Molecular mechanisms of glutamine action

Authors

  • R. Curi,

    Corresponding author
    1. Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil
    • Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, Avenue Prof. Lineu Prestes, 1524, 05508-900, Butantan, São Paulo, SP, Brazil.
    Search for more papers by this author
  • C.J. Lagranha,

    1. Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil
    Search for more papers by this author
  • S.Q. Doi,

    1. Department of Medicine, Uniformed Services University of Health Sciences, Bethesda, Maryland
    Search for more papers by this author
  • D.F. Sellitti,

    1. Department of Medicine, Uniformed Services University of Health Sciences, Bethesda, Maryland
    Search for more papers by this author
  • J. Procopio,

    1. Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil
    Search for more papers by this author
  • T.C. Pithon-Curi,

    1. Camilo Castelo Branco University, São Paulo; Faculty of Health Sciences, Methodist University of Piracicaba, Piracicaba, SP, Brazil
    Search for more papers by this author
  • M. Corless,

    1. Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland
    Search for more papers by this author
  • P. Newsholme

    1. Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland
    Search for more papers by this author

Abstract

Glutamine is the most abundant free amino acid in the body and is known to play a regulatory role in several cell specific processes including metabolism (e.g., oxidative fuel, gluconeogenic precursor, and lipogenic precursor), cell integrity (apoptosis, cell proliferation), protein synthesis, and degradation, contractile protein mass, redox potential, respiratory burst, insulin resistance, insulin secretion, and extracellular matrix (ECM) synthesis. Glutamine has been shown to regulate the expression of many genes related to metabolism, signal transduction, cell defense and repair, and to activate intracellular signaling pathways. Thus, the function of glutamine goes beyond that of a simple metabolic fuel or protein precursor as previously assumed. In this review, we have attempted to identify some of the common mechanisms underlying the regulation of glutamine dependent cellular functions. © 2005 Wiley-Liss, Inc.

Glutamine is the most abundant free amino acid in the body, and has its primary source in skeletal muscle, from where it is released into the bloodstream and transported to a variety of tissues (Young and Ajami, 2001; Newsholme et al., 2003a,b). Intracellular glutamine concentration varies between 2 and 20 mM (depending on cell type) whereas its extracellular concentration averages 0.7 mM (Newsholme et al., 2003b). Glutamine plays an essential role, promoting and maintaining function of various organs and cells such as kidney (Conjard et al., 2002), intestine (Lima et al., 1992; Ramos Lima et al., 2002), liver (de Souza et al., 2001), heart (Khogali et al., 2002), neurons (Mates et al., 2002), lymphocytes (Curi et al., 1986, 1999), macrophages (Newsholme et al., 1986), neutrophils (Garcia et al., 1999; Pithon-Curi et al., 2002a, 2003b; Pithon-Curi et al., 2003), pancreatic β-cells (Skelly et al., 1998), and white adipocytes (Curi et al., 1987; Kowalchuk et al., 1988). At the most basic level, glutamine serves as important fuel in these cells and tissues. A high rate of glutamine uptake is characteristic of rapidly dividing cells such as enterocytes, fibroblasts, and lymphocytes (Wiren et al., 1998; Curi et al., 1999) where glutamine is an important precursor of peptides and proteins, as well as of amino sugars, purines, and pyrimidines, thus participating in the synthesis of nucleotides and nucleic acids (Szondy and Newsholme, 1989; Szondy and Newsholme, 1990; Boza et al., 2000). Glutamine metabolism additionally provides precursors for the synthesis of key molecules, such as glutathione (GSH) (Higashigushi et al., 1993; Roth et al., 2002). Flaring et al. (2003) showed that glutamine supplementation attenuates glutathione depletion in human skeletal muscle following surgical trauma. Recently, Brennan et al. (2003) have demonstrated that glutamine metabolism in the pancreatic β-cells are related to optimal glutathione production via the gamma-glutamyl cycle and hence influences insulin secretion. Unpublished work by some of the authors of this review has recently demonstrated that the effect of glutamine on gene expression in the pancreatic β-cell was specific and approximately 1% of 10,000 genes assessed by micro-array techniques were altered on addition of glutamine. As changes in gene expression will impact on cell function then a change in glutamine concentration in vivo will alter many clinical parameters. For example, plasma glutamine concentration decreased by up to 50% in patients with HIV, severe burns, sepsis or post-surgery (Lacey and Wilmore, 1990; Smith and Wilmore, 1990), which was correlated with patient outcome. In cultures of neutrophils recovered from burnt and post-operative patients, addition of glutamine augmented the in vitro bacterial killing activity (Furukawa et al., 1997) and was also important to the production of reactive oxygen species (Garcia et al., 1999; Pithon-Curi et al., 2002a).

