Glutamine metabolism in advanced age

Abstract

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.

INTRODUCTION

Glutamine is a nonessential amino acid but becomes a conditionally essential amino acid in catabolic states because 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 concentrations of glutamine in plasma reflect reduced stores in muscle, and this reduced availability of glutamine in the catabolic state seems to correlate with increased morbidity and mortality. Aging, which is characterized by reduced physical activity resulting mainly from the inevitable 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 glutamine in sarcopenia because glutamine 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 utilization). Animal data are extended to elderly people, bearing 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.

Two principal enzymes regulate intracellular metabolism of glutamine.¹⁷ The enzyme glutaminase catalyzes the hydrolysis of glutamine to glutamate, while glutamine synthetase catalyzes the synthesis of glutamine 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 glutaminase than glutamine synthetase. Skeletal muscle and lung, which synthesize and release net amounts of glutamine 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 physiological 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 presented in the first section of Table 1.^3,4,17–24 The characteristics 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 of Table 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.⁵¹ in 2013.

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 glutamate, proline, glycine, and arginine	Boelens et al. (2001),18 Buchman (2001),19 Bulus et al. (1989),20 Chiba et al. (2009),21 Coster et al. (2004),22 Epler et al. (2003),23 Lacey & Wilmore (1990),3 Smith (1990),4 Souba (1991),17 Welbourne & 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 important 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 acidotic 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),28 Curi et al. (2005)29 (2007),30 Deniel et al. (2007),31 Larson et al. (2007),35 Le Bacquer et al. (2001),36 Häussinger et al. (2001),32 Kadowaki & Kanazawa (2003),33 Krause et al. (2002),34 Matés 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),42 Roth et al. (2002),45 Roth (2007),43 (2008),44 Rutten et al. (2006),46 van de Poll et al. (2007),47,52 Watford (1993),48 Xi et al. (2011),49 Ziegler 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-κB)
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-κB, nuclear factor κB.

^aApoptotic signaling mechanisms activated in response to glutamine deprivation are cell-type specific. New findings indicate that glutamine availability is strongly related to the induction of apoptosis. Glutamine 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).

Effect of glutamine starvation on cell survival and apoptosis

The consequences of glutamine deprivation on cellular survival and gene expression have led to the construction 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 deprivation are cell-type specific. New findings indicate that glutamine availability is strongly related to the induction 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 M_r heat-shock protein, which is an important factor for cell survival.⁵⁴ Consequently, glutamine-utilizing cells possess molecular 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 administration of glutamine increases tissue concentrations of reduced glutathione. Glutamine (via glutamate), cysteine, 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 influences 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 decreases production of proinflammatory interleukins 6 and 8, enhances production of anti-inflammatory interleukin 10, and reduces expression of NF-κB 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 clinical effects. Gut mucosal homeostasis is achieved through a balance between cell proliferation and apoptosis. 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 starvation induces apoptosis through caspase activation.^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-inflammatory effect via induction of nuclear degradation of the NF-κB 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.^{27,28,31,39,42,61,62} Amino acids are important 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 nutrients (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.^43,63–65 Glutamine inhibits the activation of p70 S6 kinase and the phosphorylation of eIF-4E binding protein 1 induced by arginine and leucine in rat intestinal epithelial cells. In contrast, in healthy subjects in the fed state, enteral proteins, but not glutamine, increased protein synthesis via an mTOR-independent pathway in humans.⁶⁶ Nakajo et al.³⁸ have 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.³⁸ The 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, the autophagic pathway is responsible for the bulk of proteolysis and is most sensitive to amino acid regulation.⁶⁷ Glutamine, 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 signaling.³³ Moreover, glutamine stimulates a number of metabolic pathways of intermediary metabolism in the liver. For example, glutamine activates phosphoenolpyruvate carboxykinase (PEPCK) in the liver and increases the phosphorylation state of p70 S6 kinase, a key enzyme in liver protein synthesis. Glutamine is known to induce cell swelling and to mediate the inhibition of autophagic proteolysis; glutamine’s antiproteolytic effect may be mediated through osmotic swelling and the p38^MAPK pathway.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 functions, 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 resistance in the elderly.^5,73–77

