of the ubiquitin-proteasome pathway in the diaphragm in chronic obstructive pulmonary disease[END_REF] in the level of the Ub-ligase have been reported (Table 2).

However, where elevations in MAFbx/atrogin-1 mRNA have been reported, they do not appear to have led to increased levels of the MAFbx/atrogin-1 protein [START_REF] Doucet | Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease[END_REF].

Furthermore, elevations of MAFbx/atrogin-1 mRNA were also not observed in the

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muscles of either old adults (>60 yrs; [START_REF] Whitman | Contributions of the ubiquitin-proteasome pathway and apoptosis to human skeletal muscle wasting with age[END_REF]) or very old women (~85 yrs; [START_REF] Raue | Proteolytic gene expression differs at rest and after resistance exercise between young and old women[END_REF]).

Similarly, inconsistent findings for MuRF1 mRNA levels have been observed in COPD sufferers, with both increased [START_REF] Doucet | Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease[END_REF] and basal [START_REF] Ottenheijm | Activation of the ubiquitin-proteasome pathway in the diaphragm in chronic obstructive pulmonary disease[END_REF] levels of MuRF1 mRNA reported. Increased MuRF1 mRNA levels have been observed in elderly women (~85 yrs; [START_REF] Raue | Proteolytic gene expression differs at rest and after resistance exercise between young and old women[END_REF]), but no change recorded in either amyotrophic lateral sclerosis patients [START_REF] Léger | Human skeletal muscle atrophy in amyotrophic lateral sclerosis reveals a reduction in Akt and an increase in atrogin-1[END_REF]) or elderly adults (>60 yrs; [START_REF] Whitman | Contributions of the ubiquitin-proteasome pathway and apoptosis to human skeletal muscle wasting with age[END_REF]). However, given that the age-dependent loss of skeletal muscle mass (sarcopenia) is thought to be predominantly due to the resistance of skeletal muscle protein synthesis to circulating amino acids [START_REF] Timmerman | Amino acid metabolism and regulatory effects in aging[END_REF], it is not surprising that a robust induction of MAFbx/atrogin-1 and MuRF1 was not observed in elderly individuals. Taken together, it is difficult from the limited evidence available to determine if MAFbx/atrogin-1 and MuRF1 have an active role in the UPS-mediated degradation of muscle proteins during inflammatory diseases. Given the robust induction of the UPS in ICU and cancer cachectic patients, it may be that the study of more acute and extreme inflammatory states, as seen in sepsis [START_REF] Hasselgren | Novel aspects on the regulation of muscle wasting in sepsis[END_REF] and following burn-injury [START_REF] Pereira | Post burn muscle wasting and the effects of treatments[END_REF], may provide greater insight into the purported roles of the two atrogins in humans. It is noteworthy that both sarcopenic and COPD patients have been reported to present with systemic low grade inflammation [START_REF] Ferrucci | Change in muscle strength explains accelerated decline of physical function in older women with high interleukin-6 serum levels[END_REF][START_REF] Ferrucci | Serum IL-6 level and the development of disability in older persons[END_REF][START_REF] Sin | Skeletal muscle weakness, reduced exercise tolerance, and COPD: is systemic inflammation the missing link?[END_REF] and, arguably in COPD patients, the localised levels of cytokines in the hindlimb muscles may not be notably elevated compared to controls [START_REF] Barreiro | Cytokine profile in quadriceps muscles of patients with severe COPD[END_REF]. Indeed, components of the UPS in chronic conditions such as Cushing's syndrome and Duchenne muscular dystrophy are known to be unchanged compared to acute catabolic states [START_REF] Rallière | Glucocorticoids do not regulate the expression of proteolytic genes in skeletal muscle from Cushing's syndrome patients[END_REF].

