Aerobic Exercise and Pharmacological Treatments Counteract Cachexia by Modulating Autophagy in Colon Cancer

Recent studies have correlated physical activity with a better prognosis in cachectic patients, although the underlying mechanisms are not yet understood. In order to identify the pathways involved in the physical activity-mediated rescue of skeletal muscle mass and function, we investigated the effects of voluntary exercise on cachexia in colon carcinoma (C26)-bearing mice. Voluntary exercise prevented loss of muscle mass and function, ultimately increasing survival of C26-bearing mice. We found that the autophagic flux is overloaded in skeletal muscle of both colon carcinoma murine models and patients, but not in running C26-bearing mice, thus suggesting that exercise may release the autophagic flux and ultimately rescue muscle homeostasis. Treatment of C26-bearing mice with either AICAR or rapamycin, two drugs that trigger the autophagic flux, also rescued muscle mass and prevented atrogene induction. Similar effects were reproduced on myotubes in vitro, which displayed atrophy following exposure to C26-conditioned medium, a phenomenon that was rescued by AICAR or rapamycin treatment and relies on autophagosome-lysosome fusion (inhibited by chloroquine). Since AICAR, rapamycin and exercise equally affect the autophagic system and counteract cachexia, we believe autophagy-triggering drugs may be exploited to treat cachexia in conditions in which exercise cannot be prescribed.

running distance, in both control and C26-bearing mice ( Table 1). The results show that C26-bearing mice were still capable of running, though to a lesser extent than control mice but at a similar intensity.
To quantify the effects of WR on parameters commonly used as diagnostic criteria for cachexia 8 , we compared several groups of animals: control (i.e. healthy) mice in the absence and presence of WR, and C26 tumor-bearing mice in the absence and presence of WR and of pharmacological treatments used as replacements for WR. In terms of final weight (i.e. carcass weight, which is the body weight minus tumor weight), we found no effect of WR on healthy mouse body weight; instead, all the treatments (i.e. WR and drug administrations) affected body weight by interfering with the C26-induced body weight loss (Table 2).
Since WR prevented the severe body weight loss that characterizes cachexia and since skeletal muscle mass accounts for about 50% of the body mass, a rescue of muscle homeostasis was a predicted outcome. Thus, we measured the effects of exercise on a specific representative muscle, i.e. the tibialis anterior (TA), and found that WR increased TA mass in C26-bearing mice (Fig. 1a). Histological and morphometric analyses of the muscle fiber cross-sectional area (CSA), performed by using immunostaining for laminin to visualize the myofiber perimeter, revealed a significantly bigger CSA in exercised C26-bearing mice than in sedentary C26-bearing mice (Fig. 1b,c).
Along with muscle mass, WR improved muscle function in C26-bearing mice: the extensor digitorum longus (EDL) muscle, a muscle that we previously found to be affected in cancer cachexia 29 , exhibited a shorter fatigue time (defined as the time required to halve the value of the muscle maximum strength) in C26-bearing control mice than in non-C26-bearing control mice, while no differences were detected in WR C26-bearing mice (Fig. 1d). A negative interaction between the C26-induced decrease in fatigue time and WR was found, which thus indicates that WR rescues muscle function in C26-bearing mice.
Given the many positive effects exerted by WR on skeletal muscle homeostasis and function, we sought to verify whether WR might extend the life span of C26-bearing mice. Since the presence of a wheel in the cage may represent a form of environmental enrichment 30 , we also included, as an additional control, treatments designed to test whether environmental enrichment had significant effects per se on mouse survival. Thus, we combined the use of regular or environmentally-enriched (EE) cages ( Supplementary Fig. S2a) with the absence or presence of the wheel to evaluate the survival of C26-bearing mice. WR significantly increased survival in C26-bearing mice whereas EE cages alone did not ( Supplementary Fig. S2b). We also observed that the effects of WR were maintained in EE cages (not shown in the figure), thus suggesting that WR and environmental enrichment do not affect each other.
WR activity increased the life span of C26-bearing mice by about 44%, thus indicating that voluntary physical activity had a marked beneficial effect on animal models of cancer cachexia, as previously reported in clinical studies. In order to assess any correlation between individual variations in the amount of exercise with output in terms of survival, we measured the daily distance covered by WR and the lifespan of the corresponding mouse in a subset of randomly chosen animals. This approach revealed a striking, linear correlation between km/day run on the wheel and survival time: each km/day of running activity was found to correspond to four additional days of survival in C26-bearing mice (Fig. 1f).
