Lead Article
Glutamine metabolism in advanced age
Dominique Meynial-Denis
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 gastro-intestinal 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 ad-vantages 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.
INTRODUCTION
Glutamine is a nonessential amino acid but becomes a conditionally essential amino acid in catabolic states be-cause of the body’s inability to synthesize sufficient amounts of glutamine during stress.1–4 In other words, normal plasma levels of glutamine are insufficient to meet increased demands under stress. Low concentra-tions of glutamine in plasma reflect reduced stores in muscle, and this reduced availability of glutamine in the catabolic state seems to correlate with increased morbid-ity and mortalmorbid-ity. Aging, which is characterized by reduced physical activity resulting mainly from the inev-itable age-related loss of muscle mass or to skeletal muscle atrophy, a condition referred to as sarcopenia,5–15 may be related to a “physiological” catabolic state. Consequently, it is important to discuss the role of glu-tamine in sarcopenia because gluglu-tamine synthesis can be depressed in skeletal muscle as a result of the muscle loss that occurs with aging.
The aim of this review is to present current data on the regulation of glutamine metabolism, with particular reference to animal data, in order to gain a better understanding of the cell mechanisms involved and, specifically, to determine what is currently known about the role of glutamine in nutrition (needs and utiliza-tion). Animal data are extended to elderly people, bear-ing in mind that, as reported by Welle,16 the metabolism rate is faster in rats than in humans. The potential role of glutamine in nutrition during aging, especially the relevance of glutamine supply for well-being and longevity, is highlighted.
PHYSIOLOGICAL IMPORTANCE AND BIOCHEMISTRY OF GLUTAMINE
This review covers only the enzymes responsible for the synthesis and degradation of glutamine in the cell. The data presented essentially concern skeletal muscle, liver, gut, and other rapidly dividing cells.
Affiliation: D. Meynial-Denis is with the Unit of Human Nutrition (UNH), French National Institute for Agricultural Research (INRA), Joint Research Unit (UMR) 1019, Center for Research in Human Nutrition (CRNH) Auvergne, Clermont-Ferrand, France.
Correspondence: D. Meynial-Denis. INRA, UMR 1019, UNH, CRNH Auvergne, 63009 Clermont-Ferrand, France. Email: dominique.meynial@ clermont.inra.fr. Phone:þ33-4-73-62-42-13. Fax: þ33-4-73-62-47-55.
Key words: glutaminase, glutamine synthetase, longevity, metabolism, sarcopenia.
VC The Author(s) 2016. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For
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Two principal enzymes regulate intracellular me-tabolism of glutamine.17The enzyme glutaminase cata-lyzes the hydrolysis of glutamine to glutamate, while glutamine synthetase catalyzes the synthesis of glutam-ine from glutamate and ammonia. Replicating cells, such as enterocytes, lymphocytes, endothelial cells, and tumor cells, tend to be avid consumers of glutamine and, in general, contain far greater amounts of glutami-nase than glutamine synthetase. Skeletal muscle and lung, which synthesize and release net amounts of glu-tamine into the bloodstream, contain substantial amounts of glutamine synthetase. The liver contains both enzymes because it can switch from net glutamine utilization to net production, depending on physio-logical and nutritional conditions.
OVERVIEW OF MOLECULAR AND CELLULAR ASPECTS OF GLUTAMINE METABOLISM IN MAMMALS AND
HUMANS
The major metabolic roles of glutamine (molecular and cellular mechanisms) in mammals and humans are pre-sented in the first section ofTable 1.3,4,17–24The charac-teristics of glutamine, as well as the role of glutamine in metabolic pathways in the cell, are described in this part of the table. In the second part ofTable 1, the regulatory roles of glutamine in several cell-specific processes are listed.25–52 The concept of the classical “ideal protein,” including glutamine as a nonessential amino acid, was defined according to Wu et al.51in 2013.
