J Nutr Health Aging
THE JOURNAL OF NUTRITION, HEALTH & AGING©
EFFECT OF INTERMITTENT GLUTAMINE SUPPLEMENTATION ON SKELETAL MUSCLE IS NOT LONG-LASTING IN VERY OLD RATS D. MEYNIAL-DENIS1, A.-M. BEAUFRERE2, M. MIGNON1, P. PATUREAU MIRAND1 1. INRA, UMR 1019, UNH, CRNH Auvergne, 63009 Clermont-Ferrand, France ; 2. CHU Clermont-Ferrand, Service d'Anatomie Pathologique, Hôpital Estaing, 63003 ClermontFerrand, Cedex1, France. Corresponding author: Dominique Meynial-Denis (PhD), Human Nutrition Unit, INRA and Human Nutrition Research Center, Theix 63122 - St Genes Champanelle, France. Phone: +33 (0)4 73 62 43 13; Fax: +33 (0)4 73 62 47 55; E-mail address: [email protected]
Abstract: Background and objective: Muscle 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. Design: Adult (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. Results: Glutamine 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. Conclusion: Our 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. Key words: Glutamine synthetase activity, discontinuation, tibialis anterior muscle, glutamine level, plasma, advanced age.
Introduction
In the present study, we decided to determine whether the effect of glutamine supply is long-lasting on GS activity in skeletal muscle. Before experiments, rats were exposed to glutamine supplementation for 50% of their age lifetime on 7 consecutive days a month, whatever their age, as previously described (18). However, the effect of glutamine supplementation was studied 15 days after the last supplementation, as previously reported (19). The aim of this work was to study the effects on glutamine metabolism in the skeletal muscle and the whole body of discontinuing glutamine supplementation. To address this issue, we examined the possible changes in rat body weight and in skeletal muscle mass. We measured the plasma glutamine level. We also assessed glutamine concentration and GS activity in the tibialis anterior muscle, a mixed-fiber muscle that is rich in type II fibers, representative of the whole musculature and sensitive to aging and its associated diseases (10), from 27 mo-old (very old) rats and compared results with those obtained in 8 mo-old (adult) rats.
Aging, an inevitable biological process, leads, in particular, to a progressive loss of muscle mass or sarcopenia in humans and rodents (1-6). This loss is caused by muscle protein wasting and results from an imbalance between protein synthesis and degradation rates. Aging is a complex process, with numerous inflammatory episodes (7, 8) that induce both a state of chronic, continuous stress and a general decline in physiological function leading to morbidity and mortality. Muscle throughout the body is an important producer of glutamine, which is essential for immune cell function. However, with aging muscle mass is reduced and the availability of glutamine in the elderly may subsequently be limited (1, 9). In addition, aging generally increases protein and amino acid needs above those for younger adults. Alterations in glutamine synthetase (GS) activity have been demonstrated with advanced age (> 22 months) (10). This increase in GS activity (1.5-2 fold), observed in 25-27-mo rats, may be a consequence of aging-induced stress. The stimulation of GS activity, seen in both female and male rats, was similar to the induction observed following a catabolic state in aging (6 to 24 months) such as glucocorticoid treatment (11, 12) or fasting (13). Studies have shown that glutamine per se can suppress GS activity in cultured myocytes (14, 15) and in the liver (16) and that glutamine supplementation is able to suppress the response of GS to glucocorticoids (17). Thus, we demonstrated that the effect of long-term glutamine treatment in vivo on GS regulation in skeletal muscle could prevent the up-regulation of GS with advanced age (18). Received October 3, 2012 Accepted for publication February 12, 2013
Materials and Methods Materials L-Glutamine used in supplementation experiments was purchased from Jerafrance (Jeufosse, France). [14C] Glutamate (262 mCi/mmol) was purchased from Amersham (Bucks, UK). Animals The experiments were performed in accordance with current legislation on animal experimentation in France. Wistar rats 1
J Nutr Health Aging
