Dominique Dardevet

Leucine supplementation improves muscle protein synthesis in elderly men independently of hyperaminoacidaemia

The present study was designed to assess the effects of dietary leucine supplementation on muscle protein synthesis and whole body protein kinetics in elderly individuals. Twenty healthy male subjects (70 ± 1 years) were studied before and after continuous ingestion of a complete balanced diet supplemented or not with leucine. A primed (3.6 μmol kg−1) constant infusion (0.06 μmol kg−1 min−1) of l‐[1‐13C]phenylalanine was used to determine whole body phenylalanine kinetics as well as fractional synthesis rate (FSR) in the myofibrillar fraction of muscle proteins from vastus lateralis biopsies. Whole body protein kinetics were not affected by leucine supplementation. In contrast, muscle FSR, measured over the 5‐h period of feeding, was significantly greater in the volunteers given the leucine‐supplemented meals compared with the control group (0.083 ± 0.008 versus 0.053 ± 0.009% h−1, respectively, P < 0.05). This effect was due only to increased leucine availability because only plasma free leucine concentration significantly differed between the control and leucine‐supplemented groups. We conclude that leucine supplementation during feeding improves muscle protein synthesis in the elderly independently of an overall increase of other amino acids. Whether increasing leucine intake in old people may limit muscle protein loss during ageing remains to be determined.

Lipid peroxidation and antioxidant status in experimental diabetes

Oxidative stress is currently suggested as a mechanism underlying diabetes. The present study was designed to evaluate the oxidative stress related parameters in streptozotocin-induced diabetes in rats using different complementary approaches: susceptibility to in vitro oxidation (lipid peroxidation induction in liver homogenate, red blood cells hemolysis), blood antioxidant status (total antioxidant capacity by two approaches), and plasma isoprostane measurement, a new marker of lipid peroxidation in vivo. We have shown that induced liver thiobarbituric acid reactive substances increased after 4 weeks of diabetes, in spite of increased liver vitamin E content. Red blood cells hemolysis was significantly delayed after 4 weeks of diabetes. Plasma antioxidant capacity (AOC) tended to increase after 4 weeks of diabetes and was correlated with plasma vitamin E levels. Total antioxidant activity (TAA) significantly decreased after 1 week and a significant correlation was observed with plasma albumin levels. Plasma isoprostane (8-epiprostaglandinF2alpha) concentrations were not modified significantly 1 week or 4 weeks after the induction of diabetes. Levels of vitamin E in the diet and changes in its distribution among the body seems to play an important role in the development of oxidative stress during diabetes and its consequences.

A leucine-supplemented diet restores the defective postprandial inhibition of proteasome-dependent proteolysis in aged rat skeletal muscle

We tested the hypothesis that skeletal muscle ubiquitin–proteasome‐dependent proteolysis is dysregulated in ageing in response to feeding. In Experiment 1 we measured rates of proteasome‐dependent proteolysis in incubated muscles from 8‐ and 22‐month‐old rats, proteasome activities, and rates of ubiquitination, in the postprandial and postabsorptive states. Peptidase activities of the proteasome decreased in the postabsorptive state in 22‐month‐old rats compared with 8‐month‐old animals, while the rate of ubiquitination was not altered. Furthermore, the down‐regulation of in vitro proteasome‐dependent proteolysis that prevailed in the postprandial state in 8‐month‐old rats was defective in 22‐month‐old rats. Next, we tested the hypothesis that the ingestion of a 5% leucine‐supplemented diet may correct this defect. Leucine supplementation restored the postprandial inhibition of in vitro proteasome‐dependent proteolysis in 22‐month‐old animals, by down‐regulating both rates of ubiquitination and proteasome activities. In Experiment 2, we verified that dietary leucine supplementation had long‐lasting effects by comparing 8‐ and 22‐month‐old rats that were fed either a leucine‐supplemented diet or an alanine‐supplemented diet for 10 days. The inhibited in vitro proteolysis was maintained in the postprandial state in the 22‐month‐old rats fed the leucine‐supplemented diet. Moreover, elevated mRNA levels for ubiquitin, 14‐kDa ubiquitin‐conjugating enzyme E2, and C2 and X subunits of the 20S proteasome that were characteristic of aged muscle were totally suppressed in 22‐month‐old animals chronically fed the leucine‐supplemented diet, demonstrating an in vivo effect. Thus the defective postprandial down‐regulation of in vitro proteasome‐dependent proteolysis in 22‐month‐old rats was restored in animals chronically fed a leucine‐supplemented diet.