Effect of glutamine starvation on cell survival and apoptosis
The consequences of glutamine deprivation on cellular survival and gene expression have led to the construc-tion of a new paradigm for this amino acid, namely that limited extracellular glutamine supplies modulate stress and apoptotic responses. Under these conditions, plasma glutamine levels decline and, as a result, the cells suffer from glutamine starvation. Apoptotic signaling mechanisms involved in the response to glutamine de-privation are cell-type specific. New findings indicate that glutamine availability is strongly related to the in-duction of apoptosis and that glutamine works both as a nutrient and as a signaling molecule, acting directly or indirectly on the pathways leading to programmed cell death.53 In addition, glutamine-starved cells show reduced expression of the 70 000 Mrheat-shock protein,
which is an important factor for cell survival.54 Consequently, glutamine-utilizing cells possess molecu-lar mechanisms to detect the availability of glutamine and to respond specifically to changes in extracellular glutamine concentrations.54,55
Regulative potential of glutamine: relation to glutathione metabolism
In animal studies, Roth44 demonstrated that adminis-tration of glutamine increases tissue concenadminis-trations of reduced glutathione. Glutamine (via glutamate), cyst-eine, and glycine are the precursor amino acids for glutathione, which is present within the cell in a reduced form and an oxidized form. The ratio of reduced glutathione to oxidized glutathione is the most important regulator of the cellular redox potential. Thus, glutamine in either its free or dipeptide form in-fluences this potential by enhancing, even more so in catabolic states,56,57 the formation of glutathione, the major endogenous antioxidant in mammalian cells that protects against oxidative injury and cell death.58 Glutamine signaling in the intestine
Glutamine is an essential nutrient for small intestine function. It functions as a signal to enhance cell survival in the intestine, it inhibits apoptosis in intestinal cells, it is necessary for tight junction stabilization, and it has anti-inflammatory effects in the intestine (e.g., it de-creases production of proinflammatory interleukins 6 and 8, enhances production of anti-inflammatory inter-leukin 10, and reduces expression of NF-jB protein).42 Hence, glutamine plays a key role in the metabolism of rapidly dividing cells, including enterocytes and lymphocytes, which may contribute to its beneficial clin-ical effects. Gut mucosal homeostasis is achieved through a balance between cell proliferation and apop-tosis. Whereas glutamine upregulates anti-apoptotic proteins and downregulates pro-apoptotic proteins in T cells, it prevents apoptosis in rat epithelial cell lines derived from gut mucosa; moreover, glutamine starva-tion induces apoptosis through caspase activastarva-tion.31,35 In brief, glutamine may play a role in the gut-protective effect by inhibiting apoptosis via downregulation of the transcription factor Sp3, by contributing to cell survival during physiological stress by induction of autophagy, by modulating intestinal barrier function under basal and inflammatory conditions, or by its anti-inflamma-tory effect via induction of nuclear degradation of the NF-jB p65 subunit.59,60
Glutamine is a key regulator of amino acid– controlled cell growth through the mammalian target of rapamycin (mTOR) signaling pathway in rat intestinal epithelial cells.27,28,31,39,42,61,62Amino acids are import-ant signaling regulators, especially for p70 S6 kinase and eIF-4E binding protein 1 via mTOR, which have 2 structurally and functionally distinct complexes termed mTORC1 and mTORC2. mTORC1 is activated by nu-trients (amino acids), growth factors, and cellular
energy, while mTORC2 is activated by growth factors alone. The mTOR pathway that regulates major cellular functions (growth and proliferation) plays a role in health and disease as well as in aging.43,63–65Glutamine inhibits the activation of p70 S6 kinase and the phos-phorylation of eIF-4E binding protein 1 induced by ar-ginine and leucine in rat intestinal epithelial cells. In contrast, in healthy subjects in the fed state, enteral pro-teins, but not glutamine, increased protein synthesis via an mTOR-independent pathway in humans.66 Nakajo et al.38have proposed a new concept for the biological role of phosphorylation of mTOR and intestinal cell growth: the signal induced by glutamine may stimulate cellular proliferation and increase cell number, whereas leucine or arginine induces the signal for cell growth (increase in cell size) in rat intestinal epithelial cells.
Glutamine suppresses only mTOR signaling for cell growth, and therefore it is considered an essential amino acid for cell culture.38The intracellular signaling pathways involved in controlling intestinal glutamine transport during acidosis have been studied in Caco-2 cells, a model of human enterocytes. Metabolic acidosis stimulates glutamine transport via signaling pathways that lead to transcription of the glutamine transporter gene and to intestinal glutamine absorption.23
Glutamine signaling in the liver
As in the intestine, glutamine has a direct regulatory potential in autophagic proteolysis in the liver caused by a lysosomotropic toxicity of ammonia derived from glutamine degradation. Indeed, in most visceral tissues, Table 1 Overview of the metabolic roles of glutamine
References (1)Characteristics of glutamine and its role in metabolic pathways in the cell
The most abundant free amino acid in the body. Classified among the NEAAs, which include glu-tamate, proline, glycine, and arginine
Boelens et al. (2001),18Buchman
(2001),19Bulus et al. (1989),20
Chiba et al. (2009),21Coster
et al. (2004),22Epler et al.
