A dietary supplementation with leucine and antioxidants is capable to accelerate muscle mass recovery after immobilization in adult rats.

Prolonged inactivity induces muscle loss due to an activation of proteolysis and decreased protein synthesis; the latter is also involved in the recovery of muscle mass. The aim of the present work was to explore the evolution of muscle mass and protein metabolism during immobilization and recovery and assess the effect of a nutritional strategy for counteracting muscle loss and facilitating recovery. Adult rats (6-8 months) were subjected to unilateral hindlimb casting for 8 days (I0-I8) and then permitted to recover for 10 to 40 days (R10-R40). They were fed a Control or Experimental diet supplemented with antioxidants/polyphenols (AOX) (I0 to I8), AOX and leucine (AOX + LEU) (I8 to R15) and LEU alone (R15 to R40). Muscle mass, absolute protein synthesis rate and proteasome activities were measured in gastrocnemius muscle in casted and non-casted legs in post prandial (PP) and post absorptive (PA) states at each time point. Immobilized gastrocnemius protein content was similarly reduced (-37%) in both diets compared to the non-casted leg. Muscle mass recovery was accelerated by the AOX and LEU supplementation (+6% AOX+LEU vs. Control, P<0.05 at R40) due to a higher protein synthesis both in PA and PP states (+23% and 31% respectively, Experimental vs. Control diets, P<0.05, R40) without difference in trypsin- and chymotrypsin-like activities between diets. Thus, this nutritional supplementation accelerated the recovery of muscle mass via a stimulation of protein synthesis throughout the entire day (in the PP and PA states) and could be a promising strategy to be tested during recovery from bed rest in humans.

Unilateral hindlimb casting induced a delayed generalized muscle atrophy during rehabilitation that is prevented by a whey or a high protein diet but not a free leucine-enriched diet.

Sarcopenia is the general muscle mass and strength loss associated with ageing. Muscle atrophy could be made worse by exposure to acute periods of immobilization, because muscle disuse by itself is a stimulus for atrophy. Using a model of unilateral hindlimb casting in old adult rats, we have already demonstrated that the primary effect of immobilization was atrophy in the casted leg, but was also surprisingly associated with a retarded atrophy in the non-casted leg during rehabilitation. In search of mechanisms involved in this generalized atrophy, we demonstrated in the present study that contrary to pair-fed non-immobilized control animals, muscle protein synthesis in the non-immobilized limb was unable to adapt and to respond positively to food intake. Because pair-fed control rats did not lose muscle mass, this defect in muscle protein synthesis may represent one of the explanation for the muscle mass loss observed in the non-immobilized rats. Nevertheless, in order to stimulate protein turn over and generate a positive nitrogen balance required to maintain the whole muscle mass in immobilized rats, we tested a dietary free leucine supplementation (an amino acid known for its stimulatory effect on protein metabolism) during the rehabilitation period. Leucine supplementation was able to overcome the anabolic resistance in the non-immobilized limb. A greater muscle protein synthesis up-regulation associated with a stimulation of the mTOR signalling pathway was indeed recorded but it remained inefficient to prevent the loss of muscle in the non-immobilized limb. By contrast, we demonstrated here that whey protein or high protein diets were able to prevent the muscle mass loss of the non-immobilized limb by sustaining muscle protein synthesis during the entire rehabilitation period.

Nutritional strategies to counteract muscle atrophy caused by disuse and to improve recovery.

Periods of immobilisation are often associated with pathologies and/or ageing. These periods of muscle disuse induce muscle atrophy which could worsen the pathology or elderly frailty. If muscle mass loss has positive effects in the short term, a sustained/uncontrolled muscle mass loss is deleterious for health. Muscle mass recovery following immobilisation-induced atrophy could be critical, particularly when it is uncompleted as observed during ageing. Exercise, the best way to recover muscle mass, is not always applicable. So, other approaches such as nutritional strategies are needed to limit muscle wasting and to improve muscle mass recovery in such situations. The present review discusses mechanisms involved in muscle atrophy following disuse and during recovery and emphasises the effect of age in these mechanisms. In addition, the efficiency of nutritional strategies proposed to limit muscle mass loss during disuse and to improve protein gain during recovery (leucine supplementation, whey proteins, antioxidants and anti-inflammatory compounds, energy intake) is also discussed.