GLUTAMINE SUPPLEMENTATION AND MUSCLE WITH AGE came from the experimental Unit of the Research Center of Theix. No animals were pregnant. They were housed in the animal facilities until reaching the required age for the experiment. They were acclimatized in cages in groups of five in a room with a 12 h light/12 h dark cycle (lights on at 8.00 AM) at 22 °C. The animals were fed rat pellets (AO3 "growth diet" until the age of 10 months, and AO4 "maintenance diet" from 10 to 27 months). The diets were purchased from Usine d'Alimentation Rationnelle, Villemoisson/Orge, France. The animals had free access to water and were fed ad libitum. All rats and experimental procedures were used in accordance with recommendations from the Institutional Ethics Committee of the University of Clermont-Ferrand (France). Experimental design Adult (8-month aged) and old (27-month aged) female rats, weighing 350-400 g, were used. They were exposed to preliminary treatment for 50 % of their age lifetime i.e glutamine supplementation by the addition of glutamine to drinking water on 7 consecutive days a month (20% dietary protein). During the same period, control rats received only water. Adult and very old rats were studied about 15 days after the last supplementation with glutamine as previously reported (19). The rats were randomized to the following four groups: 1) control adults without supplementation (n=10); 2) glutaminesupplemented adults (n=10); 3) control old rats without supplementation (n=10); 4) glutamine-supplemented old rats (n=10). Tissue removal Blood At the end of the experimental period, the animals were anesthetized with sodium pentobarbital (45µg/g body wt., intraperitoneally). Blood was taken from the abdominal aorta into a heparinized syringe to assay glutamine and glutamate levels. Muscle The hind limbs were removed and skinned. The tibialis anterior muscle was rapidly dissected, weighed and
immediately frozen in liquid nitrogen. It was used for determinations of intramuscular concentrations in glutamine/glutamate and glutamine synthetase activity. GS assay To investigate whether glutamine supplementation of long duration may decrease the up-regulated GS response in fed very old rats, we measured GS activity in the tibialis anterior muscle from 8- and 27-month old fed female rats. GS activity was measured by the formation of [14C]glutamine from [14C]glutamate (20). Skeletal muscle was homogenized as previously reported (21). Specific activity of GS was expressed as nmoles of glutamine formed per hour per milligram of protein. Biochemical assays Plasma and muscle glutamate and glutamine levels were determined as previously described (11). Plasma and muscle samples were deproteinized with 4% (w/v) perchloric acid, neutralized with K2CO3 (3.5 M) and stored at -20°C until analysis. This procedure was always performed the day prior to the measurement of glutamine and glutamate to ensure that no glutamine degradation occurred before the assay. Metabolite concentrations were expressed in µmol/g wet wt muscle. Statistical analysis Values are given as means ± SD. Analyses of two-way ANOVA were performed to discriminate between the effects of aging (A), supplementation (S) and their interactions. Comparisons between two means were made by Student's t test (unpaired test). The level of significance was set at P
THE JOURNAL OF NUTRITION, HEALTH & AGING©
EFFECT OF INTERMITTENT GLUTAMINE SUPPLEMENTATION ON SKELETAL MUSCLE IS NOT LONG-LASTING IN VERY OLD RATS D. MEYNIAL-DENIS1, A.-M. BEAUFRERE2, M. MIGNON1, P. PATUREAU MIRAND1 1. INRA, UMR 1019, UNH, CRNH Auvergne, 63009 Clermont-Ferrand, France ; 2. CHU Clermont-Ferrand, Service d'Anatomie Pathologique, Hôpital Estaing, 63003 ClermontFerrand, Cedex1, France. Corresponding author: Dominique Meynial-Denis (PhD), Human Nutrition Unit, INRA and Human Nutrition Research Center, Theix 63122 - St Genes Champanelle, France. Phone: +33 (0)4 73 62 43 13; Fax: +33 (0)4 73 62 47 55; E-mail address: [email protected]
Abstract: Background and objective: Muscle 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. Design: Adult (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. Results: Glutamine 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. Conclusion: Our 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. Key words: Glutamine synthetase activity, discontinuation, tibialis anterior muscle, glutamine level, plasma, advanced age.
Introduction
In the present study, we decided to determine whether the effect of glutamine supply is long-lasting on GS activity in skeletal muscle. Before experiments, rats were exposed to glutamine supplementation for 50% of their age lifetime on 7 consecutive days a month, whatever their age, as previously described (18). However, the effect of glutamine supplementation was studied 15 days after the last supplementation, as previously reported (19). The aim of this work was to study the effects on glutamine metabolism in the skeletal muscle and the whole body of discontinuing glutamine supplementation. To address this issue, we examined the possible changes in rat body weight and in skeletal muscle mass. We measured the plasma glutamine level. We also assessed glutamine concentration and GS activity in the tibialis anterior muscle, a mixed-fiber muscle that is rich in type II fibers, representative of the whole musculature and sensitive to aging and its associated diseases (10), from 27 mo-old (very old) rats and compared results with those obtained in 8 mo-old (adult) rats.
