Dominique Meynial-Denis

The role of adrenal hormones in the response of glutamine synthetase to fasting in adult and old rats.

BACKGROUND & AIMS During fasting, skeletal muscle exports increased amounts of glutamine (Gln) while increasing the production of this amino acid by glutamine synthetase (GS) in order to maintain the intramuscular Gln pool. Glucocorticoid hormones are believed to be the principal mediators of GS induction during stress conditions. The aim of this study was to evaluate (1) the effect of fasting on GS activity and expression in skeletal muscle during aging and consequently, (2) the role of glucocorticoids in fasting-induced GS activity. METHODS Male Wistar rats (6-, 22-month old) were fasted for 5 days and both the activity and expression of GS were measured in tibialis anterior muscle. To better demonstrate the role of glucocorticoids in the response of GS to fasting, we suppressed their action by RU38486 administration (a potent glucocorticoid antagonist) and their production by adrenalectomy in fed and fasted rats. RESULTS An increase in fasting-induced GS activity was observed in skeletal muscles from both adult and aged rats. Adrenalectomy, but surprisingly not RU38486, suppressed the fasting-induced increase in GS activity and expression. CONCLUSION The data clearly show that the GS responsiveness to fasting was not modified by aging in skeletal muscle.

Does long-term intermittent treatment with glutamine improve the well-being of fed and fasted very old rats?

BACKGROUND Glutamine is known to have a specific role in very old rats (>25 months of age). For this reason, we have orally supplemented female rats with glutamine (20% of diet protein) intermittently. The treatment started before animals became very old and lasted 5 months. Very old rats were studied in fed state or after 5-day fasting after the last glutamine cure. The aim of this study was to determine whether this in vivo pretreatment improves the well-being of very old rats (muscle sarcopenia decrease, gut integrity improvement, decrease of the known up-regulated glutamine synthetase observed regardless of nutrition state). METHODS Protein turnover was measured in epitrochlearis muscle, whereas glutamine synthetase (GS) activities were assessed in tibialis anterior muscle from fed and 5-days-fasted female Wistar adult (6 months) and very old (27 months) rats, pretreated or not with glutamine. Furthermore, gut was dissected and weighed. RESULTS Long-term treatment with glutamine had positive effects on very old rats: (1) it prevented the loss of body weight, but, (2) it did not prevent the inevitable sarcopenia regardless of nutrition state, and (3) it maintained the gut mass. Surprisingly, the muscle up-regulated GS activity observed in fed and fasted very old rats was only decreased in the fed state when rats were supplemented, without change in plasma and muscle glutamine concentrations. CONCLUSIONS Long-term treatment with glutamine started before advanced age had essentially a beneficial role on the gut. It may play a role in maintaining intestine integrity and intestinal immune function. Further investigations would be warranted to explore these mechanisms.

Long-term intermittent glutamine supplementation repairs intestinal damage (structure and functional mass) with advanced age: Assessment with plasma citrulline in a rodent model

OBJECTIVE Glutamine is the preferred fuel for the rat small intestine and promotes the growth of intestinal mucosa, especially in the event of gut injury. Quantitatively, glutamine is one important precursor for intestinal citrulline release. The aim of this study was to determine whether the effect of glutamine on the increase in intestinal villus height is correlated with an increase in both gut mass and citrulline plasma level in very old rats. METHODS We intermittently supplemented very old (27-mo) female rats with oral glutamine (20% of diet protein). Intestinal histomorphometric analysis of the small bowel was performed. Amino acids, in particular citrulline, were measured in the plasma, liver and jejunum. Markers of renal (creatinine, urea) and liver (alanine aminotransferase [ALT]) and aspartate aminotransferase (AST) functions were measured to evaluate renal and liver functions in relation to aging and to glutamine supplementation. Liver glutathione was also determined to evaluate cellular redox state. RESULTS Glutamine supplementation maintains the body weight of very old rats, not by limiting sarcopenia but rather by increasing the organ mass of the splanchnic area. Total intestine mass was significantly higher in glutamine-supplemented rats than in controls (15%). Measurement of villus height and crypt depth demonstrated that the difference between villus and crypt was significantly improved in glutamine pre-treated rats compared to controls (~ 11%). Plasma citrulline also increased by 15% in glutamine-supplemented rats compared to controls. CONCLUSION Citrulline appears as a biomarker of enterocyte mass in villous atrophy associated with advanced age. Non-invasive measurement of this metabolite may be useful in following the state of the gastrointestinal tract in very old people, whose numbers are increasing worldwide and the care of whom is a major public health issue. The gut may contribute to the malnutrition caused by malabsorption frequently observed in the elderly.