(2003),23Lacey & Wilmore
(1990),3Smith (1990),4Souba
(1991),17Welbourne & Joshi
(1994)24
In catabolic states, reclassified as a conditionally essential amino acid; consequently, it plays a role in nutrition and health
Skeletal muscle is the largest tissue reservoir of glutamine and is quantitatively the most im-portant site of glutamine synthesis. Cytosolic glutamine synthetase, which is a longevity-related gene, is responsible for this synthesis
Small intestine and kidneys are the major sites of glutamine utilization under normal or acid-otic conditions
Intracellular glutamine concentration generally decreases in catabolic states. In humans, this change can be prevented or attenuated by glutamine supplementation (enteral or parenteral)
The liver can synthesize or degrade glutamine, depending on the nutritional conditions
(2)Regulatory roles of glutamine in cell-specific processes Boukhettala et al. (2012),25
Brasse-Lagnel et al. (2009),26
Yi et al. (2015)27,
Brasse-Lagnel (2010),28Curi et al.
(2005)29(2007),30Deniel et al.
(2007),31Larson et al. (2007),35
Le Bacquer et al. (2001),36
Ha¨ussinger et al. (2001),32
Kadowaki & Kanazawa (2003),33Krause et al. (2002),34Mate´s et al. (2006),37 Nakajo et al. (2005),38 Naomoto et al. (2005),39 Nishikawa et al. (2007),40 Papaconstantinou (2000),41
Rhoads & Wu (2009),42Roth et al. (2002),45Roth (2007),43
(2008),44Rutten et al.
(2006),46van de Poll et al.
(2007),47,52Watford (1993),48
Xi et al. (2011),49Ziegler et al.
(2003)50
Nitrogen shuttle to form urea (via glutaminase)
Major transport form of nitrogen and carbon within the body
Oxidative fuel for rapidly dividing cells (enterocytes, fibroblasts, malignant cells, immune cells) Gluconeogenic precursor (liver and kidneys)
Lipogenic precursor (adipocytes)
Cell integrity (apoptosisa, cell proliferation)
Cell swelling (notably in the liver and brain)
Protein synthesis (in particular, contractile protein synthesis) and degradation (resulting from 3 major systems: cathepsin, calpain, and ATP-ubiquitin-dependent proteolysis)
Redox potential (glutathione synthesis from glutamine)
Respiratory burst (glutamine increases superoxide anion production) Insulin resistance or insulin secretion
Extracellular matrix synthesis Many genes related to metabolism Activation of several proteins
Enhancement of transcription factors (sp1, C/EBP, FXR/RXR, NF-jB)
Intracellular signaling pathways (e.g., glutamine is a key regulator for cell growth through the mammalian target of rapamycin pathway)
Signal transduction
Cell defense and repair (e.g., glutamine is a potent enhancer of heat-shock proteins) Neurotransmission
Immune function (cytokine production)
(3)The classical “ideal protein” concept should include both EAAs and NEAAs (including glutamine) to improve protein accretion, efficiency of food utilization, growth, and health in animals and humans
Wu et al. (2013)51
Abbreviations: ATP, adenosine triphosphate; EAAs, essential amino acids; NEAAs, nonessential amino acids; NF-jB, nuclear factor jB.
aApoptotic signaling mechanisms activated in response to glutamine deprivation are cell-type specific. New findings indicate that
glu-tamine availability is strongly related to the induction of apoptosis. Gluglu-tamine works both as a nutrient and as a signaling molecule and acts directly or indirectly on the pathways leading to programmed cell death (e.g., glutamine deprivation occurs as a protective mechanism to limit apoptosis associated with cellular stress in the intestine).