In keeping with all the aforementioned findings, WR was also found to be associated with a significant increase in food intake in WR C26-bearing mice compared with control C26-bearing mice ( Supplementary Fig. S3). Moreover, the fact that food intake in C26-bearing mice did not change over time indicates that this tumor does not induce anorexia in mice as it progresses.
Voluntary wheel running counteracts alterations in catabolic pathways in colon carcinoma (C26)-bearing mice. Cachexia is characterized by an up-regulation of the muscle specific E3-ubiquitin ligases, atrogin 1 and MuRF1 31 . When we quantified the effects of both C26 and WR on these markers in skeletal muscle, we found that both ubiquitin ligases were significantly up-regulated in muscle from C26-bearing mice when compared with control healthy mice, and that voluntary wheel running prevented the induction of these genes and consequently restored expression levels to those of controls (Fig. 2a). days. Data are shown as mean ± SEM. Two-way ANOVA (F = 5.77; df 1; p = 0.025) shows an interaction between the negative effect of C26 tumor and wheel running, which restores fatigue time to control levels; data are shown as mean ± SEM; 6 < n < 9 for each group, * p < 0.05 by Tukey's HSD test. (e) Survival curves derived from Cox model for statistical analysis of C26bearing mice in the absence (C26, circles) or presence (C26 WR, triangles) of wheel running. Wheel running significantly (p < 0.041) increased survival of C26-bearing mice. n = 9 for each group.  (c) Western blot analysis of LC3bI, LC3bII and p62 expression in control mice (CTR), or mice in the absence (-) or presence (WR) of wheel running, 19 days after C26 transplantation. Gapdh was used as a loading control. Densitometric analyses of Western blot showing LC3bII/LC3bI ratio (d) and p62/Gapdh (e) ratio in mice in the absence (-) or presence (WR) of wheel running, 19 days after C26 transplantation, compared with control mice (CTR). Data are presented as mean ± SEM. F = 4.63; df 2; p < 0.05 and F = 14.18; df 2; p < 0.02 by ANOVA; *p < 0.005 and ** p < 0.005 by Tukey's HSD test. (f) Expression of LC3b and p62 by Western blot analyses in skeletal muscle biopsies from colon carcinoma patients and healthy (CTR) subjects. Gapdh was used as a loading control. The lanes were run on the same type of gel but were non-contiguous, as indicated by the thin black lines. Densitometric analyses of Western blot showing significantly higher expression of LC3bII/LC3BI ratio (g) and p62/Gapdh ratio (h) in skeletal muscle biopsies from colon carcinoma patients (CARC), compared with healthy subjects (CTR). Data are presented as mean ± SEM. * p < 0.05 by Student's t-test.
It has recently been suggested that autophagy contributes to the pathogenesis of skeletal muscle wasting in cancer cachexia 32 . Using electron microscopy, we detected autophagosome-like structures in skeletal muscle fibers of C26-bearing mice (Supplemental Fig. S4), which is consistent with similar morphological findings observed at the ultrastructural level in other muscle pathologies 33 .
As aerobic exercise is known to trigger autophagy in skeletal muscle 34 , we decided to evaluate autophagy in our experimental conditions by immunostaining for p62 and Western blot analyses for LC3b and p62, as markers of autophagic induction. Immunofluorescence analyses on TA muscles detected accumulation of p62 in C26-bearing mice, if compared with control or WR C26-bearing mice (Fig. 2b). The activation of autophagy drives the processing of LC3bI into its lipidated, autophagosome-bound form LC3bII, with a high LC3bII/LC3bI ratio being considered to indicate an increased number of autophagosomes. We observed an increase in the levels of the LC3bII/LC3bI and p62/Gapdh ratio in C26-bearing mice (Fig. 2c-e), compared with control healthy mice, which is in agreement with findings by Penna et al. 32 . Interestingly, WR significantly reduced the LC3bII/ LC3bI and p62/Gapdh ratio in C26-bearing mice (Fig. 2d,e). These findings confirm that autophagy is activated in sedentary C26-bearing mice; nevertheless, the sustained expression of p62 suggests that the autophagic flux may, despite not being blocked completely, proceed at a slower rate. Voluntary WR exercise appears to restore basal levels of autophagy, which is beneficial to muscle homeostasis insofar as it is associated with the recovery of muscle mass and function.