Interestingly, where increases in atrogene mRNA levels have been reported

in the skeletal muscle of COPD sufferers, a parallel increase in skeletal muscle cytoplasmic protein content of phosphorylated AKT simultaneous to an increase in both Foxo1 mRNA expression and Foxo1 protein levels present in the nucleus have also been recorded [START_REF] Doucet | Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease[END_REF]. Likewise, enhanced MAFbx/atrogin-1 mRNA and protein levels in amyotrophic lateral sclerosis sufferers occurred in conjunction with a fall in AKT phosphorylation, but no change in the nuclear protein content of Foxo1 or Foxo3

A C C E P T E D M A N U S C R I P T [START_REF] Léger | Human skeletal muscle atrophy in amyotrophic lateral sclerosis reveals a reduction in Akt and an increase in atrogin-1[END_REF]. Therefore, in contrast to a proposed regulation of MAFbx/atrogin-1 and MuRF1

transcription by an AKT-forkhead transcription factor axis (Fig. 2) [103, 104], the expression of the two atrogenes appears to be dependent on another mechanism, at least in COPD or amyotrophic lateral sclerosis patients. The observation of a discord in the relationship between AKT and Foxo proteins is not without precedent. In human primary breast tumours lacking a detectable phosphorylated AKT protein, Foxo3 was predominantly confined to the cytoplasm as opposed to the nucleus [START_REF] Hu | IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a[END_REF].

Reasons for this remain unclear.

Involvement of the UPS in exercise rehabilitation

For numerous years, exercise has been prescribed as an essential element of the rehabilitation program for individuals following muscle atrophy. A limited number of studies have examined the changes that occur in the UPS following the induction of exercise-mediated rehabilitation. For example, our laboratory has shown 6 weeks of resistance exercise training to be sufficient to reverse the ~5% fall in quadriceps lean mass observed after two weeks leg immobilisation ( [START_REF] Jones | Disuse atrophy and exercise rehabilitation in humans profoundly affects the expression of genes associated with the regulation of skeletal muscle mass[END_REF]; Fig. 3). Moreover, 24 h following cast removal and the first exercise session, mRNA levels of both atrogenes were decreased, returning to basal levels when compared to immediately postimmobilisation. In addition, immobilisation induced mRNA increases in 20S

proteasome subunit HC6, returned to normal levels 1 week from the start of the exercise regime [START_REF] Jones | Disuse atrophy and exercise rehabilitation in humans profoundly affects the expression of genes associated with the regulation of skeletal muscle mass[END_REF]. Likewise, a combination of resistance exercises and aerobic exercises performed on opposing days, at an interval of once every 3 days, was able to prevent the increased protein expression of MuRF1 in soleus muscle and maintain fibre cross sectional area following 60-days bed rest [START_REF] Salanova | Molecular biomarkers monitoring human skeletal muscle fibres and microvasculature following long-term bed rest with and without countermeasures[END_REF]. These observations, in conjunction with reports that passive leg cycling in spinal cord injury patients significantly reduced mRNA levels of Ub, E2 and 20S [START_REF] Willoughby | Myosin heavy chain isoform and ubiquitin protease mRNA expression after passive leg cycling in persons with spinal cord injury[END_REF], demonstrates that exercise is sufficient to blunt the UPS in human skeletal muscle following disuse atrophy. Whilst the significance of the exercise induced depression of UPS

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components on protein content is unknown, it is interesting to note its apparent relationship with the restoration of muscle mass.

Conversely, exercise (cycle ergometry) has been shown to exacerbate the increased expression of UPS components in COPD patients [START_REF] Radom-Aizik | Effects of exercise training on quadriceps muscle gene expression in chronic obstructive pulmonary disease[END_REF]. Such changes were not observed in healthy controls undergoing the same training protocol [START_REF] Radom-Aizik | Effects of exercise training on quadriceps muscle gene expression in chronic obstructive pulmonary disease[END_REF].

Interestingly, the pathophysiology of COPD could induce mild oxidative stress during periods of exercise [START_REF] Pinho | Oxidative stress in chronic obstructive pulmonary disease patients submitted to a rehabilitation program[END_REF] and such conditions are known to be capable of increasing the expression of UPS components [START_REF] Gomes-Marcondes | Induction of protein catabolism and the ubiquitin-proteasome pathway by mild oxidative stress[END_REF]. However, it should be noted that it is unequivocal that endurance training improves exercise capacity, muscle force, quality of life and functional status of COPD sufferers [START_REF] Pitta | Are patients with COPD more active after pulmonary rehabilitation?[END_REF].

Conclusions

In comparison to the wealth of knowledge from animal models concerning the involvement of the UPS in skeletal muscle atrophy and/or remodelling, studies in humans are at a comparatively early stage, with most studies based predominantly upon the examination of transcriptional events. However, initial findings would suggest that significant differences exist in the molecular events that underpin UPS mediated protein degradation in animals and humans in response to diverse stimuli. This is most notable in non-inflammatory conditions of muscle atrophy, where the role of the UPS and that of the atrogins, appear particularly exaggerated in animals compared to that observed in humans, although this could merely be a reflection of the disparity between the severity of animal and human models of muscle disuse.