Glutamate and CO2 production from glutamine in incubated enterocytes of adult and very old rats

Glutamine is the major fuel for enterocytes and promotes the growth of intestinal mucosa. Although oral glutamine exerts a positive effect on intestinal villus height in very old rats, how glutamine is used by enterocytes is unclear. Adult (8 months) and very old (27 months) female rats were exposed to intermittent glutamine supplementation for 50% of their age lifetime. Treated rats received glutamine added to their drinking water, and control rats received water alone. Jejunal epithelial cells (~300×10(6) cells) were incubated in oxygenated Krebs-Henseleit buffer for 30 min containing [1-(13)C] glutamine (~17 M) for analysis of glutamine metabolites by (13)C nuclear magnetic resonance ((13)C NMR). An aliquot fraction was incubated in the presence of [U-(14)C] glutamine to measure produced CO2. Glutamine pretreatment increased glutamate production and decreased CO2 production in very old rats. The ratio CO2/glutamate, which was very high in control very old rats, was similar at both ages after glutamine pretreatment, as if enterocytes from very old rats recovered the metabolic abilities of enterocytes from adult rats. Our results suggest that long-term treatment with glutamine started before advanced age (a) prevented the loss of rat body weight without limiting sarcopenia and (b) had a beneficial effect on enterocytes from very old rats probably by favoring the role of glutamate as a precursor for glutathione, arginine and proline biosynthesis, which was not detected in (13)C NMR spectra in our experimental conditions.

Glutamine metabolism in advanced age

Glutamine, reviewed extensively in the last century, is a key substrate for the splanchnic bed in the whole body and is a nutrient of particular interest in gastrointestinal research. A marked decrease in the plasma glutamine concentration has recently been observed in neonates and adults during acute illness and stress. Although some studies in newborns have shown parenteral and enteral supplementation with glutamine to be of benefit (by decreasing proteolysis and activating the immune system), clinical trials have not demonstrated prolonged advantages such as reductions in mortality or risk of infections in adults. In addition, glutamine is not able to combat the muscle wasting associated with disease or age-related sarcopenia. Oral glutamine supplementation initiated before advanced age in rats increases gut mass and improves the villus height of mucosa, thereby preventing the gut atrophy encountered in advanced age. Enterocytes from very old rats continuously metabolize glutamine into citrulline, which allowed, for the first time, the use of citrulline as a noninvasive marker of intestinal atrophy induced by advanced age.

Despite similar rates of alanine release, fasting and diabetes affect de novo alanine synthesis differently

