Amino acid supplementation prevents the loss of appetite for casein in old Lou/Cjall rats.

A deleterious reduction of casein intake occurring earlier in males than in females had been previously observed in old Lou/Cjall rats. On the contrary, protein intake was observed to be maintained in old males when they were offered whey protein. Present studies were designed to investigate the effect of dietary casein modification on protein decrease. In two lifelong studies, male and female Lou/Cjall rats were tested every four months in order to study protein intake depending on the protein available: casein, whey protein or casein supplemented with an amino acid mixture (SC). In subsequent cross-sectional studies, young, adult, middle-aged and old rats were successively fed with casein, casein supplemented either with leucine or with alanine or with glycine. Supplementing casein with an amino acid mixture both globally increased protein intake and allowed old males to maintain a high rate of protein intake. In cross-sectional experiments, no effect of supplementation was seen in the young group. In older animals, the greatest effect was seen when casein was supplemented with alanine or glycine, independently of sex and age. We therefore, concluded that supplemented casein is more beneficial for old rats than casein alone, probably by increasing amino acid availability. We hypothesize that alanine could act through its effect on gluconeogenesis.

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.

Postprandial leucine deficiency failed to alter muscle protein synthesis in growing and adult rats.

OBJECTIVE: This study examined the effect of a specific acute postprandial leucine deficiency on skeletal muscle protein synthesis in growing and adult rats. Because the anabolic action of dietary leucine supplementation is controversial, except during aging, we hypothesized that the maximum leucine effect might be already achieved for a normal postprandial rise of leucine. Preventing this rise during the 1- to 3-h period after feeding may reveal the leucine regulation. METHODS: On the day of the experiment, rats were fasted (postabsorptive, PA group) or fed for 1 h a control meal (postprandial, control, PP group) or a leucine-poor meal (postprandial, PP-Leu group). Muscle protein synthesis was assessed in vivo, over the 1- to 3-h period after meal distribution, using the flooding dose method (L-1-(13)C phenylalanine). RESULTS: As expected, the postprandial increase in plasma free leucine was specifically abolished after feeding the leucine-poor meal, whereas all the other plasma free amino acids were roughly at normal postprandial levels. Plasma insulin increased after feeding in young rats but was constant in adult rats. Plasma insulin was similar whatever dietary leucine levels. Rates of muscle protein synthesis were stimulated by feeding in gastrocnemius and soleus muscles from young rats but only in gastrocnemius muscles from adult rats. The PP-Leu group did not differ from the control PP group regarding muscle protein synthesis. CONCLUSION: The rise in plasma free leucine is not required for the stimulation of muscle protein synthesis during the 1- to 3-h period after feeding young and adult rats, as previously observed in old rats.

Regulation of protein metabolism by insulin: value of different approaches and animal models.

Insulin induces protein accretion by stimulating protein synthesis and inhibiting proteolysis. However, the mechanisms of regulation of protein metabolism by insulin are complex and still not completely understood. The use of approaches combining hyperinsulinemic clamp and isotopic methods, or measurement of the activation of intracellular kinases involved in insulin signaling, in addition to the use of different animal models in a comparative physiology process, provide better understanding of the potential regulation of protein metabolism by insulin. Studies using the clamp technique in lactating goats have shown a clear inhibitory effect of insulin on proteolysis, with an interaction between the effects of insulin and amino acids. Such studies revealed that the insulin-inhibited proteolysis is improved in lactating goats, this adaptative process limiting the mobilization of body protein under the conditions of amino acid deficit which occurs during early lactation. Insulin signaling studies in growing chickens have also provided some interesting features of insulin regulation compared to mammals. Refeeding or insulin injection leads to the activation of the early steps of insulin receptor signaling in the liver but not in the muscle. Muscle p70 S6 kinase, a kinase involved in the insulin activation of protein synthesis, was found to be markedly activated in response to insulin and to refeeding, suggesting that other signaling pathways than those classically described in mammalian muscles may be involved in signal transduction. Finally, although the role of insulin has been doubtful and has long been considered to be minor in ruminants and in avian species, this hormone clearly regulates protein metabolism in both species.