the autophagic pathway is responsible for the bulk of proteolysis and is most sensitive to amino acid regula-tion.67Glutamine, like leucine, tyrosine, phenylalanine, proline, methionine, tryptophane, and histidine, may have a direct regulatory role in the liver, possibly via amino acid receptors or sensors that enable recognition of these amino acids at the plasma membrane, leading subsequently to their involvement in intracellular sig-naling.33 Moreover, glutamine stimulates a number of metabolic pathways of intermediary metabolism in the liver. For example, glutamine activates phosphoenolpyr-uvate carboxykinase (PEPCK) in the liver and increases the phosphorylation state of p70 S6 kinase, a key en-zyme in liver protein synthesis. Glutamine is known to induce cell swelling and to mediate the inhibition of autophagic proteolysis; glutamine’s antiproteolytic ef-fect may be mediated through osmotic swelling and the p38MAPKpathway.68
IMPACT OF AGING ON REGULATION OF GLUTAMINE METABOLISM
Aging is defined as the progressive changes that occur after maturity in various organs, leading to a decrease in their functional ability and possible alterations in metabolic pathways.69–72 Because protein ingestion is crucial for the maintenance of a variety of body func-tions, the requirements of protein in the elderly are a major factor in maintaining skeletal muscle mass; the amount of protein ingested that induces maximal muscle protein synthesis must be higher in elderly than in young individuals in order to combat anabolic resist-ance in the elderly.5,73–77
Dysregulation of autophagy78–80 contributes not-ably to aging. Autophagy, a lysosomal process involved in the maintenance of cellular homeostasis, is inhibited by the insulin-amino acid-mTOR signaling pathway that controls both protein synthesis and longevity (see next paragraph). During aging, autophagy declines and insulin resistance can develop.81 Thus, autophagy can provide protection against aging and cell death. Indeed, the best way to increase autophagy in vivo is by restrict-ing calorie intake, which may promote longevity durrestrict-ing aging.82–84Moreover, glutamine inhibits autophagy and regulates cell growth.85The role of glutamine metabol-ism in autophagy is related to the activation of mTOR by leucine, which is an activator of glutaminolysis.86
Aging is also characterized by protein wasting (see next paragraph), and glutamine, which is the most abundant amino acid in the blood, may be a hallmark of catabolic states. Indeed, a low concentration of glu-tamine in plasma reflects reduced stores in muscle, and this reduced availability of glutamine in the catabolic state seems to correlate with increased morbidity and
mortality.18 Moreover, in catabolic states, glutamine may be replenished by supplementation of branched-chain amino acids. Branched-branched-chain amino acids, especially leucine, stimulate protein synthesis, inhibit proteolysis (in cell culture and animals), and promote glutamine synthesis.87,88Depletion of plasma glutamine may result in loss of branched-chain amino acids and increased protein wasting.89When the “glutamine trap” (e.g., phenylbutyrate) is used to deplete plasma glutam-ine,90skeletal muscle glutamine is not depleted, regard-less of the age of rats, and muscle glutamine synthetase is not increased.91 Despite this, there are few docu-mented data in the literature on the role of glutamine in aging.
Glutamine: the potential cornerstone of protein turnover in skeletal muscle during aging
The potential regulatory effect of glutamine in aging has implications for the development and treatment of age-related muscle loss and strength loss (sarcopenia). There is a progressive decrease in lean body mass with aging and, consequently, in total body protein, due largely to a loss of skeletal muscle protein.77,92 Sarcopenia is a highly significant public health problem. These changes in skeletal muscle are attributed largely to various molecular mediators that affect fiber size, mitochondrial homeostasis, and apoptosis and highlight the mTORC1 pathway as a key therapeutic target to prevent sarcopenia.93–97Indeed, in skeletal muscle, the activation of mTORC1 is involved in the regulation of protein synthesis and controls skeletal muscle mass.98 This progressive loss of skeletal muscle with aging is attributed to a disruption in the regulation of protein turnover in skeletal muscle99and may be considered as a “physiological” catabolic state. For this reason, protein and amino acids, notably glutamine, as dietary supple-mentation may have potential usefulness in fighting against muscle atrophy during aging. Thus far, however, dietary supplementation with proteins or amino acids seems inefficient in limiting the atrophy processes, and neither leucine100–102 (or essential amino acids103,104) nor glutamine105 fights against the loss of skeletal muscle. Protein supplementation increased muscle mass gain only during resistance-type exercise training in elderly people.106,107Essential amino acids combined with resistance exercise also increased protein synthesis via mTORC1 signaling.108 During a 5-week exercise program, cysteine-rich whey protein increased lean body mass and decreased fat mass in comparison with a control diet, but the authors made no mention of the ef-fect on skeletal muscle mass per se.109Thus, neither glu-tamine nor essential amino acids such as leucine are able to fight against sarcopenia; the increase in protein
synthesis is not sufficient to increase muscle mass
(Table 2).