Thus the importance of glutamine for cell function, first recognized by Prof. Hans Krebs (reviewed in Brosnan, 2001), is now firmly established. However, Kreb's early assumptions that glutamine provided a source of respiratory fuel and nitrogen for biosynthetic reactions has been replaced by a realization that this amino acid plays diverse regulatory roles in the relevant target cells. The mechanisms underlying these diverse actions of glutamine are only now becoming clear and are discussed in the present review.

MECHANISMS OF GLUTAMINE ACTION ON CELL METABOLISM AND FUNCTION

A summary of the glutamine-regulated cell functions and the possible mechanisms involved are shown in Figure 1.

Figure 1.

Schematic representation of the glutamine-regulated cell functions and the corresponding changes in gene expression and protein activation (target molecules). PEPCK, phosphoenolpyruvate carboxykinase; ASS, argininosuccinate synthase; JNK, c-Jun-N-terminal kinase; SAPK, stress-activated protein kinase; QRS, glutaminyl-tRNA synthetase; ASK1, apoptosis signal-regulating kinase 1; α-SMA, smooth muscle cell-α-actin; α-MHC, alpha myosin heavy chain; p70S6K, p70 ribosomal protein S6 kinase; CPT-1, carnitine palmitoyl transferase I; ADSS-1, adenylosuccinate synthase; HSP25, heat shock protein 25 kDa; HSP70, heat shock protein 70 kDa; HSP72, heat shock protein 72 kDa; ERK, extracellular signal-regulated kinases; AP-1, activating protein 1; IL-6, interleukin-6; IL-1β, interleukin-1β; Fas, cell death receptor 95 kDa; FasL, cell death receptor 95 kDa ligand; CD45RO, lymphocyte cell surface marker; Bcl-2, b-cell lymphoma-2; TNF-α, tumor necrosis factor-α; IFN-γ, interferon gamma; ASCT2, transporter sodium-dependent systems isoform 2; bcl-xL, b-cell lymphoma-2-associated x protein long; bax, b-cell lymphoma-2-associated x protein membrane; bcl-xS, b-cell lymphoma-2-associated x protein short.

Cell metabolism

Respiratory fuel

Glutamine is quantitatively the most important fuel for a number of rapidly dividing cells and tissues including intestinal tissue (Windmueller, 1982; Ardawi and Newsholme, 1985, 1990; Newsholme and Carrie, 1994; Newsholme et al., 2003a,b). It is metabolized to L-alanine in intestinal epithelial cells by a route involving conversion to glutamate, then 2-oxoglutarate via glutaminase and glutamate dehydrogenase respectively, then TCA cycle conversion to malate (2-oxoglutarate, succinate, fumarate, and finally malate) followed by the action of NADP+-dependent malic enzyme to create pyruvate which undergoes amination to produce L-alanine via the action of alanine aminotransferase. In other cells, pyruvate may be converted to lactate via lactate dehydrogenase and released, or it may enter the TCA cycle via pyruvate dehydrogenase, resulting in complete oxidation of glutamine-derived carbon.

The NADH and FADH2 generated via these pathways are used for electron donation to the electron transport chain in the mitochondria and thus they promote ATP synthesis. The L-alanine produced in the intestinal epithelial cell pathway is exported to the hepatic portal vein for transport to the liver (Brosnan et al., 2001; Brosnan, 2003; Newsholme et al., 2003b).