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%α2‐macroglobulin, +12.6% albumin, −59.6% IL6 and −45.9% IL1β 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.

Skeletal muscle proteolysis in aging

Purpose of reviewTo understand age-related changes in proteolysis and apoptosis in skeletal muscle in relation to oxidative stress and mitochondrial alterations. Recent findingsDuring aging, a progressive loss of muscle mass (sarcopenia) has been described in both human and rodents. Sarcopenia is attributable to an imbalance between protein synthesis and degradation or between apoptosis and regeneration processes or both. Major age-dependent alterations in muscle proteolysis are a lack of responsiveness of the ubiquitin–proteasome-dependent proteolytic pathway to anabolic and catabolic stimuli and alterations in the regulation of autophagy. In addition, increased oxidative stress leads to the accumulation of damaged proteins, which are not properly eliminated, aggregate, and in turn impair proteolytic activities. Finally, the mitochondria-associated apoptotic pathway may be activated. These age-induced changes may contribute to sarcopenia and decreased ability of old individuals to recover from stress. SummaryAlterations in proteasome-dependent or lysosomal proteolysis, increased oxidative stress, mitochondrial dysfunction, and apoptosis presumably contribute to the development of sarcopenia.

Antioxidant Supplementation Restores Defective Leucine Stimulation of Protein Synthesis in Skeletal Muscle from Old Rats

Aging is characterized by a progressive loss of muscle mass that could be partly explained by a defect in the anabolic effect of food intake. We previously reported that this defect resulted from a decrease in the protein synthesis response to leucine in muscles from old rats. Because aging is associated with changes in oxidative status, we hypothesized that reactive oxygen species-induced oxidative damage may be involved in the impairment of the anabolic effect of leucine with age. The present study assessed the effect of antioxidant supplementation on leucine-regulated protein metabolism in muscles from adult and old rats. Four groups of 8- and 20-mo-old male rats were supplemented or not for 7 wk with an antioxidant mixture containing rutin, vitamin E, vitamin A, zinc, and selenium. At the end of supplementation, muscle protein metabolism was examined in vitro using epitrochlearis muscles incubated with increasing leucine concentrations. In old rats, the ability of leucine to stimulate muscle protein synthesis was significantly decreased compared with adults. This defect was reversed when old rats were supplemented with antioxidants. It was not related to increased oxidative damage to 70-kDa ribosomal protein S6 kinase that is involved in amino acid signaling. These effects could be mediated through a reduction in the inflammatory state, which decreased with antioxidant supplementation. Antioxidant supplementation could benefit muscle protein metabolism during aging, but further studies are needed to determine the mechanism involved and to establish if it could be a useful nutritional tool to slow down sarcopenia with longer supplementation.

Role of Physical Bolus Properties as Sensory Inputs in the Trigger of Swallowing

Background Swallowing is triggered when a food bolus being prepared by mastication has reached a defined state. However, although this view is consensual and well supported, the physical properties of the swallowable bolus have been under-researched. We tested the hypothesis that measuring bolus physical changes during the masticatory sequence to deglutition would reveal the bolus properties potentially involved in swallowing initiation. Methods Twenty normo-dentate young adults were instructed to chew portions of cereal and spit out the boluses at different times in the masticatory sequence. The mechanical properties of the collected boluses were measured by a texture profile analysis test currently used in food science. The median particle size of the boluses was evaluated by sieving. In a simultaneous sensory study, twenty-five other subjects expressed their perception of bolus texture dominating at any mastication time. Findings Several physical changes appeared in the food bolus as it was formed during mastication: (1) in rheological terms, bolus hardness rapidly decreased as the masticatory sequence progressed, (2) by contrast, adhesiveness, springiness and cohesiveness regularly increased until the time of swallowing, (3) median particle size, indicating the bolus particle size distribution, decreased mostly during the first third of the masticatory sequence, (4) except for hardness, the rheological changes still appeared in the boluses collected just before swallowing, and (5) physical changes occurred, with sensory stickiness being described by the subjects as a dominant perception of the bolus at the end of mastication. Conclusions Although these physical and sensory changes progressed in the course of mastication, those observed just before swallowing seem to be involved in swallowing initiation. They can be considered as strong candidates for sensory inputs from the bolus that are probably crucially involved in the triggering of swallowing, since they appeared in boluses prepared in various mastication strategies by different subjects.