Dysregulation of autophagy^78–80 contributes notably 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 restricting calorie intake, which may promote longevity during aging.^82–84 Moreover, glutamine inhibits autophagy and regulates cell growth.⁸⁵ The role of glutamine metabolism 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 glutamine 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-chain amino acids, especially leucine, stimulate protein synthesis, inhibit proteolysis (in cell culture and animals), and promote glutamine synthesis.^87,88 Depletion of plasma glutamine may result in loss of branched-chain amino acids and increased protein wasting.⁸⁹ When the “glutamine trap” (e.g., phenylbutyrate) is used to deplete plasma glutamine,⁹⁰ skeletal muscle glutamine is not depleted, regardless of the age of rats, and muscle glutamine synthetase is not increased.⁹¹ Despite this, there are few documented 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.^93–97 Indeed, 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 muscle⁹⁹ and may be considered as a “physiological” catabolic state. For this reason, protein and amino acids, notably glutamine, as dietary supplementation 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 leucine^100–102 (or essential amino acids^103,104) nor glutamine¹⁰⁵ fights against the loss of skeletal muscle. Protein supplementation increased muscle mass gain only during resistance-type exercise training in elderly people.^106,107 Essential 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 effect on skeletal muscle mass per se.¹⁰⁹ Thus, neither glutamine 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).

Table 2.

Effect of glutamine and other amino acids on protein turnover in skeletal muscle with aging

Supplementation	Physical activity	Whole-body improvement or preservation of muscle functionality	Change in fat body mass	Changed in lean body mass	Skeletal muscle atrophy	References
Leucine-rich whey protein or 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	Dröge (2005)109

Abbreviation: NR, not reported.

Glutamine deficiency, which stimulates cell apoptosis, may stimulate sarcopenia, thereby triggering a low-level inflammatory process.⁴³ Moreover, aging is known to induce a dysregulation of immune and inflammation functions that may affect protein synthesis rates in lymphoid tissues and plasma proteins.¹¹⁰ Aging 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 2 provides 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 transaminated to glutamate in order to increase the synthesis of glutamine, which is a substrate highly consumed by inflammatory cells such macrophages.¹¹¹ But in old rats, both the production and the release of glutamine in muscle are reduced in relation to the increased splanchnic sequestration of leucine and the reduced renal and intestinal glutamine uptake in order to maintain whole-body glutamine homeostasis.¹¹² Thus, glutamine depletion has a role in the sarcopenia and low-grade inflammation observed with aging.

Effect of aging on whole-body glutamine metabolism

A link can be made between aging and physical inactivity (experimental bed rest in healthy volunteers) since, as previously reported, inactivity contributes to sarcopenia.¹¹³ Physical activity generally decreases with age. Physical inactivityreduces whole-body glutamine availability due to downregulated de novo synthesis and, more generally, to anabolic resistance.^114,115 This alteration also results in decreased cytosolic ratios of glutamine 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.¹¹⁶ However, 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 (0.59±0.13 vs 0.55±0.15 mM in plasma and 3.21±0.60 vs 3.18±0.85 µmol/g in muscle).¹¹⁷ Advanced age in rats corresponds to an age of about 75–80 years in humans. Glutamine deficiency 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 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 using ¹³C 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 requirements of the cell. These requirements are dependent on age, as evidenced by the increase in glutamine synthetase activity with aging, reported below. Under normal healthy conditions, glutamine synthetase activity 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 significant 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, glutamine synthetase in muscle is a longevity-related gene and increases with caloric restriction in mice.²¹ Because glutamine synthetase activity increases with fasting, whatever the age of animals,¹¹⁸ glutamine synthesis was compared directly in skeletal muscle from 2-[¹³C] acetate 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, unpublished data, 2000). In old rats, glutamine and glutamate 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 glutamate through skeletal muscle were too quick to be detected 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.

Figure 1.