Gluconeogenesis

Glutamine plays an important role in gluconeogenesis in liver and kidney. It can function as a substrate (Start and Newsholme, 1970; Curi, 1988) and also controls the expression and activity of phosphoenolpyruvate carboxykinase (PEPCK), a key regulatory enzyme of gluconeogenesis (Lavoinne et al., 1996). Glutamine, at low concentration (up to 5 mM) causes cell swelling and decreases PEPCK mRNA expression. However, at higher concentrations glutamine induced an increase in PEPCK transcript level (Lavoinne et al., 1996), in support of earlier work (Newsome et al., 1994) in perfused rat liver.

Several studies in humans have shown that in the post-absorptive state, glutamine is an important glucose precursor and makes a significant contribution in the addition of new carbon to the glucose carbon pool (for review see Stumvoll et al., 1999). The contribution of renal gluconeogenesis to whole-body glucose production is in the order of 20–25%, but this rate increased in humans with type II diabetes (Stumvoll et al., 1999). Alanine can additionally contribute to the supply of new carbon for gluconeogenesis. It appears that glutamine is a major gluconeogenic substrate predominantly in the kidney, whereas alanine dependent gluconeogenesis is essentially confined to the liver (de Souza et al., 2001). In support of this observation, Ikeda and Iwata (2003) confirmed gluconeogenesis from glutamine in the kidney had a significant role in whole body glucose homeostasis.

Conjard et al. (2002), in a study with renal cortex isolated from fed and starved mice and incubated with glutamine, showed that the uptake of this amino acid and production of ammonium ions are similar in both groups. However, in starved mice, glucose production from glutamine was greatly stimulated as shown by the increase in activity and mRNA levels of glucose-6-phosphatase and especially of PEPCK, but not of fructose-1,6-biphosphatase. A study to address the effect of glutamine on expression of important enzymes of gluconeogenesis in renal cortex still remains to be carried out.

Urea cycle

Glutamine is an important substrate of urea cycle. This amino acid, via glutaminase, provides the first “N” atom for urea synthesis. Quillard et al. (1996, 1997) showed that glutamine increases argininosuccinate synthase (ASS) activity and expression in cultured hepatocytes from fetal and adult rats. ASS converts citrulline to arginosuccinate. As demonstrated by Husson et al. (2003), glutamine can increase ASS mRNA expression in Caco-2 cells (a human colon intestine cell line) and this effect does not involve cell swelling. Glutamate derived from glutamine can give rise to N-acetyglutamate formation, an allosteric activator of carbamoyl phosphate synthetase (CPS) in the liver. This is a key enzyme of urea cycle that converts ATP, bicarbonate and ammonium ions into carbamoyl-phosphate.

Lipogenesis

Glutamine carbon is utilized as a precursor for lipid synthesis in adipocytes (Kowalchuk et al., 1988). Fatty acids produced from glutamine are incorporated in to triacylglycerol in incubated adypocytes (Curi et al., 1999). Recently, Rumberger et al. (2003) showed that glutamine (16 mM) potentiated the glucose dependent increase in fatty acid synthase (FAS) and glycerophosphate dehydrogenase (GPDH) mRNA in cultured primary rat adipocytes. These authors suggested that products of glutamine metabolism, such as glucosamine-6-phosphate, were important for glucose regulation of FAS and GPDH. Thus, glutamine is an important substrate for lipid synthesis and contributes to the regulation of the expression of key enzymes of this biosynthetic pathway.

Stimulation of anabolic pathways

Hickson et al. (1995) have shown that glutamine infusion is an effective therapy in counteracting glucocorticoid-induced muscle atrophy by preventing the decline in myosin heavy chain synthesis.

Evidence was obtained by Boza et al. (2001) that glutamine supplied as a free amino acid in the diet can induce muscle protein synthesis after dexamethasone treatment. The authors postulated that free glutamine or glutamine-rich protein supplementation were equally effective in increasing protein synthesis in the jejunum, and suggested that this is the main benefit of glutamine supplementation in enteral nutrition formulas. Other researchers, however, did not find the same effects. Gore and Wolfe (2002) studying the effect of enterally administered glutamine on muscle protein synthesis in critically ill patients found that glutamine kinetics was not affected. Incorporation, release, and rate of de novo synthesis of glutamine in muscle were not altered by exogenous glutamine, suggesting a possible restriction in the transport of glutamine into muscles of those patients.