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.

Whole body leucine flux in HIV-infected patients treated with or without protease inhibitors.

The present study was carried out to assess the effects of protease inhibitor (PI) therapy on basal whole body protein metabolism and its response to acute amino acid-glucose infusion in 14 human immunodeficiency virus (HIV)-infected patients. Patients treated with PIs (PI+, 7 patients) or without PIs (PI-, 7 patients) were studied after an overnight fast during a 180-min basal period followed by a 140-min period of amino acid-glucose infusion. Protein metabolism was investigated by a primed constant infusion of l-[1-(13)C]leucine. Dual-energy X-ray absorptiometry for determination of fat-free mass (FFM) and body fat mass measured body composition. In the postabsorptive state, whole body leucine balance was 2.5 times (P < 0.05) less negative in the PI+ than in the PI- group. In HIV-infected patients treated with PIs, the oxidative leucine disposal during an acute amino acid-glucose infusion was lower (0.58 +/- 0.09 vs. 0.81 +/- 0.07 micromol x kg FFM(-1) x min(-1) using plasma [(13)C]leucine enrichment, P = 0.06; or 0.70 +/- 0.10 vs. 0.99 +/- 0.08 micromol x kg FFM(-1) x min(-1) using plasma [(13)C]ketoisocaproic acid enrichment, P = 0.04 in PI+ and PI- groups, respectively) than in patients treated without PIs. Consequently, whole body nonoxidative leucine disposal (an index of protein synthesis) and leucine balance (0.50 +/- 0.10 vs. 0.18 +/- 0.06 micromol x kg FFM x (-1) x min(-1) in PI+ and PI- groups respectively, P < 0.05) were significantly improved during amino acid-glucose infusion in patients treated with PIs. However, whereas the response of whole body protein anabolism to an amino acid-glucose infusion was increased in HIV-infected patients treated with PIs, any improvement in lean body mass was detected.

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.

Altered responses in skeletal muscle protein turnover during aging in anabolic and catabolic periods.

One of the most important effects of aging is sarcopenia, which is associated with impaired locomotion and general weakness. In addition, there is increased susceptibility to illness in aging, which often results in muscle wasting episodes. In such instances, the mobilization of muscle proteins provides free amino acids that are used for energetic purpose, the synthesis of acute phase proteins, and the immune response. However, since muscle protein mass is already depleted, the ability of the aged organism to recover from stress is impaired. Therefore, elucidating the mechanisms that result in sarcopenia is of obvious importance. Age-related changes in protein synthesis and proteolysis are rather small and our current methodology does not enable one to establish unequivocally whether sarcopenia results from depressed protein synthesis, increased proteolysis or both. By contrast, in anabolic and catabolic periods, a number of dysregulations in muscle protein turnover became clearly apparent. The aim of this review is to provide an overview of such altered responses to nutrients and catabolic treatments, which may ultimately contribute to explain sarcopenia. This includes impaired recovery in catabolic states, impaired anabolic effects of nutrients, in particular leucine, and a lack of regulation of the ubiquitin-proteasome proteolytic system. These alterations are discussed with respect to modifications in the insulin/IGF-1 axis and glucocorticoid related effects.

Insulin-dependent glycogen synthesis is delayed in onset in the skeletal muscle of food-deprived aged rats.