Glutamine deficiency, which stimulates cell apop-tosis, may stimulate sarcopenia, thereby triggering a low-level inflammatory process.43 Moreover, aging is known to induce a dysregulation of immune and in-flammation functions that may affect protein synthesis rates in lymphoid tissues and plasma proteins.110Aging has been also described as a condition characterized by anabolic resistance to nutrients, especially amino acids, which impairs muscle protein synthesis and contributes to muscle wasting.Table 2provides an overview of the effect of amino acids on protein turnover in skeletal muscle. Under inflammatory conditions, branched-chain amino acids, notably leucine, can be transami-nated to glutamate in order to increase the synthesis of glutamine, which is a substrate highly consumed by in-flammatory cells such macrophages.111But in old rats, both the production and the release of glutamine in muscle are reduced in relation to the increased splanch-nic sequestration of leucine and the reduced renal and intestinal glutamine uptake in order to maintain whole-body glutamine homeostasis.112Thus, glutamine deple-tion has a role in the sarcopenia and low-grade inflam-mation observed with aging.
Effect of aging on whole-body glutamine metabolism A link can be made between aging and physical inactiv-ity (experimental bed rest in healthy volunteers) since, as previously reported, inactivity contributes to sarcope-nia.113 Physical activity generally decreases with age. Physical inactivityreduces whole-body glutamine avail-ability due to downregulated de novo synthesis and, more generally, to anabolic resistance.114,115This alter-ation also results in decreased cytosolic ratios of glu-tamine to branched-chain amino acids (leucine, valine, isoleucine) and glutamine to aromatic amino acids (tyrosine, phenylalanine). These changes in the profiles of free amino acids may be caused by sedentary lifestyle, nutrition, and immobilization, as commonly observed in the aging process.116However, in very old rats aged 25 months (female rats, whose mean life expectancy is 27 months, with a maximum life span of 32 months), aging does not induce changes either in plasma or muscle glutamine concentrations (0.5960.13 vs 0.556 0.15 mM in plasma and 3.2160.60 vs 3.1860.85 mmol/g in muscle).117 Advanced age in rats corresponds to an age of about 75–80 years in humans. Glutamine defi-ciency in bed rest in humans also alters monocyte or macrophage activity, decreases the formation of heat-shock proteins, stimulates cell apoptosis, and shifts the cellular redox potential by altering glutathione synthesis
in lymphocytes. However, glutamine is able to preserve Table
2 Ef fe ct of glutamin e a nd o ther a mi no ac ids o n p ro te in turno ver in ske leta l mu sc le with a g ing Supplementation Physical activity W hole-body improvement or preservation of muscle functi onality Change in fat body mass Changed in lean body mass Skeletal muscle atrophy References Leucine-rich whey protein o r leucine alone or essential amino acids Bed rest or no physical activity Yes Decreased or maintained Increased or maintained No effective change Koopman et al. (2008), 101 Dillon et al. (2009), 103 Ferrando et al. (2010), 104 Walker et al. (2011), 108 Casperson et al. (2012), 100 Magne et al. (2013) 102 Glutamine None Yes NR NR No effective change Mignon et al. (2007) 105 Protein (2 15 g) 24 wk of progressive resistance exercise Yes NR Increased No effective change Tieland et al. (2012), 107 Evans et al. (2013) 106 Cysteine-rich whey protein 5 wk of exercise program Yes Decreased Increased NR Dro ¨g e (2005) 109 Abbreviation :NR, not reported.
hepatic glutathione after hepatic injury,44 as measured in the liver of very old female rats.59
Impact of aging on glutamine synthesis in skeletal muscle: in vivo study using13C magnetic resonance spectroscopy
Skeletal muscle is the principal organ of the synthesis of glutamine, which is exported to other tissues such as the liver, gut, and immune cells according to the re-quirements of the cell. These rere-quirements are depend-ent on age, as evidenced by the increase in glutamine synthetase activity with aging, reported below. Under normal healthy conditions, glutamine synthetase activ-ity measured in skeletal muscle is higher in female and male rats of advanced age (about 25–27 months) than in 8- to 22-month-old rats. In contrast, very old age (29–32 months, which corresponds to >85-year-old humans, including centenarians) is associated with sig-nificant muscle wasting, resulting in 2 subpopulations of low or high glutamine synthetase activity. The low glutamine synthetase activity may reflect a state of lesser stress, as if these survivors may be physiologically younger than their age would indicate. High levels, such as those reported in the 25- to 27-month-old rats, may be considered an indicator of stress and frailty and, thus, the need for more glutamine.117 Moreover, glu-tamine synthetase in muscle is a longevity-related gene and increases with caloric restriction in mice.21Because glutamine synthetase activity increases with fasting, whatever the age of animals,118glutamine synthesis was compared directly in skeletal muscle from 2-[13C] acet-ate by magnetic resonance spectroscopy in fasted 25-month-old female Wistar rats and adult rats (8 months) (V. Mezzarobba, D. Meynial-Denis, and J.P. Renou, un-published data, 2000). In old rats, glutamine and glu-tamate were undetectable in muscle in vivo but were detected in muscle extract at the end of the experiment