Glutamine transport into the human hepatoma cell line HepG2 is catalyzed primarily by an ASCT2-type transporter (Broer et al., 1999; Pollard et al., 2002). Bungard and McGivan (2004) found that variation in cell growth rate did not affect ASCT2 expression, but both growth rate and ASCT2 expression were significantly reduced by glutamine deprivation. Expression of a number of other proteins was shown to be unaffected under these conditions. The authors postulated that both ASCT2 promoter activity and ASCT2 protein expression in these cells are dependent on glutamine availability.

In rat hepatocytes, Na+-co-transported amino acids like glutamine stimulates lipogenesis and glycogen synthesis by activation of acetyl-CoA carboxylase (ACC) and glycogen synthase (GS), respectively (Lavoinne et al., 1987). Krause et al. (2002) have documented a time dependent activation of ACC and GS as well as an increase in phosphorylation of p70S6K in hepatocytes, and suggested that the activation of ACC, GS and the p70S6K resulted from an anabolic response of the liver to glutamine.

Cell swelling

The mechanism through which glutamine activates some enzymes of anabolic pathways may involve glutamine-induced cell swelling. The transport of glutamine into hepatocytes via a sodium-dependent transporter causes an osmotic swelling of the cells (Lavoinne et al., 1998). In fact, cell swelling is now regarded as a novel regulatory element of hepatic metabolism (Husson et al., 1996; Lavoinne et al., 1998).

Stoll et al. (1992) have shown that glutamine stimulated synthesis of DNA, RNA, and proteins in hepatocytes, and that these anabolic effects are regulated by changes in cell volume. Specifically, glutamine exerted a dose-dependent regulation of β-actin gene expression at the transcriptional level, while regulation of PEPCK gene expression was achieved by stabilizing its mRNA.

Cell proliferation

Glutamine plays an important role in cell proliferation. This effect of glutamine has been observed in a variety of cell types including lymphocytes (Szondy and Newsholme, 1991; Newsholme and Calder, 1997; Yaqoob and Calder, 1997; Calder and Yaqoob, 1999), enterocytes (Wiren et al., 1998; Boza et al., 2000; Yamauchi et al., 2002), and tumor cells (Obrador et al., 2001; Mates et al., 2002).

Yamauchi et al. (2002) showed that the proliferation of Caco-2 cells was increased by nucleoside, nucleotide, and glutamine supplementation, but not by glutamate. Arginine potentiated the effect of glutamine. The level of nucleotide synthesis from glutamine, as indicated by N15 incorporation from L-[5-N15]-glutamine, was increased by arginine supplementation and decreased by nucleoside and nucleotide supplementation. These findings suggest that the effects of glutamine and arginine on Caco-2 cell proliferation are mediated by the stimulation of nucleotide synthesis, and that the major role of glutamine in this process was not energy supply.

The synthesis of both purines and pyrimidines was stimulated by the administration of extracellular glutamine. The conversion of glutamine phosphorybosylpyrophosphate by amidophosphorybosyltransferase from ribose-5-phosphate is the first step of the de novo synthesis of purines. De novo synthesis of pyrimidines begins with the production of carbamyl phosphate from glutamine, carbon dioxide, and adenosine triphosphate. Carbamyl phosphate is then converted to carbamyl aspartate by aspartate transcarbamylase (Engstrom and Zetterberg, 1984).

Rhoads et al. (1997) have shown that glutamine activates both ERKs and JNKs (extracellular signal regulated kinase and jun kinase, respectively), proteins involved in signal transduction pathways stimulated by growth factors in IEC-6 (epithelial cells from rat small intestine) and IPEC-J2 (porcine intestine epithelial cell line) cells, resulting in an increase in AP-1 dependent gene transcription and c-Jun mRNA levels. AP-1 and c-Jun are transcription factors that regulate the expression of genes involved in cell division. Glutamine has, therefore, been postulated to potentiate the effects of growth factors on cell proliferation and repair.