Dear Sir, Diabetes and fasting have long been known to induce a substantial release of alanine from muscle whereas the intramuscular alanine pool remains unchanged [1, 2]. The substantial alterations of alanine metabolism in fasting and diabetes are not completely understood, especially with regard to the contribution of leucine as nitrogen donor in alanine de novo synthesis. Alanine remains an important gluconeogenic precursor in humans [3, 4], although glutamine was recently reported as a major source of carbon for gluconeogenesis in diabetic or postabsorptive humans [5, 6]. For this reason, we recently assessed, by using [15N]leucine, the capacity of skeletal muscle to synthesise alanine de novo through leucine transamination in experimental diabetic rats. Indeed, few studies have addressed either the direct effect of leucine supply on both synthesis (within the muscle) and release of alanine (from the muscle) or the contribution of proteolysis and de novo synthesis to alanine production in muscle. For this purpose, we used 15N/1H NMR combined with GC-MS. We have previously demonstrated that streptozotocin-induced diabetes in growing rats is associated with: 1) an increase in nitrogen exchange between leucine and alanine leading to newly synthesised [15N]alanine; and 2) an increase in total alanine release from muscle originating from both proteolysis and de novo synthesis [7]. In the present letter, we would like to report some data obtained with extensor digitorum longus muscles from fasted rats in order to compare nitrogen exchange between leucine and alanine in 48 h-fasting and in experimental diabetes. We observed that the size of [15N]alanine pool within the muscle was smaller in fasted than in diabetic and control extensor digitorum longus muscles (Table 1). Yet, in order to evaluate the true nitrogen transfer between leucine and alanine, both the labelled intramuscular alanine pool (ML) and the labelled alanine released (RL) at the end of experiment should be taken into account. For this reason, we calculated the sum of these two components under each experimental condition (see Table 1). Surprisingly, nitrogen exchange was less intense in fasted than in diabetic rats. Moreover, it should be pointed out that the intramuscular total alanine pool was severely depressed by 48 h-fasting. This could be related to the extensive alanine utilisation in the whole body during the period of fasting before the superfusion experiment. By contrast, a similar increase in total alanine release from muscle of fasted and streptozotocin-diabetic rats (Table 1) was observed. Even though 48 h-fasting had a greater effect on proteolysis than did streptozotocin-diabetes (as reflected by the measurement of net tyrosine release, e. g. 292 ± 25 vs 186 ± 12 nmol tyrosine × g-1 × 2 h-1), the percentage of alanine release originating from proteolysis was the same in fasted muscle as it is in streptozotocin-diabetic extensor digitorum longus muscles (approximatively 60 %). Consequently, leucine contributed to approximately 40 % of alanine a-amino N released in both control and diabetic rats. Only a concomitant increase in proteolysis and de novo synthesis can explain the increase in alanine released from fasted extensor digitorum longus muscles, as previously reported in experimental diabetes [7]. In conclusion, these results confirm the interest of studying both muscle and medium superfusion compartments to better understand alanine regulation within the muscle of fasted rats. Surprisingly, leucine appears less efficient as a donor of a-amino N for the synthesis of alanine in skeletal muscle from fasted than in streptozotocin-diabetic rat even though leucine transamination has been previously described as being increased to a similar extent by fasting or diabetes [8]. These data confirm that alanine synthesis within the muscle may be limited by the availability of amino group acceptors [2]. Whereas a-ketoglurate is added to the medium of superfusion (3 mmol/l) regardless of the animals conditions, glucose is presumably extensively oxidized in superfused extensor digitorum longus muscles from fasted rats, as reflected by the high pH value (7±7.3) in comparison to fed muscles [9]. Consequently, decreased availability of pyruvate may be a limiting factor in glucose-superfused extensor digitorum longus muscles from fasted rats, explaining a lower rate of alanine synthesis in fasted rats than in diabetic rats.

Effect of intermittent glutamine supplementation on skeletal muscle is not long-lasting in very old rats

Background and objectiveMuscle is the major site for glutamine synthesis via glutamine synthetase (GS). This enzyme is increased 1.5–2 fold in 25–27-mo rats and may be a consequence of aging-induced stress. This stimulation is similar to the induction observed following a catabolic state such as glucocorticoid treatment (6 to 24 months). Although oral glutamine supply regulates the plasma glutamine level, nothing is known if this supplementation is interrupted before the experiment.DesignAdult (8-mo) and very old (27-mo) female rats were exposed to intermittent glutamine supplementation for 50 % of their age lifetime. Treated rats received glutamine added to their drinking water and control rats water alone but the effect of glutamine supplementation was only studied 15 days after the last supplementation.ResultsGlutamine pretreatment discontinued 15 days before the experiment increased plasma glutamine to ∼ 0.6 mM, a normal value in very old rats. However, it failed to decrease the up-regulated GS activity in skeletal muscle from very old rats.ConclusionOur results suggest that long-term treatment with glutamine started before advanced age but discontinued 15 days before rat sacrifice is effective in increasing plasma glutamine to recover basal adult value and in maintaining plasma glutamine in very old rats, but has no long-lasting effect on the GS activity of skeletal muscle with advanced age.