Chronic α-hydroxyisocaproic acid treatment improves muscle recovery after immobilization-induced atrophy.

Muscle disuse atrophy is observed routinely in patients recovering from traumatic injury and can be either generalized resulting from extended bed rest or localized resulting from single-limb immobilization. The present study addressed the hypothesis that a diet containing 5% α-hydroxyisocaproic acid (α-HICA), a leucine (Leu) metabolite, will slow the loss and/or improve recovery of muscle mass in response to disuse. Adult 14-wk-old male Wistar rats were provided a control diet or an isonitrogenous isocaloric diet containing either 5% α-HICA or Leu. Disuse atrophy was produced by unilateral hindlimb immobilization ("casting") for 7 days and the contralateral muscle used as control. Rats were also casted for 7 days and permitted to recover for 7 or 14 days. Casting decreased gastrocnemius mass, which was associated with both a reduction in protein synthesis and S6K1 phosphorylation as well as enhanced proteasome activity and increased atrogin-1 and MuRF1 mRNA. Although neither α-HICA nor Leu prevented the casting-induced muscle atrophy, the decreased muscle protein synthesis was not observed in α-HICA-treated rats. Neither α-HICA nor Leu altered the increased proteasome activity and atrogene expression observed with immobilization. After 14 days of recovery, muscle mass had returned to control values only in the rats fed α-HICA, and this was associated with a sustained increase in protein synthesis and phosphorylation of S6K1 and 4E-BP1 of previously immobilized muscle. Proteasome activity and atrogene mRNA content were at control levels after 14 days and not affected by either treatment. These data suggest that whereas α-HICA does not slow the loss of muscle produced by disuse, it does speed recovery at least in part by maintaining an increased rate of protein synthesis.

Contrarily to whey and high protein diets, dietary free leucine supplementation cannot reverse the lack of recovery of muscle mass after prolonged immobilization during ageing.

During ageing, immobilization periods increase and are partially responsible of sarcopaenia by inducing a muscle atrophy which is hardly recovered from. Immobilization-induced atrophy is due to an increase of muscle apoptotic and proteolytic processes and decreased protein synthesis. Moreover, previous data suggested that the lack of muscle mass recovery might be due to a defect in protein synthesis response during rehabilitation. This study was conducted to explore protein synthesis during reloading and leucine supplementation effect as a nutritional strategy for muscle recovery. Old rats (22­24 months old) were subjected to unilateral hindlimb casting for 8 days (I8) and allowed to recover for 10­40 days (R10­R40). They were fed a casein (±leucine) diet during the recovery. Immobilized gastrocnemius muscles atrophied by 20%, and did not recover even at R40. Amount of polyubiquitinated conjugates and chymotrypsin- and trypsin-like activities of the 26S proteasome increased. These changes paralleled an 'anabolic resistance' of the protein synthesis at the postprandial state (decrease of protein synthesis, P-S6 and P-4E-BP1). During the recovery, proteasome activities remained elevated until R10 before complete normalization and protein synthesis was slightly increased. With free leucine supplementation during recovery, if proteasome activities were normalized earlier and protein synthesis was higher during the whole recovery, it nevertheless failed in muscle mass gain. This discrepancy could be due to a 'desynchronization' between the leucine signal and the availability of amino acids coming from casein digestion. Thus, when supplemented with leucine-rich proteins (i.e. whey) and high protein diets, animals partially recovered the muscle mass loss.

Curcumin treatment prevents increased proteasome and apoptosome activities in rat skeletal muscle during reloading and improves subsequent recovery.