Glutamine can stimulate expression of adenylosuccinate synthase (ADSS-1), which can regulate cell proliferation via activation of protein kinase A and mTOR in neonatal rat cardiomyocytes (Xia et al., 2003). The latter is an important intracellular transducer of a growth-related signaling pathway, which is rapamycin-sensitive and dependent on activation of a 70 kDa S6 kinase (p70S6K) (Xia et al., 2003). The S6 phosphorylation is known to be required for the translation of the terminal oligopyrimidine family of RNAs that contain an oligopyrimidine tract upstream of their transcription-initiation site. These messengers encode proteins belonging to the protein-translation machinery (Van Sluijters et al., 2000).

Protein synthesis and degradation

Contractile protein synthesis

Glutamine may serve as an energy source also for the heart. Xia et al. (2003) have demonstrated that glutamine can induce cardiomyocyte growth and maturation accompanied by an increase in mRNA levels of contractile proteins including α-myosin heavy chain (α-MHC) and cardiac α-actin. This was associated with physiological hypertrophy. No effect of glutamine on markers of pathological hypertrophic response such as β-myosin heavy chain (β-MHC), skeletal α-actin, and atrial natriuretic factor was observed.

Extracellular matrix (ECM) formation

Bellon et al. (1987) have shown that glutamine increases the synthesis of collagen in human fibroblasts by a direct stimulatory effect and as a precursor of proline and hydrooxyproline residues. The direct effects of glutamine on collagen biosynthesis included a dose-dependent increase in transcription and mRNA steady-state of α 1(I) and α 1(III) collagen (Bellon et al., 1995). The response of fibroblasts with respect to collagen synthesis and mRNA reached a maximal level at glutamine concentrations between 0.15 and 0.25 mM, and did not change further up to 10 mM. The authors postulated that the selection of glutamine for protein synthesis takes place in close conjunction with the amino acid transport system. The effect of glutamine on collagen gene expression appeared to be specific as analogs and/or derivatives of glutamine, such as acivicin, homoglutamine, ammonium chloride, and glutamate were unable to produce the same effect. Karna et al. (2001) showed that intermediates of glutamine interconversion, glutamate and pyrroline-5-carboxylate (P5C), stimulate collagen biosynthesis in cultured skin fibroblast cells. P5C was found to be a potent stimulator of collagen biosynthesis, whereas glutamate stimulated type I procollagen expression.

Routh et al. (2002) studied the effect of troglitazone, a peroxisome proliferator-activated receptor-gamma agonist that has been shown to inhibit mesangium expansion in experimental type 2 diabetes. They found that the reduction in glutamine utilization and alanine formation induced by troglitazone is associated with a decrease in monolayer collagen-glycosaminoglycan content. In spite of the reduced glutamine uptake, ammonium formation did not decrease. This is consistent with an increased glutamate flow through the deamination pathway.

Pithon-Curi et al. (2001) have investigated the role of glutamine in the synthesis of ECM proteins in cultured mesangial cells. Glutamine at 2 mM elicited an increase in α1-type IV collagen and fibronectin transcripts compared to control cells in absence of glutamine. A concomitant marked increase in smooth muscle cell α-actin (α-SMA) transcripts accompanied by an increase in α-SMA stress fibers was detected. Elevated expression of α-SMA in mesangial cells has been proposed as a marker of cell activation, and frequently precedes the increase in ECM protein production. These findings support the hypothesis that glutamine leads mesangial cells to produce pro-sclerotic markers.

Hyperglycemia induces marked changes in mesangial cell function and ECM protein accumulation as seen in diabetic glomerulopathy (Isono et al., 2000; Singh et al., 2004). The hexosamine biosynthesis pathway is implicated in mediating several metabolic effects of high glucose and glutamine in cells. Singh et al. (2001) showed that metabolism of glucose through the hexosamine biosynthesis pathway mediates the effects of glucose on ECM (fibronectin) synthesis and transcription factor phosphorylation in SV-40-transformed rat mesangial cells. UDP-N-acetyl-glucosamine is the end product of the hexosamine biosynthesis pathway and serves as a precursor for O-linked serine/threonine glycosylation of cytoplasmic and nuclear proteins.