Glucocorticoids regulate mRNA levels for subunits of the 19 S regulatory complex of the 26 S proteasome in fast-twitch skeletal muscles.

Circulating levels of glucocorticoids are increased in many traumatic and muscle-wasting conditions that include insulin-dependent diabetes, acidosis, infection, and starvation. On the basis of indirect findings, it appeared that these catabolic hormones are required to stimulate Ub (ubiquitin)-proteasome-dependent proteolysis in skeletal muscles in such conditions. The present studies were performed to provide conclusive evidence for an activation of Ub-proteasome-dependent proteolysis after glucocorticoid treatment. In atrophying fast-twitch muscles from rats treated with dexamethasone for 6 days, compared with pair-fed controls, we found (i) increased MG132-inhibitable proteasome-dependent proteolysis, (ii) an enhanced rate of substrate ubiquitination, (iii) increased chymotrypsin-like proteasomal activity of the proteasome, and (iv) a co-ordinate increase in the mRNA expression of several ATPase (S4, S6, S7 and S8) and non-ATPase (S1, S5a and S14) subunits of the 19 S regulatory complex, which regulates the peptidase and the proteolytic activities of the 26 S proteasome. These studies provide conclusive evidence that glucocorticoids activate Ub-proteasome-dependent proteolysis and the first in vivo evidence for a hormonal regulation of the expression of subunits of the 19 S complex. The results suggest that adaptations in gene expression of regulatory subunits of the 19 S complex by glucocorticoids are crucial in the regulation of the 26 S muscle proteasome.

Muscle Wasting and Resistance of Muscle Anabolism: The “Anabolic Threshold Concept” for Adapted Nutritional Strategies during Sarcopenia

Skeletal muscle loss is observed in several physiopathological situations. Strategies to prevent, slow down, or increase recovery of muscle have already been tested. Besides exercise, nutrition, and more particularly protein nutrition based on increased amino acid, leucine or the quality of protein intake has generated positive acute postprandial effect on muscle protein anabolism. However, on the long term, these nutritional strategies have often failed in improving muscle mass even if given for long periods of time in both humans and rodent models. Muscle mass loss situations have been often correlated to a resistance of muscle protein anabolism to food intake which may be explained by an increase of the anabolic threshold toward the stimulatory effect of amino acids. In this paper, we will emphasize how this anabolic resistance may affect the intensity and the duration of the muscle anabolic response at the postprandial state and how it may explain the negative results obtained on the long term in the prevention of muscle mass. Sarcopenia, the muscle mass loss observed during aging, has been chosen to illustrate this concept but it may be kept in mind that it could be extended to any other catabolic states or recovery situations.

Effect of glucocorticoid excess on skeletal muscle and heart protein synthesis in adult and old rats

This study was carried out to analyse glucocorticoid-induced muscle wasting and subsequent recovery in adult (6-8 months) and old (18-24 months) rats because the increased incidence of various disease states results in hypersecretion of glucocorticoids in ageing. Adult and old rats received dexamethasone in their drinking water for 5 or 6 d and were then allowed to recover for 3 or 7 d. As dexamethasone decreased food intake, all groups were pair-fed to dexamethasone-treated old rats (i.e. the group that had the lowest food intake). At the end of the treatment, adult and old rats showed significant increases in blood glucose and plasma insulin concentrations. This increase disappeared during the recovery period. Protein synthesis of different muscles was assessed in vivo by a flooding dose of [13C]valine injected subcutaneously 50 min before slaughter. Dexamethasone induced a significant decrease in protein synthesis in fast-twitch glycolytic and oxidative glycolytic muscles (gastrocnemius, tibialis anterior, extensor digitorum longus). The treatment affected mostly ribosomal efficiency. Adult dexamethasone-treated rats showed an increase in protein synthesis compared with their pair-fed controls during the recovery period whereas old rats did not. Dexamethasone also significantly decreased protein synthesis in the predominantly oxidative soleus muscle but only in old rats, and increased protein synthesis in the heart of adult but not of old rats. Thus, in skeletal muscle, the catabolic effect of dexamethasone is maintained or amplified during ageing whereas the anabolic effect in heart is depressed. These results are consistent with muscle atrophy occurring with ageing.