To validate previous observations yielded by the C26-bearing mouse model, we compared the levels of expression of the aforementioned autophagy-related proteins with data obtained from skeletal muscle biopsies of colon carcinoma patients. Remarkably, accumulation of the lipidated form of LC3b, a high LC3bII/LCb3I ratio and significantly higher p62 levels were detected in skeletal muscle of colon carcinoma patients compared with healthy subjects (Fig. 2f-h). The pathological classification of tumors, gender, age, BMI were recorded for this cohort of patients, together with the levels of the following parameters: total proteins, albumin and transthyretin, creatine kinase, C-reactive protein and leukocytes (Supplementary Tables 1 and 2). Other events and chemotherapy treatments were also taken into account. No significant correlation between p62 levels and any of these parameters was found. The lack of significant differences between patients suggests that it is possible to pool patients for the p62 analysis.
By showing that both tumor-bearing mice and patients display altered autophagic markers, our results indicate that such alterations in the autophagic system may play a role in the development of cachexia. This prompted us to further investigate the efficacy of pharmacological treatments designed to trigger autophagy in cancer-induced cachexia.
Drugs promoting autophagy counteract body weight loss in cancer cachexia. As described above, AICAR is a pharmacological activator of AMPK 17 that has been shown to recapitulate the molecular response of skeletal muscle to aerobic exercise and to promote autophagy 14,35 . We therefore treated, on a daily basis, C26-bearing mice with AICAR, or a vehicle for controls, by means of intraperitoneal injections. We confirmed the activation of AMPK and its downstream target ACC in skeletal muscles of AICAR-treated mice by Western blot analyses (Fig. 3a). Rapamycin, a specific inhibitor of mTOR, is another drug known to trigger autophagy in many cell types, including skeletal muscle 36 . In parallel experiments, we treated, on a daily basis, C26-bearing mice with rapamycin, or a vehicle for controls, by means of intraperitoneal injections, and confirmed the inhibition of mTOR in skeletal muscles of rapamycin-treated mice, by Western blot analyses (Fig. 3b). Treatment with AICAR prevented weight loss in C26-bearing mice, compared with vehicle-treated C26-bearing mice (Table 2). Accordingly, AICAR blocked skeletal muscle wasting in C26-bearing mice by restoring both muscle mass and myofiber size (Fig. 3c-e). Similarly, rapamycin treatment significantly affected cancer cachexia in C26-bearing mice by preventing a drop in body weight (Table 2), which in turn resulted in heavier and larger TA muscles than in vehicle-treated mice (Fig. 3c-e). These drugs were found to exert a significant effect on both the TA weight and muscle fiber CSA, thereby showing that pharmacological treatments are highly effective in counteracting muscle wasting in C26-bearing mice. Interestingly, no difference was detected between AICAR and rapamycin treatment in terms of body weight rescue in tumor-bearing mice ( Table 2). AICAR and rapamycin counteract muscle atrophy and affect autophagy. We compared the transcriptional response to cancer cachexia in C26-bearing mice treated with either AICAR or rapamycin with that in vehicle-treated mice 19 days after tumor transplantation. Strikingly, both AICAR and rapamycin treatment significantly reduced atrogin 1 and MuRF1 expression levels, which were instead up-regulated in vehicle-treated C26-bearing mice (Fig. 4a). Similarly to voluntary wheel running, we found that both AICAR and rapamycin treatments modulated autophagy in C26-bearing mice by significantly reducing C26-mediated p62 accumulation, as revealed by immunofluorescence analyses on TA muscles (Fig. 4b). Moreover, quantification of LC3bII/LC3bI and p62/Gapdh protein levels by Western blot analyses confirmed the effects of both AICAR and rapamycin treatments on these autophagic markers (Fig. 4c-e).

AICAR and rapamycin affect skeletal muscle cells in vitro.