Mesangial cells cultured in the presence of high glucose and glucosamine show high levels of O-N-acetylglucosamine in several cytoplasmic and nuclear proteins (Singh et al., 2003). Inhibition of O-glycosylation by benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside blocks both high glucose and glucosamine-induced fibronectin synthesis and CREB phosphorylation. In addition, mesangial cells exposed to high glucose exhibited an elevated GFAT expression, and became more sensitive to the glucose effects on fibronectin expression and CREB phosphorylation than control cells. The authors then postulated that the hexosamine biosynthesis pathway works as a glucose sensor and mediates at least some of the effects of hyperglycemia in the diabetic kidney (Singh et al., 2003).

Protein degradation

In organs such as the liver and the muscle, protein degradation results from the activity of the three major systems: the lysosomal (cathepsins), the Ca2+-activated (calpains), and the ATP-ubiquitin-dependent proteolytic pathways (Samuels et al., 1996) These pathways have also been reported to occur in rat intestinal mucosa. Enteral glutamine stimulates mucosal protein synthesis and attenuates ubiquitin-dependent proteolysis improving protein balance in human gut. The mRNA level of ubiquitin was significantly decreased by glutamine supplementation (0.8 mmol kg−1 h−1) while cathepsin D and m-calpain mRNA levels were not affected (Coeffier et al., 2003).

Cell defense and repair

Heat shock proteins (HSP)

Heat shock at 43°C induces intestinal epithelial cell death, as reflected by a marked increase in floating cell count. In the absence of glutamine supplementation, the percentage of floating cells reached 90%. Supplementation of glutamine at concentrations higher than 1 mM causes a dose-dependent decrease in the percentage of floating cells (Chow and Zhang, 1998).

Glutamine is a potent enhancer of heat shock protein 72 (HSP72) expression in vitro and in vivo (Wischmeyer, 2002). The induction of a heat shock response can attenuate pro-inflammatory cytokine release (Cahill et al., 1996; Yoo et al., 2000). HSP may downregulate cytokine expression binding to the heat shock element present in the promoter region of interleukin-1β (IL-1β) and potentially of other cytokines, a process that results in downregulation of cytokine expression (Cahill et al., 1996).

Constitutive shock cognate protein 73 is not altered by glutamine, demonstrating that this amino acid exerts a specific effect on inducible stress proteins rather than increasing overall protein synthesis. In contrast, glutamine in a concentration equivalent to that found in normal plasma markedly increases HSP72 expression in mononuclear cells following LPS treatment (Wischmeyer et al., 2003).

Naka et al. (1996) examined the effect of an intravenous glutamine dipeptide administration on septic rats and found that mortality was significantly lower in the glutamine-total parenteral nutrition group than in animals receiving conventional diet. Human studies have reported that glutamine-treated patients experience fewer clinical infections and shorter hospital stays (Houdijk et al., 1998; Morlion et al., 1998). Therefore, some investigators have suggested that glutamine may be useful in the treatment of established infections or inflammation (Wilmore and Shabert, 1998).

Glutathione depletion in skeletal muscle is pronounced following major trauma and sepsis in intensive care unit patients (Tischler and Fagan, 1982; Luo et al., 1996) Flaring et al. (2003) have shown that intravenous glutamine supplementation attenuates glutathione depletion in skeletal muscle in humans following standardized surgical trauma.

Preoperative administration of glutamine induces HSP70 expression and attenuates cyclic AMP response element-binding protein (CREBP)-induced inflammation by regulating nitric oxide synthase (NOS) activity (Hayashi et al., 2002). This may be a useful strategy to confer tolerance to CREBP-induced inflammatory response through a self-protective mechanism (Hayashi et al., 2002). Wischmeyer et al. (2001) showed that in addition to HSP72 glutamine also enhances HSP25 expression in multiple organs both stressed and unstressed animals. These authors suggested that glutamine could be utilized to induce a protective stress response and to prevent organ injury under stressful conditions.

Redox potential

Glutamine is required for glutathione synthesis as it can be metabolized by the gamma-glutamyl cycle to produce glutathione. Glutathione is produced from glutamate, glycine, and cysteine (Mates et al., 2002). Glutathione is present in the cell in both reduced (GSH) and oxidized (GSSG) forms. The ratio of GSH to GSSG is the main regulator of the cellular redox potential (Wernerman and Hammarqvist, 1999; Mates et al., 2002). Addition of glutamine to cells in vitro can lead to an increase in total glutathione concentration (Mates et al., 2002; Brennan et al., 2003). Glutamine metabolism via entry into the TCA cycle may allow action of malic enzyme (NADP+ dependent), which will result in an increase in NADPH production. This will subsequently increase the GSH/GSSG ratio. Studies performed by Roth et al. (2002) have shown that mice fed with glutamine exhibit an increase in the cellular content of reduced glutathione (GSH).

