Michelle Mignon

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.

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.

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]