Since both AICAR and rapamycin treatments were administered systemically, to prove that they directly affect skeletal muscle cells, we reproduced our experimental model in vitro by treating C2C12 myotubes with conditioned media derived from C26 cells. Forty-eight hours of treatment with C26-conditioned media induced approximately 30% of myotube atrophy in vitro, as quantified by measuring myotube diameter, whereas both AICAR and rapamycin treatments counteracted C26-induced muscle atrophy (Fig. 5). The effects of these treatments in vitro suggest that autophagy plays a role in controlling myotube size; to prove that autophagy is involved in the control of the myotube mass, we interfered with the autophagic flux by treating cells with chloroquine, an agent that prevents the fusion between Scientific RepoRts | 6:26991 | DOI: 10.1038/srep26991 autophagosomes and lysosomes 37 . A six-hour treatment with 50 microM chloroquine induced an accumulation of LC3b and p62 in myotubes ( Supplementary Fig. S5), which is indicative of a block in the autophagic flux, and worsened myotube atrophy in all the conditions analyzed (Fig. 5).

Discussion
Cachexia most commonly presents in lung-or gastrointestinal tract-cancer patients 38 , who can lose as much as 30% of their initial body weight. Since loss of strength and muscle mass are, together with involuntary loss of body weight 39 , the main features associated with cachexia, physical activity has proved to be a good therapeutic strategy to fight cachexia, thanks to its effects on both strength and muscle mass 40 . The molecular mechanisms underlying the physical activity-mediated rescue of cachexia are poorly understood. We show that all the major diagnostic features of C26-induced cachexia, such as loss of body weight, skeletal muscle mass and fiber size, can be counteracted by voluntary wheel running in mice. Voluntary running also improves skeletal muscle function in C26-bearing mice by raising the fatigue threshold, a relevant finding if we consider that fatigue is one of the most frequently reported complaints of cachectic patients 41 . It is noteworthy that C26-bearing mice did not develop anorexia and, thus, represent a pure cachexia model 42 . Conventional nutritional support cannot fully reverse cachexia, though a limited beneficial effect of appetite stimulants has been observed in mouse models 43 . We observed a significant increase in appetite in C26-bearing running mice associated with an improvement in muscle homeostasis, which suggests that exercise might have contributed to the improvement in muscle homeostasis by providing the aminoacids required for protein synthesis. Resistance exercise has traditionally been proposed to fight cachexia and other forms of muscle atrophy because it is a type of exercise that increases muscle protein content balance, mass, strength and resistance to fatigue. Here, we show that wheel running, which shares the features of endurance training 44 , effectively counteracts muscle wasting; as opposed to activating pro-hypertrophic signaling pathways 14 , this type of exercise counteracts pro-atrophic NF-κ B-dependent signaling 45 . We observed a significant increase in life span in wheel running C26-bearing mice. Strikingly, life span directly correlates with the amount of voluntary running in C26-bearing mice. Exercise prolongs survival in both tumor-bearing mice and humans. However, since cancer patients, particularly the elderly, are often unable to perform voluntary exercise, any therapy based on voluntary running or other physical aerobic activities has certain limitations for cancer patients with progressive muscular atrophy. It is hence important i) to experimentally test alternative interventions, such as functional neuromodulation (i.e. electrical stimulation training of muscles) and ii) to identify the molecular pathways involved in voluntary running-mediated muscle rescue in cachexia. The latter is not merely due to an exercise-mediated shift toward a more oxidative fiber phenotype, as is shown by the fact that 19 days of wheel running did not significantly increase oxidative fibers 44 , which are considered to be more resistant to cancer cachexia 46 . Molecular analyses showed that wheel running reduces tumor-mediated atrogin 1 and MuRF1 gene expression, thus demonstrating that aerobic physical activity counteracts muscle atrophy by targeting the catabolic pathways of protein degradation. Pioneering analyses of the effect of a bout of wheel running (more than 5 km during the first night) demonstrated that muscle damage/regeneration/repair are accompanied by structural and molecular events associated with muscle apoptosis 47,48 . Apoptosis and inflammation share underlying healing (short-term) and/or damaging (long-term) processes: exercise may induce muscle adaptation to damage, but exposing severely cachectic tumor-bearing mice to exercise protocols might be deleterious. Consequently, pharmacological alternatives to physical activity should be considered.