The synthesis of a number of pro-inflammatory cytokines depends on the activation of the transcription factor NF-κB, which in turn depends on the cellular redox potential and consequently is regulated by the intracellular GSH:GSSG ratio. The possible involvement of glutamine in NF-κB regulated cytokine synthesis, however, remains to be clarified.

Apoptosis

Evidence has been accumulated that glutamine influences apoptosis-related cellular mechanisms. Hyperosmotic Fas (APO-1/CD95) targeting to the plasma membrane is diminished by glutamine and taurine in a dose-dependent manner (Reinehr et al., 2002). Fas belong to the subfamily of death receptors and play a major role in activation-induced cell death. In rat hepatocytes exposed to normal osmosis, Fas is found predominantly in intracellular localization, whereas under hyperosmotic conditions Fas is transferred to the plasma membrane.

Apoptosis can be induced in HeLa cells by treatment with anti-Fas antibody. In glutamine-free medium, HeLa cell apoptosis increases in a dose-dependent manner with anti-Fas antibody, whereas cells in the presence of glutamine (4 mM) are not sensitive to Fas ligand (Ko et al., 2001). MAPK/JNK pathways is involved in anti-Fas induced HeLa cell apoptosis. In fact, phosphorylation of ERK occurs at 10 min following anti-Fas antibody treatment regardless the presence of glutamine. However, Fas ligand does not activate JNK/SAPK cascade in the presence of glutamine. In glutamine-starved HeLa cells, JNK/SAPK activity is markedly increased by Fas stimulation (Ko et al., 2001). JNK/SAPK induction by Fas ligand is mediated through ASK1 (a critical protein kinase in apoptosis; Chang et al., 1998), which is activated after Fas ligand treatment only in the absence of glutamine. These observations suggest that glutamine suppresses ASK1 and JNK/SAPK activation by Fas ligand (Ko et al., 2001).

Human glutaminyl-tRNA synthetase (QRS) is one of the enzymes that utilize free glutamine (Ko et al., 2001). QRS is not only a key enzyme for cell proliferation but also plays a regulatory role in cell death through an antagonistic interaction with ASK1 (Ko et al., 2001). The authors studied the effect of glutamine on the molecular interaction of QRS with ASK1 in HEK-293 cells (human embryo kidney cell line). The expression level of QRS and ASK1 was not affected by glutamine, but the molecular interaction between these two proteins was significantly increased in cells cultured in the presence of glutamine. QRS and ASK1 interaction can also be intensified by addition of 20 mM glutamine to the immuno precipitation buffer, even when cells were cultured in absence of glutamine.

T cell death is considered to be critically important for maintenance of T-cell homeostasis and deletion of self-reactive T-cells. This pathway requires interaction between Fas and Fas ligand (FasL/CD95L) (Van Parijs and Abbas, 1996). On the other hand, expression of the Bcl-2 (an anti-apoptotic protein) can rescue T cells from apoptosis (Van Parijs and Abbas, 1998). Chang et al. (2002) have shown that glutamine significantly down-regulates the expression of Fas and FasL but up-regulates the expression of CD45RO and Bcl-2 in Jurkat T cells (human T-lymphocyte cell line). In addition, glutamine significantly decreased both caspase-3 and caspase-8 activities in PMA-ionomycin stimulated Jurkat T cells. These results suggest that glutamine may protect activated T cells from apoptosis partially by up-regulating the expression of Bcl-2 and inhibiting Fas.

Voehringer et al. (1998) have found that T lymphocytes undergoing apoptosis are depleted of reduced glutathione coinciding with the onset of chromatin fragmentation. In contrast, augmentation of intracellular GSH is sufficient to reduce the Fas-triggered increase in apoptosis. Overexpression of Bcl-2 causes accumulation of glutathione in the nucleus, thereby altering the nuclear redox state and blocking caspase activity and other nuclear features of apoptosis.