Thus, in more global terms, the role of muscle protein breakdown in these conditions remains equivocal. Furthermore, several discrepancies in the proposed regulation of the atrogenes between animals/cell lines and that of humans have started to emerge, suggesting alternative regulatory pathways that have yet to be described.

Collectively, these findings highlight the pivotal importance for detailed human studies in determining the pathways regulating skeletal muscle mass in conditions of

  Heavy resistance training can induce substantial increases in muscle mass, predominantly the result of increases in fibre cross-sectional area (hypertrophy) as opposed to fibre number (hyperplasia). Moreover, resistance training can increase the cross-sectional area of all three major fibre types (I, IIa and IIb), with a 20-week training program sufficient to increase the cross-sectional areas of type IIab and IIb fibres by 47%, type IIa by 39% and type I by 17%[START_REF] Staron | Strength and skeletal muscle adaptations in heavy-resistance-trained women after detraining and retraining[END_REF]. The hypertrophic response to strength training appears predominantly the result of enhanced rates of muscle protein synthesis simultaneous to increases in muscle protein breakdown, albeit the change in the latter being of smaller magnitude than the former thus leading to an overall positive accumulation of muscle protein content[START_REF] Rennie | Protein and amino acid metabolism during and after exercise and the effects of nutrition[END_REF]. Two groups have reported an elevation of MuRF1 mRNA levels up to 24 h[START_REF] Louis | Time course of proteolytic, cytokine, and myostatin gene expression after acute exercise in human skeletal muscle[END_REF][START_REF] Yang | Proteolytic mRNA expression in response to acute resistance exercise in human single skeletal muscle fibers[END_REF][START_REF] Raue | Proteolytic gene expression differs at rest and after resistance exercise between young and old women[END_REF][START_REF] Mascher | Repeated resistance exercise training induces different changes in mRNA expression of MAFbx and MuRF-1 in human skeletal muscle[END_REF], and MAFbx/atrogin-1 mRNA levels up to 4 h[START_REF] Louis | Time course of proteolytic, cytokine, and myostatin gene expression after acute exercise in human skeletal muscle[END_REF][START_REF] Deldicque | Effects of resistance exercise with and without creatine supplementation on gene expression and cell signaling in human skeletal muscle[END_REF], following the induction of an acute bout of resistance exercise (

A

  C C E P T E D M A N U S C R I P T failing to coincide with transient elevations of the atrogenes and/or that only transcriptional changes in the atrogenes have mainly thus far been examined. Work examining the temporal expression of the atrogins (mRNA and protein) during disuse in conjunction with measurements of protein degradation in humans should resolve this conundrum.

A

  C C E P T E D M A N U S C R I P T health and disease, utilising temporal measures of muscle protein turnover and biomolecular techniques across the spectrum of transcriptional, translational and post-translational events. A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

Figure 1 :

 1 Figure 1: UPS mediated protein degradation. Diagrammatical representation of

Figure 2 :

 2 Figure 2: AKT regulated signalling events. Representation of the effects of

Figure 3 :

 3 Figure 3: Effect of leg immobilisation and rehabilitation on the UPS and muscle

Table 1

 1

	ACCEPTED MANUSCRIPT
	M A N U S C R I P T
	A C C E P T E D

). However, mRNA quantification of the atrogenes a few hours subsequent to these measurements have shown either reduced or basal levels

[START_REF] Louis | Time course of proteolytic, cytokine, and myostatin gene expression after acute exercise in human skeletal muscle[END_REF][START_REF] Yang | Proteolytic mRNA expression in response to acute resistance exercise in human single skeletal muscle fibers[END_REF][START_REF] Raue | Proteolytic gene expression differs at rest and after resistance exercise between young and old women[END_REF][START_REF] Mascher | Repeated resistance exercise training induces different changes in mRNA expression of MAFbx and MuRF-1 in human skeletal muscle[END_REF][START_REF] Deldicque | Effects of resistance exercise with and without creatine supplementation on gene expression and cell signaling in human skeletal muscle[END_REF][START_REF] Coffey | Interaction of contractile activity and training history on mRNA abundance in skeletal muscle from trained athletes[END_REF][START_REF] Kostek | Gene expression responses over 24 h to lengthening and shortening contractions in human muscle: major changes in CSRP3, MUSTN1, SIX1, and FBXO32[END_REF][START_REF] Churchley | Influence of preexercise muscle glycogen content on transcriptional activity of metabolic and myogenic genes in welltrained humans[END_REF]