Insulin resistance with aging may be responsible for impaired glycogen synthesis in the skeletal muscle of aged rats and contribute to the well-known decreased ability to respond to stress with aging. For this reason, to assess the ability of the skeletal muscle to utilize glucose for glycogen synthesis during aging, the time course of glycogen synthesis was continuously monitored by 13C nuclear magnetic resonance for 2 h in isolated [13C] glucose-perfused gastrocnemius-plantaris muscles of 5-day food-deprived adult (6-8 months; n=10) or 5-day food-deprived aged (22 months; n=8) rats. [13C] glucose (10 mmol/L) perfusion was carried out in the presence or absence of an excess of insulin (1 micromol/L). Food deprivation only decreased glycogen level in adult rats (8.9+/-2.4 micromol/g in adults vs. 35.6+/-2.4 micromol/g in aged rats; P<.05). In the presence of an excess of insulin, muscle glycogen synthesis was stimulated in both adult and aged muscles, but the onset was delayed with aging (40 min later). In conclusion, this study highlights the important role of glycogen depletion in stimulating glycogen synthesis in muscles. Consequently, the absence of glycogen depletion in response to starvation in aged rats may be the origin of the delay in insulin-stimulated glycogen synthesis in the skeletal muscle. Glycogen synthesis clearly was not impaired with aging.

Differential effects of insulin and dietary amino acids on muscle protein synthesis in adult and old rats.

The potential roles of insulin and dietary amino acids in the regulation of skeletal muscle protein synthesis were examined in adult and old rats. Animals were fed over 1 h with either a 25% or a 0% amino acid/protein meal. In each nutritional condition, postprandial insulin secretion was either maintained or blocked with diazoxide injections. Protein synthesis in gastrocnemius and soleus muscles was assessed in vivo using the flooding dose method. Insulin suppression decreased protein synthesis in both muscles irrespective of the nutritional condition and age of the rats. Moreover, reduced insulinaemia was associated with 4E-BP1 dephosphorylation, enhanced assembly of the 4E-BP1-eIF4E inactive complex and hypophosphorylation of eIF4E, p70S6k and protein kinase B, key intermediates in the regulation of translation initiation and protein synthesis. Old rats did not differ from adult rats. The lack of amino acids in the meal of insulin-suppressed rats did not result in any additional decrease in protein synthesis. In the presence of insulin secretion, dietary amino acid suppression significantly decreased gastrocnemius protein synthesis in adult but not in old rats. Amino acid suppression was associated with reduced phosphorylation of 4E-BP1 and p70S6k in adults. Along with protein synthesis, only the inhibition of p70S6k phosphorylation was abolished in old rats. We concluded that insulin is required for the regulation of muscle protein synthesis irrespective of age and that the effect of dietary amino acids is blunted in old rats.

The reduction of protein intake observed in old rats depends on the type of protein.

Previous experiments have shown in Lou/c/jall rats growing old a deleterious reduction of protein intake, which occurs earlier in males than in females. We previously showed that this decrease could not be attributed to a loss of regulation of protein intake with age. Present studies were designed to investigate if the age-related decrease of protein intake was dependent on the type of protein used. In a first sectional study, adult, middle and old-aged Lou/c/jall rats were submitted to a self-selection procedure. They were fed successively with casein, whey protein and fish flour as protein. In a second longitudinal study, self-selected males and females were tested each 4 months (at 3, 7, 11, 15, 19, 23 and 27 months of age) with only casein and whey protein as protein. In the two experiments, the type of dietary protein had an influence on the protein intake: when casein is offered, the well-established decrease in protein consumption was seen after 15 months of age in male groups. The introduction of whey protein induced maintenance of protein intake in old male groups at the level of female's protein intake. Moreover, young females showed an obvious preference for casein versus whey protein. This preference disappeared in old ages. These data showed that casein, even if it was an appropriate protein for young animals, could become inadequate for old animals and could result in a protein aversion in old rats. On the contrary, whey protein seemed to be a more appropriate protein than casein for old rats.

Glucocorticoid excess induces a prolonged leucine resistance on muscle protein synthesis in old rats.