Aging, an inevitable biological process, leads, in particular, to a progressive loss of muscle mass or sarcopenia in humans and rodents (1-6). This loss is caused by muscle protein wasting and results from an imbalance between protein synthesis and degradation rates. Aging is a complex process, with numerous inflammatory episodes (7, 8) that induce both a state of chronic, continuous stress and a general decline in physiological function leading to morbidity and mortality. Muscle throughout the body is an important producer of glutamine, which is essential for immune cell function. However, with aging muscle mass is reduced and the availability of glutamine in the elderly may subsequently be limited (1, 9). In addition, aging generally increases protein and amino acid needs above those for younger adults. Alterations in glutamine synthetase (GS) activity have been demonstrated with advanced age (> 22 months) (10). This increase in GS activity (1.5-2 fold), observed in 25-27-mo rats, may be a consequence of aging-induced stress. The stimulation of GS activity, seen in both female and male rats, was similar to the induction observed following a catabolic state in aging (6 to 24 months) such as glucocorticoid treatment (11, 12) or fasting (13). Studies have shown that glutamine per se can suppress GS activity in cultured myocytes (14, 15) and in the liver (16) and that glutamine supplementation is able to suppress the response of GS to glucocorticoids (17). Thus, we demonstrated that the effect of long-term glutamine treatment in vivo on GS regulation in skeletal muscle could prevent the up-regulation of GS with advanced age (18). Received October 3, 2012 Accepted for publication February 12, 2013
Materials and Methods Materials L-Glutamine used in supplementation experiments was purchased from Jerafrance (Jeufosse, France). [14C] Glutamate (262 mCi/mmol) was purchased from Amersham (Bucks, UK). Animals The experiments were performed in accordance with current legislation on animal experimentation in France. Wistar rats 1
J Nutr Health Aging
GLUTAMINE SUPPLEMENTATION AND MUSCLE WITH AGE came from the experimental Unit of the Research Center of Theix. No animals were pregnant. They were housed in the animal facilities until reaching the required age for the experiment. They were acclimatized in cages in groups of five in a room with a 12 h light/12 h dark cycle (lights on at 8.00 AM) at 22 °C. The animals were fed rat pellets (AO3 "growth diet" until the age of 10 months, and AO4 "maintenance diet" from 10 to 27 months). The diets were purchased from Usine d'Alimentation Rationnelle, Villemoisson/Orge, France. The animals had free access to water and were fed ad libitum. All rats and experimental procedures were used in accordance with recommendations from the Institutional Ethics Committee of the University of Clermont-Ferrand (France). Experimental design Adult (8-month aged) and old (27-month aged) female rats, weighing 350-400 g, were used. They were exposed to preliminary treatment for 50 % of their age lifetime i.e glutamine supplementation by the addition of glutamine to drinking water on 7 consecutive days a month (20% dietary protein). During the same period, control rats received only water. Adult and very old rats were studied about 15 days after the last supplementation with glutamine as previously reported (19). The rats were randomized to the following four groups: 1) control adults without supplementation (n=10); 2) glutaminesupplemented adults (n=10); 3) control old rats without supplementation (n=10); 4) glutamine-supplemented old rats (n=10). Tissue removal Blood At the end of the experimental period, the animals were anesthetized with sodium pentobarbital (45µg/g body wt., intraperitoneally). Blood was taken from the abdominal aorta into a heparinized syringe to assay glutamine and glutamate levels. Muscle The hind limbs were removed and skinned. The tibialis anterior muscle was rapidly dissected, weighed and
immediately frozen in liquid nitrogen. It was used for determinations of intramuscular concentrations in glutamine/glutamate and glutamine synthetase activity. GS assay To investigate whether glutamine supplementation of long duration may decrease the up-regulated GS response in fed very old rats, we measured GS activity in the tibialis anterior muscle from 8- and 27-month old fed female rats. GS activity was measured by the formation of [14C]glutamine from [14C]glutamate (20). Skeletal muscle was homogenized as previously reported (21). Specific activity of GS was expressed as nmoles of glutamine formed per hour per milligram of protein. Biochemical assays Plasma and muscle glutamate and glutamine levels were determined as previously described (11). Plasma and muscle samples were deproteinized with 4% (w/v) perchloric acid, neutralized with K2CO3 (3.5 M) and stored at -20°C until analysis. This procedure was always performed the day prior to the measurement of glutamine and glutamate to ensure that no glutamine degradation occurred before the assay. Metabolite concentrations were expressed in µmol/g wet wt muscle. Statistical analysis Values are given as means ± SD. Analyses of two-way ANOVA were performed to discriminate between the effects of aging (A), supplementation (S) and their interactions. Comparisons between two means were made by Student's t test (unpaired test). The level of significance was set at P