¹³C 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] acetate perfusion (V. Mezzarobba, D. Meynial-Denis, and J.P. Renou, unpublished data, 2000). Abbreviations: Ac, acetate; βOHC2, β-hydroxybutyrate; 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.

Figure 2.

Hypothetical fluxes of both glutamine and glutamate through the skeletal muscle to the plasma. Abbreviations: αKG, α-ketoglutarate; Gln, glutamine; Glu, glutamate; GS, glutamine synthetase; TCA, tricarboxylic acid.

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 affect 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 synthetase 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 animals, 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 homeostasis. 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 synthesize glutamine because the enzymes responsible, which are located in different hepatocyte populations, are active simultaneously. In the liver acinus (functional unit of the liver), ureagenesis and glutaminase (L-glutamine amidohydrolase; EC 3.5.1.2) activity are localized predominantly in the periportal area, whereas glutamine synthetase (L-glutamate:ammonia ligase [ADP-forming]; EC 6.3.1.2) activity is found exclusively in a small perivenous hepatocyte population (≈7% of all hepatocytes of an acinus). The pericentral expression pattern of glutamine synthetase in the liver is due to the upstream 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 physiological and nutritional conditions. For this reason, it is interesting to study the regulation of glutamine metabolism during aging. Although a large proportion of proteases (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 active. Indeed, Sahakian¹²⁸ demonstrated that hepatocyte glutamine synthetase decreased by 40%–50% between 3 and 26 months of age, irrespective of gender. By contrast, 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 increases with age (2–24 months) to become significantly different at 24 months (Figure 3).¹²⁹ In 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 agreement with the findings of Pinel et al.¹¹⁷ It 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 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.,¹²⁹ Pinel et al.,¹¹⁷ Meynial-Denis et al.,⁹¹ and Mezzarobba et al.¹¹⁸ Abbreviations: Gln, glutamine; Glu, glutamate; NH4+, ammonium; TCA, tricarboxylic acid.

Impact of aging on glutamine synthesis in the gut

Glutamine may have gut-protective effects. Since the expression of Schlafen 3, a negative regulator of cellular proliferation, decreases by 8- to 10-fold in the colonic mucosa of aged rats,¹³¹ it 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 synthetic pathway with aging in jejunal epithelial cells.¹³²

In healthy humans, approximately 10%–15% of glutamine taken up by the intestines is converted to citrulline.^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 dependent on glutamine supply. Because glutamine breakdown 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 differentiation 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 sulfoximine, 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 survival, and to regulate intestinal barrier function.¹³⁹ Thus, glutamine holds great promise in protecting the 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 phenotype of human endothelial cells are related to glutaminolysis, which is an important target for in vitro induction of senescence. Indeed, a prerequisite for glutaminolysis 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 glutaminase (demonstrated by the use of a glutaminase inhibitor 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 innate immune system.¹⁴¹ Programmed cell death pathways 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 supplementation 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 synthetase 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 prevent the inevitable sarcopenia, regardless of nutritional state (inefficient in modifying the rates of protein turnover); and (3) it decreased upregulated glutamine synthetase 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, maintaining 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 synthetase 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 supplementation is not interrupted before the study begins. Long-term treatment with glutamine before advanced age but discontinued 15 days before rat sacrifice is effective in increasing plasma glutamine to the same levels as those in adult rats and in maintaining plasma glutamine 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, 0.8±0.1 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 preventing 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 interrelation in the gut in vivo in humans. Intestinal atrophy with advanced age (reduction in the jejunal surface area), which has been widely documented, may contribute to the frailty syndrome and, consequently, have implications for public health.⁵⁹

Supplementation in humans

Although the beneficial effects of glutamine supplementation 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 population and are of interest primarily to nutritional researchers rather than medical doctors. Medical researchers are more interested in glutamine supplementation as a means of improving patient health. They tend to focus on the illness in humans, whereas nutritional scientists place greater emphasis on prevention. The aging of the population will become an important societal question 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 sarcopenia. 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.,¹⁸ and 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 published report about collagen synthesis after supplementation 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 nutritional 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 metabolic fuel or protein precursor, as previously assumed, but instead is both a nutrient and a signaling molecule.