Within this context, the results of our study show that one of the molecular pathways involved in cancer cachexia, i.e. autophagy, is directly responsible for the induction of skeletal muscle atrophy. It may thus be possible to use pharmacological treatments designed to trigger this cellular signal to improve muscle mass maintenance in cancer patients. We observed similar patterns of LC3bII and p62 protein levels in skeletal muscle biopsies from both colon carcinoma patients and C26-bearing mice, which were characterized by an accumulation of the lipidated form LC3b and the p62 protein. The fact that autophagocytosis normally degrades p62, whose levels thus become negligible, suggests that the autophagic flux is altered in the skeletal muscle of both animal models and human beings. In keeping with our findings, a recent study proved that autophagy is over-induced in skeletal muscle and contributes to muscle atrophy in cancer cachexia 32 . In that study, however, colchicine treatment, which blocks the autophagic flux after autophagosome formation 49 , anticipated the death of C26-bearing mice, thereby suggesting that a complete block in the autophagic flux is deleterious in cancer cachexia. The results of our study confirm the activation of the autophagic pathway, demonstrated by increased levels of the lipidated form of LC3b, and a delay in autophagosome clearance, demonstrated by the accumulation of p62, in the skeletal muscle of C26-bearing mice. Our in vitro observations with chloroquine also confirmed that a block in the autophagic flux is detrimental to skeletal muscle mass. The novelty of our findings stems from the fact that drugs promoting, and not inhibiting, the autophagic flux are effective against cancer cachexia. Despite being counter-intuitive, since autophagy may appear to be an additional catabolic pathway leading to sarcomere dismantling in cachexia, the model we propose suggests that it is of paramount importance to maintain balanced and efficient autophagy, which is dysregulated in cachexia, to improve muscle homeostasis. Autophagy is required at physiological levels for the removal of misfolded proteins and damaged organelles, and to prevent the accumulation of protein aggregates 50 . Autophagy has also been reported to play a key role in skeletal muscle homeostasis 27 . Skeletal muscles of autophagy-deficient mice progressively develop myopathy with age, and upon autophagy-triggering stresses, such as denervation or fasting, they severely degenerate owing to the accumulation of ubiquitinated proteins and damaged organelles 51 . Many studies have shown that an incorrect autophagic flux correlates with, and sometimes contributes to, skeletal muscle diseases such as Bethlem myopathy 52 , various muscular dystrophies 53,54 and cancer cachexia 32 . The fact that treatment with two different drugs known to trigger autophagy, i.e. AICAR and rapamycin, improved muscle homeostasis in cancer cachexia strongly suggests that cachexia can be counteracted by triggering autophagy. AICAR has already been used to recover the autophagic flux in skeletal muscle and to counteract Duchenne muscular dystrophy in mice 55 . Rapamycin has instead been used as an immunosuppressant and anti-cancer drug 46 , though its effect on skeletal muscle wasting has never been investigated. Since AICAR and rapamycin, as well as other drugs that target AMPK such as metformin 56 , are currently being tested in cancer clinical trials 57 , these pharmacological treatments may be readily available for the translational applications deriving from our study. To prove that skeletal muscle cells are directly affected by AICAR, rapamycin and factors derived from C26 tumor cells, we reproduced cancer-derived muscle atrophy in vitro by treating myotubes with C26-conditioned media in the absence or presence of pharmacological treatments. Treatment with AICAR or rapamycin prevented C26-induced C2C12 myotube atrophy, thus proving that skeletal muscle cells are targeted by these drugs. Moreover, a block in the autophagic flux prevented the rescue of C26-induced myotube atrophy mediated by AICAR or rapamycin, thus indicating that both drugs regulate muscle homeostasis by promoting the autophagic flux.
In conclusion, the pharmacological treatments used in our study and exercise share the ability to counteract cachexia and restore autophagic levels to those found in control healthy muscles. In vitro studies show that muscle cells are direct targets of both tumor-derived factors and exercise mimetics involved in the rescue of muscle mass homeostasis through a process involving the autophagic flux. A graphical abstract of our findings is shown in Fig. 6. To date, we cannot establish a causative role between the accumulation of autophagic markers and worsened cachexia, nor can we rule out the possibility that exercise and pharmacological treatments ameliorate C26-induced cachexia through indirect effects on muscle. In this regard, voluntary running was recently demonstrated to directly counteract tumor growth in tumor-bearing mice by modulating the inflammation of the tumor microenvironment 58 However, our study provides unequivocal evidence showing that physical activity or exercise In physiological conditions, a balance between autophagosome production and clearance maintains an adequate autophagic flux (red arrow) in the muscles, mirrored by basal levels of LC3bII (black dots) and p62 (green dots) expression. In cachectic muscles from C26-bearing mice (B), a strong accumulation of both LC3bII and p62 proteins points to an unbalanced autophagosome production/ clearance ratio. This correlates with the pathophysiological features observed in cancer-related muscle wasting, including overexpression of Atrogin1 and Murf1 genes, as well as a decline in body weight (BD), muscle weight (MW), fiber size and muscle function. Spontaneous wheel running, or treatment with AICAR or rapamycin (C) counteracts cancer-related muscle wasting in tumor-bearing mice and induces a decrease in both LC3bII and p62 accumulation. Atrogin1 and Murf1 gene expression was restored to the basal levels observed in healthy muscles. This is associated with increased body and muscle weight and improved muscle function.