(Figure 1). Consequently, flows of glutamine and
glu-tamate through skeletal muscle were too quick to be de-tected by magnetic resonance spectroscopy in vivo. A metabolic schema is shown in Figure 2. This method was well described and assessed by Hwang and Choi119. Impact of aging on glutamine synthetase in skeletal muscle: in vitro biochemistry study
Glutamine synthetase, a gluco/corticoid-induced enzyme that plays a key role in glutamine synthesis, is preserved in skeletal muscle during aging but not in self-contracted muscle such as the heart.120 In contrast, other steroids, such as sex steroids (progesterone, estradiol), do not af-fect glutamine synthetase activity in either the muscle or the heart, whatever the age of animals. The activity of
glutamine synthetase in the heart is not regulated by glucocorticoids but is dependent on mineralocorticoid hormones (aldosterone).121 In brief, glutamine synthe-tase is regulated by glucocorticoids in skeletal muscle but not in the heart. An acute depletion of plasma glutamine does not modify the upregulation of muscle glutamine synthetase activity in response to fasting in either adult or aged rats.91 In old rats, there is increased glutamine synthetase activity (in 25- to 27-month-old female and 24- to 27-month-old male rats, which are very old ani-mals, since mortality at this age is about 60%), suggesting a greater need for glutamine. In some very old female rats, low glutamine synthetase activity may be associated with longevity or may reflect a limitation of glutamine production due to extremely advanced age per se.117 Impact of aging on glutamine synthesis in the liver The liver is an important organ in glutamine homeosta-sis. So, it has a particular role in nitrogen salvage, and urea production, and glutamine metabolism.122–126 It has the ability both to degrade glutamine and to synthe-size glutamine because the enzymes responsible, which are located in different hepatocyte populations, are ac-tive simultaneously. In the liver acinus (functional unit of the liver), ureagenesis and glutaminase (L-glutamine amidohydrolase; EC 3.5.1.2) activity are localized pre-dominantly in the periportal area, whereas glutamine synthetase (L-glutamate:ammonia ligase [ADP-form-ing]; EC 6.3.1.2) activity is found exclusively in a small perivenous hepatocyte population (7% of all hepato-cytes of an acinus). The pericentral expression pattern of glutamine synthetase in the liver is due to the up-stream region of this enzyme.127 Because ammonium ions at low concentration are effectively removed by glutamine synthetase, but not by urea synthesis, both pathways contribute to ammonia detoxification in the liver acinus. The liver may switch from net glutamine utilization to net production, depending on physio-logical and nutritional conditions. For this reason, it is interesting to study the regulation of glutamine metab-olism during aging. Although a large proportion of pro-teases (to which glutamine synthetase belongs) are known to be oxidatively modified with aging in the liver and neutral or alkaline proteolytic activity is maintained during aging, native glutamine synthetase remains ac-tive. Indeed, Sahakian128 demonstrated that hepatocyte glutamine synthetase decreased by 40%–50% between 3 and 26 months of age, irrespective of gender. By con-trast, it has been reported that, in fed male Wistar rats, liver glutamine synthetase activity remained constant, whatever the age of animals (2–24 months) (Figure 3). Moreover, glutaminase slightly and continuously in-creases with age (2–24 months) to become significantly
different at 24 months (Figure 3).129In contrast to these findings, Spindler130 demonstrated that glutaminase, which is a key enzyme in liver nitrogen disposal, increased in caloric-restricted aged mice. This author also reported that glutamine synthetase decreased by
about 40% in these conditions, which is in good agree-ment with the findings of Pinel et al.117It is noteworthy that glutaminase was shown to be significantly increased only at 24 months in male Wistar rats, but at this age, 50% of them died.117
Figure 2 Hypothetical fluxes of both glutamine and glutamate through the skeletal muscle to the plasma. Abbreviations: aKG, a-keto-glutarate; Gln, glutamine; Glu, glutamate; GS, glutamine synthetase; TCA, tricarboxylic acid.
Figure 113C magnetic resonance spectrometry study of skeletal muscle from adult (8 mo) and aged (25 mo) rats: (A) Kinetics of in vivo spectra between 24 and 144 min of [2-13C] acetate perfusion, and (B) in vitro spectrum obtained at the end of [2-13C] acet-ate perfusion (V. Mezzarobba, D. Meynial-Denis, and J.P. Renou, unpublished data, 2000). Abbreviations: Ac, acetacet-ate; bOHC2, b-hydroxybuty-rate; Glu, glutamate; Gln, glutamine; PCr/Cr, ratio of phosphocreatine to creatine; ppm, parts per million (MRS unit); S, unknown resonance; MRS, magnetic resonance spectroscopy; T, unknown resonance.