Immobilization is characterized by activation of the ubiquitin (Ub)-proteasome-dependent proteolytic system (UPS) and of the mitochondrial apoptotic pathway. Increased oxidative stress and inflammatory response occur in immobilized skeletal muscles. Curcumin exhibits antioxidant and anti-inflammatory properties, blocked proteasome activation in intact animals, and may favor skeletal muscle regeneration. We therefore measured the effects of curcumin on immobilization-induced muscle atrophy and subsequent recovery. Rats were subjected to hindlimb immobilization for 8 days (I8) and allowed to recover for 10 days (R10). Fifty percent of the rats were injected daily with either curcumin or vehicle. Proteolytic and apoptotic pathways were studied in gastrocnemius muscles. Curcumin treatment prevented the enhanced proteasome chymotrypsin-like activity and the trend toward increased caspase-9-associated apoptosome activity at I8 in immobilized muscles. By contrast, the increase of these two activities was blunted by curcumin at R10. Curcumin did not reduce muscle atrophy at I8 but improved muscle recovery at R10 and the cross-sectional area of muscle fibers of immobilized muscles. Curcumin reduced the increased protein levels of Smac/DIABLO induced by immobilization and enhanced the elevation of X-linked inhibitory apoptotic protein levels at R10. Ub-conjugate levels and caspase-3 activity increased at I8 and were normalized at R10 without being affected by curcumin treatment. Altogether, the data show that curcumin treatment improved recovery during reloading. The effect of curcumin during the atrophic phase on proteasome activities may facilitate the initiation of muscle recovery after reloading. These data also suggest that this compound may favor the initial steps of muscle regeneration.

Lack of muscle recovery after immobilization in old rats does not result from a defect in normalization of the ubiquitin-proteasome and the caspase-dependent apoptotic pathways.

Immobilization periods increase with age because of decreased mobility and/or because of increased pathological episodes that require bed-rest. Then, sarcopaenia might be partially explained by an impaired recovery of skeletal muscle mass after a catabolic state due to an imbalance of muscle protein metabolism, apoptosis and cellular regeneration. Mechanisms involved during muscle recovery have been little studied and in elderly they remain almost unknown. We show, in rats, that a short immobilization period during ageing initiated muscle atrophy that was indeed not recovered after 40 days. Immobilization was associated with an activation of both the ubiquitin-proteasome and the mitochondria-associated apoptotic pathways and the inflammatory and redox processes, and a decrease of cellular regeneration. We show that the lack of muscle recovery during ageing is not due to a defect in proteolysis or apoptosis down-regulation. These observations lead us to hypothesize that muscle protein synthesis activation after immobilization was altered during ageing.

Reduction of low grade inflammation restores blunting of postprandial muscle anabolism and limits sarcopenia in old rats.

Ageing is characterized by a decline in muscle mass that could be explained by a defect in the regulation of postprandial muscle protein metabolism. Indeed, the stimulatory effect of food intake on protein synthesis and its inhibitory effect on proteolysis is blunted in old muscles from both animals and humans. Recently, low grade inflammation has been suspected to be one of the factors responsible for the decreased sensitivity of muscle protein metabolism to food intake. This study was undertaken to examine the effect of long-term prevention of low grade inflammation on muscle protein metabolism during ageing. Old rats (20 months of age) were separated into two groups: a control group and a group (IBU) in which low grade inflammation had been reduced with a non-steroidal anti inflammatory drug (ibuprofen). After 5 months of treatment, inflammatory markers and cytokine levels were significantly improved in treated old rats when compared with the controls: -22.3% fibrinogen, -54.2% alpha2-macroglobulin, +12.6% albumin, -59.6% IL(6) and -45.9% IL(1beta) levels. As expected, food intake had no effect on muscle protein synthesis or muscle proteolysis in controls whereas it significantly increased muscle protein synthesis by 24.8% and significantly decreased proteolysis in IBU rats. The restoration of muscle protein anabolism at the postprandial state by controlling the development of low grade inflammation in old rats significantly decreased muscle mass loss between 20 and 25 months of age. In conclusion, the observations made in this study have identified low grade inflammation as an important target for pharmacological, nutritional and lifestyle interventions that aim to limit sarcopenia and muscle weakness in the rapidly growing elderly population in Europe and North America.