Scientific RepoRts | 6:26991 | DOI: 10.1038/srep26991 mimetics help to preserve muscle mass in the presence of a tumor and should be taken into consideration when planning therapies to treat cancer cachexia.

Methods
Mice and patients. Cachexia was induced by subcutaneous grafting of a 0.5 mm 3 fragment of colon carcinoma (C26, obtained from the National Cancer Institute) in the dorsal region of 7-week-old BALB/c female mice (Charles River, Wilmington, MA), as previously described 29 . Unless otherwise specified, animals were sacrificed 19 days following tumor transplantation, on the basis of our evidence from survival curves showing that the vast majority of animals are still alive at this time point, even in the presence of overt cachexia. Mice were treated strictly according to the guidelines of the Institutional Animal Care and Use Committee, and to relevant national and European legislation, throughout the experiments. All the experimental protocols were approved by the Organ for Prevention and Wellbeing of Animals (OPBA) of the University of Palermo and by the Charles Darwin ethics committee n°5 in Paris and sent to the respective Ministries of Research in the two countries. Informed consent was obtained from patients for muscle biopsies.
Exercise protocols and environmental enrichment. Wheels for rodents, with a diameter of 15 cm, were purchased in general customer pet shops. DC-9 tachometers were purchased from Decathlon. The mice were kept individually in cages that were identical, the only exception being that those of the WR mice were equipped with a wheel, as described in 44 , and were allowed to exercise ad libitum from the first day of the experiment, i.e. from the day of transplantation in tumor-bearing mice so as to mimic clinical studies in which physical activity started at the time of diagnosis. Since wheel-running activity may represent an environmental enrichment 30 , we set up additional, custom-made cages, characterized by the presence of novel objects, separate food reserves and tunnels, designed in such a way as to represent an environment enrichment 59 , to which a wheel was or was not added.

Drug administration. Mice were treated daily by means of an intraperitoneal (IP) injection of 250 mg/Kg
AICAR water solution (9 mice) or of a vehicle (18 mice), or of 2 mg/kg rapamycin in physiologic solution containing 1% ethanol (12 mice), or of a physiologic solution containing 1% ethanol (8 mice, which were subsequently pooled with the other vehicle group since no statistically significant difference between these two groups was detected in any parameter throughout the study). Treatments with both AICAR and rapamycin started on the day after tumor transplantation and stopped at sacrifice. All the groups in a given experiment were sacrificed on the same day, which was selected depending on the extent of cachexia developed by C26-bearing, non-treated animals. The latter were considered cachectic when they had lost at least 10% of their body weight.
Histological and immunofluorescence analyses. TA muscles were dissected, embedded in tissue-freezing medium (Leica, Wetzlar, Germany) and frozen in liquid nitrogen-cooled isopentane. Cryosections (8 μ m) were obtained using a Leica cryostat. Transverse cryosections of TA muscles were fixed in 4% paraformaldehyde for 10 min at room temperature. For p62 immunofluorescence analyses, muscles were permeabilized with 0.2% Triton in PBS for 30 min. After incubation with 1% BSA (Sigma, St. Lous, MO) for 30 min, samples were incubated with a 1:100 dilution in 1% BSA of polyclonal rabbit anti-laminin antibody (Sigma) or a 1:50 dilution in 1% BSA of anti-p62 guinea pig antibody (Progen) overnight at 4 degrees, followed by incubation with a 1:500 dilution in BSA of anti-rabbit-Alexa 488 or anti-guinea pig-Alexa 488 (Life Technologies) secondary antibodies for 1 hour at room temperature. 0.5 ug/ml Hoechst 33342 (Sigma) was used to stain nuclei.

Morphometric analyses.
Cross-sectional areas of TA myofibers were measured by using ImageJ software, freely availabe at http://imagej.nih.gov/ij/docs/intro.html. The whole muscle cross-sections from seven AICAR-treated mice or from seven vehicle-treated mice, and from five rapamycin-treated mice or from seven vehicle-treated mice were analyzed.
