Differential effect of long-term leucine supplementation on skeletal muscle and adipose tissue in old rats: an insulin signaling pathway approach.

Leucine acts as a signal nutrient in promoting protein synthesis in skeletal muscle and adipose tissue via mTOR pathway activation, and may be of interest in age-related sarcopenia. However, hyper-activation of mTOR/S6K1 has been suggested to inhibit the first steps of insulin signaling and finally promote insulin resistance. The impact of long-term dietary leucine supplementation on insulin signaling and sensitivity was investigated in old rats (18 months old) fed a 15% protein diet supplemented (LEU group) or not (C group) with 4.5% leucine for 6 months. The resulting effects on muscle and fat were examined. mTOR/S6K1 signaling pathway was not significantly altered in muscle from old rats subjected to long-term dietary leucine excess, whereas it was increased in adipose tissue. Overall glucose tolerance was not changed but insulin-stimulated glucose transport was improved in muscles from leucine-supplemented rats related to improvement in Akt expression and phosphorylation in response to food intake. No change in skeletal muscle mass was observed, whereas perirenal adipose tissue mass accumulated (+45%) in leucine-supplemented rats. A prolonged leucine supplementation in old rats differently modulates mTOR/S6K pathways in muscle and adipose tissue. It does not increase muscle mass but seems to promote hypertrophy and hyperplasia of adipose tissue that did not result in insulin resistance.

Leucine supplementation in rats induced a delay in muscle IR/PI3K signaling pathway associated with overall impaired glucose tolerance.

Although activation of the mammalian target of rapamycin complex/p70 S6 kinase (S6K1) pathway by leucine is efficient to stimulate muscle protein synthesis, it can also exert inhibition on the early steps of insulin signaling leading to insulin resistance. We investigated the impact of 5-week leucine supplementation on insulin signaling and sensitivity in 4-month old rats fed a 15% protein diet supplemented (LEU) or not (C) with 4.5% leucine. An oral glucose tolerance test was performed in each rat at the end of the supplementation and glucose transport was measured in vitro using isolated epitrochlearis muscles incubated with 2-deoxy-d-[(3)H]-glucose under increasing insulin concentrations. Insulin signaling was assessed on gastrocnemius at the postabsorptive state or 30 and 60 min after gavage with a nutrient bolus. Tyrosine phosphorylation of IRß, IRS1 and PI3 kinase activity were reduced in LEU group 30 min after feeding (-36%, -36% and -38% respectively, P<.05) whereas S6K1, S6rp and 4EBP1 phosphorylations were similar. Overall glucose tolerance was reduced in leucine-supplemented rats and was associated with accumulation of perirenal adipose tissue (+27%, P<.05). Conversely, in vitro insulin-response of muscle glucose transport tended to be improved in leucine-supplemented rats. In conclusion, dietary leucine supplementation in adult rats induced a delay in the postprandial stimulation in the early steps of muscle insulin signaling without muscle resistance on insulin-induced glucose uptake. However, it resulted in overall glucose intolerance linked to increased local adiposity. Further investigations are necessary to clearly define the beneficial and/or deleterious effects of chronic dietary leucine supplementation in healthy subjects.

Antioxidant supplementation had positive effects in old rat muscle, but through better oxidative status in other organs.

OBJECTIVE: Aged muscle is characterized by a defect in the ability of leucine to stimulate protein synthesis. We showed previously that antioxidant supplementation improved the anabolic response to leucine of old muscle and reduced inflammation. The aim of the present study was to determine if the positive effects observed in muscle could be related to an improvement of local muscle oxidative status. METHODS: Two groups of 20-mo-old male Wistar rats were supplemented or not with rutin, vitamin E, vitamin A, zinc, and selenium during 7 wk. We measured body weight, food intake, oxidative status in muscle and other tissues, gastrocnemius muscle proteolytic activities, and liver glutathione metabolism. RESULTS: Antioxidant supplementation had no effect on muscle antioxidant capacity, superoxide dismutase activities, and myofibrillar protein carbonyl content and induced an increase in muscle cathepsin activities. In other tissues, antioxidant supplementation increased liver glutathione (reduced plus oxidized glutathione) content, reduced oxidative damage in the liver and spleen (as measured by γ-keto-aldehyde content), and reduced heart thiobarbituric acid-reactive substances. CONCLUSION: Our results showed that the positive effects of antioxidant supplementation observed previously on the anabolic response to leucine of old muscle were not directly related to an improvement of in situ muscle oxidative status. It could result from reduced systemic inflammation/oxidative stress. The dialog between muscle and other organs should be studied more thoroughly, especially during aging.

Long-term effects of leucine supplementation on body composition.

PURPOSE OF REVIEW: Leucine does not only serve as a substrate for protein synthesis but is also recognized as a potent signal nutrient that regulates protein metabolism. Accordingly, leucine supplementation has been suggested to develop muscle mass or prevent protein loss in several conditions characterized by muscle protein wasting. In the present review, we reported the recent results related to the effect of dietary leucine or leucine-rich amino acid mixture and proteins on whole body composition. RECENT FINDINGS: Although recent studies corroborate that increasing plasma leucine concentration generally induces an increase in muscle protein synthesis, long-term dietary leucine supplementation has been poorly investigated. Chronic free leucine supplementation alone did not improve lean body or muscle mass during resistance training or in elderly, whereas it was able to limit the weight loss induced by malnutrition. Contradictory data were also reported concerning the effect of leucine supplementation for weight management in obese patients. Leucine-rich amino acid mixture or proteins appeared more efficient than leucine alone to improve muscle mass and performance, suggesting the efficacy of leucine depends nevertheless on the presence of other amino acids. SUMMARY: Until now, there is no evidence that chronic leucine supplementation is efficient in promoting muscle mass or preventing protein loss during catabolic states. Further studies are required to determine the duration and nutritional conditions of long-term leucine supplementation and to establish whether such nutritional interventions can help to prevent or treat muscle loss in various pathological or physiological conditions.