Impact of aging on glutamine synthesis in the gut Glutamine may have gut-protective effects. Since the ex-pression of Schlafen 3, a negative regulator of cellular proliferation, decreases by 8- to 10-fold in the colonic mucosa of aged rats,131it would be interesting to study the effect of glutamine on the expression of the Schlafen 3 gene. Glutamine also contributes to the suppression of the tricarboxylic acid cycle as an oxidative and syn-thetic pathway with aging in jejunal epithelial cells.132
In healthy humans, approximately 10%–15% of glu-tamine taken up by the intestines is converted to citrul-line.133,134 Quantitatively, glutamine is considered a major precursor of intestinal citrulline release.47,52,135,136 The intestines consume glutamine at a rate that is de-pendent on glutamine supply. Because glutamine break-down in the gut produces citrulline, there is a good relation between the amount of metabolically active gut tissue and the production of citrulline in this tissue.46,137,138
To investigate whether the regulation of glutamine metabolism remains the same throughout aging, the production of citrulline from glutamine in the gut of
old female rats was studied. Glutamine was used as long-term intermittent supplementation to evaluate the state of the gastrointestinal tract with advanced age (see section below, Long-term supplementation in rats).59
De novo glutamine synthesis plays a major role in the maintenance of intracellular glutamine pools in Caco-2 cells. In this model, glutamine availability affects the rates of protein synthesis. Caco-2 cells represent a model of human intestine enterocytes because the cell line is of human origin and originally derives from a colon carcinoma; these cells undergo enterocytic differ-entiation in vitro and share many characteristics with normal human enterocytes. Glutamine deprivation in Caco-2 cells is achieved either by maintaining the cell in glutamine-free medium or by using methionine sulfoxi-mine, an inhibitor of glutamine synthetase.36
In brief, recent studies highlighted a critical role for glutamine in enterocytes, namely the activation of the mTOR signaling pathway. In catabolic states, glutamine has been reported to enhance intestinal and whole-body growth, to promote enterocyte proliferation and sur-vival, and to regulate intestinal barrier function.139 Thus, glutamine holds great promise in protecting the
Figure 3 Effect of advanced age on interorgan glutamine flow between muscle, blood, and liver in Wistar rats. Glutamine orientation toward the gut and immune cells was not studied. Young rats were aged 2 months and very old rats 27 months. Muscle is the principal organ of glutamine synthesis. Glutamine is released from muscle into the blood. The liver uptakes glutamine and metabolizes it in urea, which is released into the blood. Adapted from Mouchard et al.,129Pinel et al.,117Meynial-Denis et al.,91and Mezzarobba et al.118Abbreviations: Gln,
glutamine; Glu, glutamate; NH41, ammonium; TCA, tricarboxylic acid.
gut from atrophy and injury in mammals and in humans during aging.
Impact of aging on glutamine metabolism at the cellular level
Senescence-associated changes in the metabolic pheno-type of human endothelial cells are related to glutami-nolysis, which is an important target for in vitro induction of senescence. Indeed, a prerequisite for glu-taminolysis is the overexpression of glutaminase, the first enzyme within the glutaminolytic pathway. Cell proliferation was found to correlate with glutaminase overexpression. Thus, premature senescence of human endothelial cells is induced by the inhibition of glutami-nase (demonstrated by the use of a glutamiglutami-nase inhibi-tor as 6-diaso-5-oxo-L-norleucine).140 Senescence and apoptosis act as parallel pathways by which severely damaged cells are eliminated from the body by the in-nate immune system.141 Programmed cell death path-ways are promising targets for interventions in aging and aging-related diseases. But to inhibit them, muscle atrophy that occurs with aging must be prevented.142 EFFECT OF GLUTAMINE SUPPLEMENTATION ON AGING Can glutamine supplementation have an effect on age-related muscle wasting and immunity, consequently playing a role in intestinal activity? In other words, can glutamine contribute to the well-being or the longevity of the elderly?