This experiment was undertaken to examine leucine responsiveness of muscle protein synthesis during dexamethasone treatment and the subsequent recovery in young (4-5 weeks), adult (10-11 months) and old rats (21-22 months). Rats received dexamethasone in their drinking water. The dose and length of the treatment was adapted in order to generate the same muscle atrophy. Protein synthesis was assessed in vitro by incorporation of radiolabelled phenylalanine into proteins at the end of the treatment and after 3 or 7-day recovery. Results showed that dexamethasone did not alter muscle protein synthesis stimulation by leucine in young rats. In contrast, muscles from adult and old rats became totally resistant to leucine. Furthermore, the recovery of leucine responsiveness after dexamethasone withdrawal was slowed down in old rats when compared to younger rats. We concluded that glucocorticoids exert their catabolic action in adult and old rats partly through antagonising the stimulatory effect of leucine and may contribute to sarcopenia in old rats.

Impaired anabolic response of muscle protein synthesis is associated with S6K1 dysregulation in elderly humans.

Age-related loss of muscle protein may involve a decreased response to anabolic factors of muscle protein synthesis through dysregulation of translation factors. To verify this hypothesis, we simultaneously investigated muscle protein synthesis and expression of some factors implicated in insulin signal transduction during hyperinsulinemia and hyperaminoacidemia in 6 young (25+/-1 year; mean+/-sem) and 8 elderly subjects (72+/-2 year). Incorporation of L-[1-13C] leucine in muscle proteins (fractional synthesis rate, FSR) was measured in vastus lateralis, before and during a euglycemic hyperinsulinemic hyperaminoacidemic clamp, together with Western blot analysis of protein kinase B (PKB), mTOR, 4E-BP1, and S6K1 phosphorylation. In basal state, muscle protein FSR was reduced in elderly in comparison with young subjects (0.061+/-0.004% per hour) vs 0.082+/-0.010% per hour, elderly vs. young, P<0.05). During clamp, muscle protein FSR was stimulated in young (0.119+/-0.006% per hour; P<0.05), but this response was significantly lower in elderly subjects (0.084+/-0.005% per hour, P<0.05 vs young subjects). Phosphorylation of PKB, mTOR, and 4E-BP1 were similarly increased by insulin and amino acid in both groups, except for S6K1 phosphorylation, which was not stimulated in elderly subjects. In conclusion, 1) response of muscle protein synthesis to insulin and amino acid is impaired in elderly humans; 2) a defect in S6K1 pathway activation may be responsible for this alteration. This modification is a mechanistic basis of sarcopenia development during aging.

Insulin and amino acids both strongly participate to the regulation of protein metabolism.

PURPOSE OF REVIEW: The application of tracer kinetic methods, combined with measurements of the activity of components of the cellular signaling pathways involved in protein synthesis and degradation, affords new insights into the regulation of skeletal muscle protein metabolism in vivo in humans. Feeding is associated with an increase in protein synthesis and a decrease in proteolysis. These changes are mediated by feeding-induced increases in plasma concentrations of both nutrients and hormones. RECENT FINDINGS: Recent studies definitely demonstrated that insulin and amino acids directly interacted in promoting postprandial anabolism. However, the contribution of amino acids was abolished in old individuals in whom only insulin action persisted. There was a line of evidence that the effect of amino acids originates from leucine, which should not be viewed simply as a building block for protein synthesis, but as a signal in the regulation of cell functions. Although their cellular signaling pathways do not completely overlap, insulin and amino acids both activate the translation initiation of protein synthesis. Insulin presumably inhibits skeletal muscle protein degradation through a decrease in the activity of the ubiquitin proteasome-dependent pathway. SUMMARY: Whether or not amino acids modify insulin action and have specific effects on proteolysis has not yet been documented. At the molecular level, amino acids such as insulin modulate gene expression. Such studies are needed to gain a better understanding of the interactions between insulin and amino acids in the regulation of skeletal muscle protein anabolism.

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.

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.

Leucine: a key amino acid in ageing-associated sarcopenia?