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.

Increased availability of leucine with leucine-rich whey proteins improves postprandial muscle protein synthesis in aging rats.

OBJECTIVE: We previously found that aging was characterized by a decreased sensitivity of muscle protein synthesis to leucine and that a free leucine-supplemented diet corrected this defect in old rats and elderly humans. The present experiment was undertaken to evaluate the efficiency of selected leucine-rich proteins to stimulate postprandial muscle protein synthesis in old rats to optimize nutritional protein support in the elderly. METHODS: Sixty rats (22 mo old) received an experimental meal for the first hour of feeding and a standard diet for the rest of the day for 30 d. Experimental meals contained milk proteins that differed in leucine content: beta-lactoglobulin (14.5% leucine), Prolacta (13.4%), alpha-lactalbumin (10.9%), and casein (10%). As a control, a fifth group was added that received herring flour protein (7.3% leucine). Muscle protein synthesis was determined in vivo in the postprandial state at the end of the 30-d nutritional period using the flooding dose method (1-(13)C phenylalanine). RESULTS: Leucine intake and plasma leucine concentrations were significantly increased in rats fed meals containing the leucine-rich proteins (i.e., beta-lactoglobulin and Prolacta). As previously observed with free leucine-supplemented meals, postprandial muscle protein synthesis was significantly improved in rats fed the meals containing the leucine-rich proteins. Interestingly, the beneficial effect was maintained after the 30-d supplementation. CONCLUSION: The results indicated that leucine-rich proteins were efficient in improving muscle protein synthesis in old rats. Thus, nutritional supplements containing such proteins may be efficient in preventing sarcopenia in the elderly and would represent a safe and optimized nutritional strategy. However, further experiments are necessary to determine the duration of such nutritional support to obtain a significant protein gain in muscle.

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 micromol kg(-1)) constant infusion (0.06 micromol 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.

Effect of medroxyprogesterone acetate on the efficiency of an oral protein-rich nutritional support in HIV-infected patients.

We have examined the effect of a medroxyprogesterone therapy in HIV-infected patients under appropriate nutrition for anabolism. The experiments were performed on 12 men (mean age 40 y), HIV seropositive but free of any clinically active opportunistic infection for at least one month. The patients underwent a 2-week baseline diet period (1.2 g protein x kg(-1) body weight (BW) x d(-1)) and then a 5-week experimental period with again the baseline diet in conjunction with supplements including Tonexis HP (0.7 g protein x kg(-1) BW) x d(-1)), L-threonine (0.018 g x kg(-1) BW x d(-1)) and L-methionine (0.013 g x kg(-1) BW x d(-1)). Indeed HIV-infected patients showed deficiencies in these amino acids. They were randomly divided into groups I and II under double-blinded condition. Group II was given medroxyprogesterone acetate (0.4 g x d(-1)) during the last 3 weeks whereas group I received a placebo. All the patients significantly increased their body weight (P < 0.05) during the experimental periods. Those under medroxyprogesterone tended to show a higher but not significant weight gain (+3.1 +/- 1.0 kg in group II and +1.9 +/- 0.3 kg in group I). Blood free amino acids were used as rough indicators of amino acid utilization and were analyzed prior and during acute 150 min intravenous infusion of a complete glucose-amino acid mixture. This test was done before and at the end of the experimental periods. Basal essential blood free amino acids were similar in the two groups and did not change during the experimental period. Most essential amino acids increased following glucose-amino acid infusions. The incremental increase was of less magnitude after the experimental period than before when medroxyprogesterone was present (P < 0.05 for valine, leucine, lysine, threonine and methionine). This was not the case in the absence of the hormone. We concluded that medroxyprogesterone might improve the efficacy of an oral protein-rich nutritional support in HIV-infected patients.

Leucine-supplemented meal feeding for ten days beneficially affects postprandial muscle protein synthesis in old rats.

Acute leucine supplementation of the diet has been shown to blunt defects in postprandial muscle protein metabolism in old rats. This study was undertaken to determine whether the effect of leucine persists in a 10-d experiment. For this purpose, adult (9 mo) and old (21 mo) rats were fed a semiliquid 18.2 g/100 g protein standard diet during the 8-h dark period for 1 mo. Then, each group was given either a leucine-supplemented meal or an alanine-supplemented meal (as the control meal) for 1 h and the standard diet the rest of the feeding period. On d 10, rats were fed either no food (postabsorptive group) or the supplemented meal for 1 h. Muscle protein synthesis was assessed in vivo 90-120 min after meal distribution using the flooding dose method (1-(13)C phenylalanine). Leucinemia was similar in rats of both ages in the postabsorptive state. Postprandial plasma leucine concentrations were one- to twofold greater after the leucine meal than after the control meal. In the postabsorptive state, leucine supplementation did not modify the muscle protein synthesis rate in old rats but enhanced it to the postprandial rate in adult rats. As expected, muscle protein synthesis was stimulated by the control meal in adult rats but not in old rats. The leucine meal restored this stimulation in old rats but did not further stimulate muscle protein synthesis in adult rats. In conclusion, the beneficial effect of leucine supplementation on postprandial muscle protein anabolism persists for at least 10 d. The long-term utilization of leucine-rich diets may therefore limit muscle protein wasting during aging.