. Collectively, these observations are in accordance with findings of a 31% increase in the fractional breakdown rate of skeletal muscle proteins 3 h postresistance exercise, declining to an 18% increase by 24 h, and a return to resting values by 48 h

[START_REF] Phillips | Mixed muscle protein synthesis and breakdown after resistance exercise in humans[END_REF]

. The transient elevation of the fractional breakdown rate concomitant to an increase in MuRF1 and MAFbx/atrogin-1 mRNA levels is consistent with the suggestion that the enhanced leg proteolysis observed is in part MAFbx/atrogin-1 and MuRF1 dependent. However, measurements of the protein expression for the two atrogins are currently lacking and would represent a remodelling stimulus driving a change in fibre-type expression to that of a faster phenotype. Despite the extensive remodelling that occurs following a chronic resistance exercise regime, levels of MAFbx/atrogin-1 protein and MuRF1 mRNA remain elevated

[START_REF] Léger | Akt signalling through GSK-3beta, mTOR and Foxo1 is involved in human skeletal muscle hypertrophy and atrophy[END_REF]

. The consequences of the sustained elevation of the atrogins in response to chronic resistance exercise remains unknown, although suggestions levied of an increased basal rate of protein breakdown in the context of increased protein turnover remain speculative in the absence of measures of muscle protein turnover itself.

Increases in muscle protein synthesis are observed hours after a bout of resistance exercise paralleled by increases in the phosphorylation state of key components of the insulin-signalling pathway, in most cases including AKT

[START_REF] Cuthbertson | Anabolic signaling and protein synthesis in human skeletal muscle after dynamic shortening or lengthening exercise[END_REF]

. synthesis

[START_REF] Wilkinson | Differential effects of resistance and endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle[END_REF][START_REF] Greenhaff | Disassociation between the effects of amino acids and insulin on signalling, ubiquitin-ligases and protein turnover in human muscle[END_REF]

, reinforcing the requirement of measures of muscle protein turnover in study designs. What is also not clear is the impact of Foxo inhibition (phosphorylation) by AKT on MAFbx/atrogin-1 and MuRF1 transcription in human

  R I P T is particularly pertinent given the recent observations of Vary et al.[START_REF] Vary | Acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA without increasing proteolysis in skeletal muscle[END_REF] who demonstrated acute alcohol intoxication in rodents to increase MAFbx/atrogin-1 and MuRF1 mRNA levels without resulting in a concomitant rise in muscle proteolysis.

	Such observations could suggest that MAFbx/atrogin-1 and MuRF1 do not
	necessitate muscle protein breakdown; but without direct measures of
	MAFbx/atrogin-1 and MuRF1 protein levels, it is difficult to ascertain the true
	significance of such findings. However, it should be noted that a few reports of
	muscle proteolysis occurring independently of changes in MAFbx/atrogin-1 and/or
	MuRF1 mRNA levels have recently been published, which would question the role of
	the atrogins in muscle proteolysis [50, 119-121]. Further work is required to
	rationalise these important observations.

Table legends Table 1 :

 legends1 Exercise and the UPS. Data obtained from human studies investigating

	changes in skeletal muscle UPS components following acute eccentric or acute
	resistance exercise bouts in non-previously resistance trained individuals. Time
	denotes period(s) elapsed from completion of exercise bout and recorded
	measurement. KE = Knee extension, LE = Leg extension, LP = Leg press, 1-RM = 1-
	Rep max. * = previously endurance trained.

Table 2 : The UPS in inflammatory and non-inflammatory states characterised by

 2

	Amyotrophic lateral sclerosis; VL = Vastus lateralis, GAS = Gastrocnemius, SOL =
	Soleus, TA = Tibialis Anterior, RA = Rectus abdominus, B = Bicep, D = Diaphragm

muscle atrophy. Data obtained from human studies investigating changes in skeletal muscle UPS components following non-inflammatory and inflammatory states of muscle atrophy. COPD = Chronic obstructive pulmonary disease, ALS =