Short-term supplementation in rats. Glutamine was added to drinking water for 7 consecutive days each month (20% of dietary protein) after 5-day fasting to determine whether glutamine synthetase enhancement induced by fasting disappeared with glutamine supply irrespective of animal age. This process was associated with re-feeding. The effect of glutamine supplementa-tion was compared with the effect of alanine and glycine supplementation. Only glutamine supplementation was able to significantly decrease glutamine synthetase activity in the skeletal muscle of very old rats (27 months), whereas supplementation with other amino acids decreased upregulated glutamine synthe-tase activity in adult rats.143
Five-month supplementation in rats. Supplementation with glutamine was the same as that given previously but was an intermittent oral supplementation given during the last 5 months of life (7 consecutive days each month). Long-term treatment with glutamine (started before advanced age was reached) had several effects on very old rats (in good agreement with Neu et al.144): (1)
it prevented the loss of body weight; (2) it did not pre-vent the inevitable sarcopenia, regardless of nutritional state (inefficient in modifying the rates of protein turn-over); and (3) it decreased upregulated glutamine syn-thetase activity only in the fed state. Whole-body glutamine requirements in the rat may be satisfied in the fed state but would not be met during catabolic states (fasting) with advanced age. Glutamine may also have an essentially beneficial role for the gut, maintain-ing both intestinal integrity and intestinal immune function.104
Long-term supplementation in rats
Muscle. Supplementation was the same as above but was given for 50% of the rats’ lifetime. Glutamine syn-thetase activity increased in skeletal muscle with aging in very old fed and fasted animals. The enhancement of glutamine synthetase activity (1.5- to 2-fold) in 25- to 27-month-old rats may be a consequence of aging-induced stress104 and will occur if glutamine supple-mentation is not interrupted before the study begins. Long-term treatment with glutamine before advanced age but discontinued 15 days before rat sacrifice is effec-tive in increasing plasma glutamine to the same levels as those in adult rats and in maintaining plasma gluta-mine in very old rats, but it has no long-lasting effect on the glutamine synthetase activity of skeletal muscle with advanced age.145
Intestine. Glutamine was added to the drinking water of very old (27 months) female rats for 10 months of their life span for 7 consecutive days a month (20% of dietary protein; average of the 10 glutamine treatments, 0.860.1 g/rat/d).59
Long-term treatment with glutamine initiated before advanced age maintains rat body weight and has a beneficial effect on enterocytes by increasing gut mass and improving the villus height of mucosa, thereby pre-venting the gut atrophy encountered in advanced age. The mucosal enzyme activities required for citrulline synthesis in the gut are preserved in the gut of very old rats, as reported by Crenn et al.137 in humans. Therefore, citrulline can be used, for the first time, as a noninvasive marker of intestinal atrophy induced by advanced age.
Further investigations are warranted to explore the effect of very old age on this glutamine–citrulline inter-relation in the gut in vivo in humans. Intestinal atrophy with advanced age (reduction in the jejunal surface area), which has been widely documented, may contrib-ute to the frailty syndrome and, consequently, have implications for public health.59
Supplementation in humans
Although the beneficial effects of glutamine supplementa-tion were demonstrated as early as 1990, few data from healthy humans were reported.4,146 Healthy elderly subjects account for a very small part of the general popu-lation and are of interest primarily to nutritional research-ers rather than medical doctors. Medical researchresearch-ers are more interested in glutamine supplementation as a means of improving patient health. They tend to focus on the ill-ness in humans, whereas nutritional scientists place greater emphasis on prevention. The aging of the popula-tion will become an important societal quespopula-tion as the number of very old individuals increases worldwide.
The interest in glutamine has been altered in the last century because this amino acid does not combat muscle-wasting during disease or age-related sarcope-nia. For this reason, few data on the healthy elderly are reported in the literature. Nevertheless, glutamine is of continuing interest in medical research because of its potential role in improving the well-being of ill humans (see the reviews of Boelens et al.,18and Neu et al.144). Gut function in both healthy and ill old humans is incompletely understood and needs to be more fully investigated. As far as is known, there is only 1 pub-lished report about collagen synthesis after supplemen-tation with glutamine.147
CONCLUSION
Preventive nutrition should be developed in France and other countries to maintain the well-being and good health of humans as long as possible, particularly as aging progresses. Glutamine added to classical amino acid nutri-tional supplementation may contribute to the preservation of the gut by decreasing villus atrophy and maintaining gut function. Glutamine may therefore constitute an essential factor in the well-being and good health of very old individuals. In short, this review demonstrates that the function of glutamine goes beyond that of a simple meta-bolic fuel or protein precursor, as previously assumed, but instead is both a nutrient and a signaling molecule.
Acknowledgments
The author extends special thanks to Dennis Bier and Morey Haymond for their advice and encouragement.
Declaration of interest. The authors have no relevant interests to declare.
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