During ageing, a progressive loss of muscle mass has been well described in both man and rodents. This loss of proteins results from an imbalance between protein synthesis and degradation rates. Although some authors have shown a decrease of myofibrillar protein synthesis rates in human volunteers, this imbalance is not clearly apparent when basal rates of protein turnover are measured. A decrease in muscle protein synthesis stimulation was detected nevertheless in ageing rats during the postprandial period, suggesting that the 'meal signal' was altered during ageing. Many results now suggest that aged muscle is less sensitive to the stimulatory effect of amino acids at physiological concentrations but is still able to respond if the increase in aminoacidaemia is sufficiently large. Indeed amino acids play an important role in regulating muscle protein turnover both in vitro and in vivo. At the molecular level, amino acids modulate gene expression. Amino acid response elements have been characterised in the promoter of transcriptional factor CCAAT-enhancer binding protein homologous protein and asparagine synthetase genes. Among amino acids, leucine seems to play the major role in regulating the metabolic function. It inhibits proteolysis and stimulates muscle protein synthesis independently of insulin. Leucine has been shown to act as a real mediator by modulating specifically the activities of intracellular kinases linked to the translation of proteins such as phosphatidylinosinol 3' kinase and mammalian target of rapamycin-70 kDa ribosomal protein S6 (p70S6K) kinases. We recently demonstrated in vitro that protein synthesis of ageing rat muscles becomes resistant to the stimulatory effect of leucine in its physiological concentration range. However, when leucine concentration was increased greatly above its postprandial level, protein synthesis was stimulated normally. Moreover, we studied the effect of meal leucine supplementation on in vivo protein synthesis in adult and ageing rats. Leucine supplementation had no additional effect on muscle protein synthesis in adults but totally restored its stimulation in ageing rats. Whether chronic oral leucine supplementation would be beneficial for maintaining muscle protein mass in elderly men and women remains to be studied.

Dexamethasone-mediated regulation of death and differentiation of muscle cells. Is hydrogen peroxide involved in the process?

The hypothetical involvement of H2O2 in dexamethasone-mediated regulation of muscle cell differentiation and elimination was studied. Rat L6 myoblasts and mouse C2C12 satellite cells were chosen for acute (24 h) and chronic (5 or 10 day) experiments. Mitogenicity and anabolism were both affected by H2O2. Micromolar concentrations of H2O2 inhibited DNA while stimulating protein synthesis. At the millimolar level, H2O2 led to cell death by apoptosis. Synthetic glucocorticoi - dexamethasone (Dex) was shown to effect muscle cell fate similarly to H2O2. Chronic treatment with H2O2 or Dex dose-dependently accelerated either the formation of myotubes or cell elimination. Dex-induced cell death slightly differed from classical apoptosis and was featured by the symptoms of cell senescence such as extensive cytoplasm vacuolisation, accumulation of inclusion-bodies and lack of low molecular weight oligonucleosomal DNA fragmentation but chromatin condensation. Antioxidants (sodium ascorbate, N-acetyl-L-cysteine, catalase) abrogated Dex-dependent cell death. We conclude that H2O2 directly influences myogenesis and muscle cell elimination. Moreover, H2O2 can be considered as the potent mediator of glucocorticoid-dependent effects on muscle cells.

Short-term mild hyperglycemia enhances insulin-stimulated glucose disposal in lactating goats.

This work was designed to study the effect of a 3-day mild hyperglycemia (5.3 vs. 3.3 mM) on the regulation of glucose metabolism in lactating goats. Glucose was intravenously infused at variable rates simultaneously with a constant potassium-amino acid infusion. Diet plus substrate infusion maintained net energy but not protein supply. Milk yield did not change. Skeletal muscle glucose transporter (GLUT-4) was analyzed before and after hyperglycemia. In addition, the acute effect of medium and high insulin doses on glucose turnover was measured in vivo during euglycemic and hyperglycemic hyperinsulinemic clamps under potassium and amino acid replacement. Hyperglycemia reduced the endogenous glucose appearance but increased glucose disposal. It decreased the total membrane-associated GLUT-4 protein in skeletal muscle. In contrast, it improved the acute insulin-stimulated glucose disposal. Both the level and duration (3 days) of hyperglycemia contributed to this improvement. We conclude that short-term mild hyperglycemia has similar effects in lactating goats as those already observed in nonlactating rodents or humans.