Myotube atrophy was quantified by measuring myotube diameter using ImageJ software. For each replicate sample, ten photomicrographs, each containing approximately 100 myotubes, were analyzed and the median myotube diameter (measured at the level of the maximum diameter displayed by each myotube) was calculated.
Functional analyses. Fatigue time was measured as previously described. Briefly, the EDL muscle was mounted vertically in an ASI 300 b Dual-Mode actuator/transducer. To induce isometric fatigue, muscles were subjected to a series of closer trains of pulses (0.4 s train of 120 Hertz pulses) and the time required to halve the value of their own maximum strength was calculated.

RNA extraction and Real-time PCR analyses.
Total RNA was isolated and purified from 30-50 mg of TA muscle using Trizol Reagent (Invitrogen), according to the manufacturer's protocol. One microgram of total RNA was converted to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR was performed with the SDS-ABI Prism 7500 (Applied Biosystem, Life Technologies, Monza, IT), using the Sybr Green reaction mix (Applied Biosystem).
The following primer sequences were used: GAPDH for ACC CAG AAG ACT GTG GAT GG GAPDH rev CAC ATT GGG GGT AGG AAC AC Five control healthy mice, six C26-bearing mice, six C26 WR, six vehicles, and five mice treated with either AICAR or rapamycin were analyzed.

Statistical analyses.
For each set of data, the first step entailed verifying the normal distribution of the data and their homoscedasticity by means of Levene's test. If the data were homoscedastic, one-or two-way analysis of variance (ANOVA) was used, followed by Tukey's HSD as a post-hoc test. If data were not homoscedastic (and in case of an unsuccessful transformation attempt), the data were analyzed using non-parametric tests, such as the Kruskal-Wallis test. Student's t-test was used when only two treatments were compared. A Cox model was used to treat and analyze the survival curves. The specific test used and the global effects are indicated in the Results, while the specific post-hoc test used and the significance level of specific comparisons are indicated in the Figure legends, which also indicate the number of samples analyzed. All values are expressed as mean ± standard error of the mean (SEM) or as median ± semi-interquartile range, as appropriate.Both ISSP and VassarStats, a statistical computation website that is available for free at http://vassarstats.net/, were used as softwares for the statistical analysis.

In vitro experiments. C26-conditioned medium was obtained by culturing confluent C26 cells (CLS Cell
Lines Service GmbH, Eppelheim, Germany) for 2 days in serum-free DMEM (this C26 cell-conditioned medium was diluted 1 to 5 (20% final concentration) to treat C2C12 cultures (DMEM stored for 2 days at 37 °C used for controls). C2C12 cells were seeded at 20000 cells/cm2 and cultured in growing conditions (GM, DMEM containing 1% glutamine, 2% HEPES, 0.5% gentamicin, supplemented with 10% FBS, SIgma); the following day the cells were shifted to DMEM supplemented with 2% horse serum (DM, Sigma) and induced to differentiate for 4 days. C2C12 myotubes were cultured for 2 additional days with DM, in the absence or presence of 20% of C26-conditioned medium, with or without 1 mM AICAR (Sellckchem) or 50 nM rapamycin (Sellckchem) in 0.0025 DMSO solution. To block the autophagic flux, 50microM cloroquine (SIGMA) was added for 6 hours at the beginning of the treatments described above to 4-day myotube cultures; the samples were then extensively washed and incubated with the corresponding treatment for the remaining time until the end of the experiment. Immunofluorescence analyses. Cells were fixed in 4% PFA for 10 min at RT, permeabilized with 1% BSA plus 0.2% Triton in phosphate-buffered saline (PBS) and blocked in 5% goat serum (SIGMA) in PBS for 1 hour. Cells were incubated with a 1:50 dilution of p62 (Progen), or 1:50 dilution of LC3b (Cell Signaling), or 1:10 dilution of MF20 (Hybridoma Bank) in 1% BSA in PBS overnight at 4 degrees. Primary antibody incubation was followed by incubation with a 1:500 dilution in 1% BSA of secondary antibody anti-guinea pig, anti-rabbit or anti-mouse Alexa (Life technologies) for 1 hour at room temperature. 0.5 μ g/mL Hoechst 33342 (SIGMA) was used to stain nuclei.