Contribution of proteolysis and de novo synthesis to alanine production in diabetic rat skeletal muscle : a 15N/1H nuclear magnetic resonance study

Summary To assess the role of leucine as a precursor of alanine α-amino nitrogen in skeletal muscle during diabetes, extensor digitorum longus muscles from control (n = 7 experiments) and streptozotocin-diabetic rats (n = 8 experiments) were isolated and superfused with [15N]leucine (3 mmol/l) in the presence of glucose (10 mmol/l) for 2 h. Muscle perchloric acid extraction was performed at the end of superfusion in order to quantify newly synthesized alanine by 15N/1H nuclear magnetic resonance. Release of [15N]alanine in the superfusion medium was also measured. The pool of newly synthesized [15N]alanine was significantly increased ( ∼ 40 %) in extensor digitorum longus muscles from streptozotocin-diabetic rats. Whereas a significant enhancement of total alanine release from muscle was induced by diabetes (20 %), only a slight increase in [15N]alanine release was detectable under our experimental conditions. Consequently, we conclude that streptozotocin-diabetes in growing rats induces in skeletal muscle: 1) an increase in nitrogen exchange between leucine and alanine leading to newly synthesized [15N]alanine; and 2) an increase of total alanine release from muscle originating from both proteolysis and de novo synthesis. [Diabetologia (1997) 40: 1159–1165]

New Strategies to Fight against Sarcopenia at Old Age

The focus of this special issue is to determine new strategies to fight against sarcopenia in old age. Aging is associated with a progressive loss of muscle mass and strength, a process called sarcopenia. The most evident metabolic explanation for muscle decline in elderly people is an imbalance between protein synthesis and breakdown rates, but other causes, such as neurodegenerative processes, reduction in anabolic hormone production or sensitivity (e.g., insulin, growth, and sex hormones) and in capacity to respond to anabolic stimuli (e.g., amino acids, exercise), dysregulation of cytokine secretion, modifications in the response to inflammatory events, inadequate nutritional intake and sedentary lifestyle, are involved. The sequelae of sarcopenia often contribute to frailty, decreased independence, and subsequently increased health care costs. In this issue, a few aspects of new strategies to fight against sarcopenia are presented, including role of disease in sarcopenia, new strategies and/or therapeutics in sarcopenia, muscle performance and sarcopenia/physical activity and sarcopenia, antioxidants and aging /sarcopenia. In a clinical study, Domanski and Ciechanowski explain how the whole-body protein-energy deficiency, called protein-energy wasting, aggravates sarcopenia in elderly patients suffering from chronic kidney disease. Novel strategies attenuating sarcopenia are presented in two reviews by Robinson et al. (optimising diet and nutrition throughout life, e.g., sufficient protein intake, vitamin D, and antioxidant nutrients combined with resistance exercise training interventions) and by Sakuma and Yamaguchi (myostatin inhibition, supplementation with eicosapentaenoic acid or ursolic acid, activation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) by exercise). Kemmler et al. present a review on the benefits of physical exercise in preventing sarcopenia and related muscle malfunction. They underline the lack of motivation or the physical limitations of elderly subjects and present two new exercise technologies, whole-body vibration and whole-body electromyostimulation, which are also feasible in unmotivated or handicapped persons. However, while these methods increase strength and power parameters, they do not improve muscle mass. In clinical studies, Sayers and Gibson examine whether high-speed power training (HSPT) improves muscle performance and braking speed using a driving stimulator. They demonstrate that HSPT exerts a wider effect and improves speed compared to slow-speed strength training. Marcus et al., in another clinical study, evidence that thigh intramuscular adipose tissue plays a potent role in reducing mobility function and physical activity in older adults. Cerullo et al. present a review on the benefits of oral antioxidants in the prevention and treatment of sarcopenia. They demonstrate that oral antioxidant supplementation may contribute to reducing indices of oxidative stress in both animal and human models by reinforcing the natural endogenous defences. This special issue provides the opportunity to discuss the challenges of sarcopenia and proposes creative solutions to fight against it in old age and to contribute to “aging well.” Dominique Meynial-Denis Olivier Guerin Stephane Michel Schneider Dorothee Volkert Cornel Christian Sieber