The endogenous concentration of various metabolites was determined in human neutrophils undergoing apoptosis (Nunn et al., 1996). The endogenous concentration of lactate and glutamine was reduced, whereas that of arginine, glycine, alanine, aspartate, and glutamate was not modified (Nunn et al., 1996). The authors postulated that glutamine utilization might be increased in apoptotic neutrophils.

We have investigated nuclear, mitochondrial, and plasma membrane events associated with apoptosis in rat and human neutrophils cultured in the presence or absence of glutamine (Pithon-Curi et al., 2003). Condensation of chromatin assessed by Hoechst 33342 staining was reduced in neutrophils cultured in the presence of glutamine. Annexin V binding to externalized phosphatidylserine was reduced in the presence of glutamine. In the absence of glutamine, neutrophils exhibited a marked reduction in the uptake of rhodamine 123, which was restored by the addition of glutamine. Rhodamine 123 uptake is used to monitor loss of mitochondrial transmembrane potential (Green and Reed, 1998). Similar effect was found in human neutrophils by measuring DNA fragmentation and mitochondrial transmembrane potential. Therefore, glutamine protects from events associated with triggering and executing apoptosis in both rat and human neutrophils. This protective effect of glutamine against neutrophils apoptosis was accompanied by an increase in Bcl-2 expression (Pithon-Curi et al., 2003).

The intensity and duration of exercise plays a key role in determining responses to exercise (Fielding et al., 2000; Matthews et al., 2002). In Nieman's “J-shaped model” for upper respiratory tract infection it was postulated that exercise could enhance or reduce immunity depending on the frequency, duration, and intensity of the exercise (Nieman, 1994). In the same direction, Pedersen and Ullum (1994) have proposed that there is an open window period following intensive exercise that makes the athletes susceptible to infections. Frequent intense exercise and training has been shown to impair the immune response and might increase the susceptibility to infections (Castell and Newsholme, 1996). Some authors explain the increase in susceptibility to infections due to a decrease in plasma glutamine concentration, which impairs some neutrophil functions (Smith and Wilmore, 1990; Parry-Billings et al., 1992; Keast et al., 1995; Lehmann et al., 1995; Pedersen and Hoffman-Goetz, 2000). In support of this hypothesis, Nieman (1997) showed that glutamine supplementation decreased upper respiratory tract infections in athletes.

We have found (Lagranha et al., 2004) that acute exercise leads to marked changes in expression of pro- and anti-apoptotic genes of neutrophils in mature rats (90 days old). The alterations induced by acute exercise include an increase in the expression of bax and bcl-xS expression and a significant decrease in bcl-xL expression. The effect of exercise on gene expression was not observed in neutrophils obtained from immature rats (60 days old). This suggests that the changes in the pro- and anti-apoptotic genes expression induced by exercise are dependent on sexual maturation (Lagranha et al., 2004). The same was observed for the effect of glutamine administration. Glutamine treatment (1 g kg−1 body weight) decreased bax and bcl-xS expression in neutrophils from mature rats but had not effect on cells of immature rats.

Modulation of immune function

Cytokine production

Glutamine is known to modulate immune cell function and cytokine production both in vitro and in vivo. A requirement for glutamine was also observed for the expression of key lymphocyte cell surface markers such as CD25, CD45RO, CD71, and for the production of interferon-γ and tumor necrosis factor-α (Roth et al., 2002). This topic has been reviewed by Newsholme (2001). Expression and production of TNF-α by cultured mononuclear cells stimulated with lipopolyssacharide (LPS) can be suppressed by glutamine (2–10 mM) (Pithon-Curi et al., 2002; Wischmeyer et al., 2003). Opposite effects were found for the synthesis and secretion of IL-1β and IL-6 in LPS-stimulated rat peritoneal macrophages. LPS induced a parallel increase in mRNA and synthesis of IL-1β and IL-6. The addition of glutamine increased the synthesis of both cytokines (Yassad et al., 2000). Additionally, glutamine (10 mM) increases mRNA of α-2 macroglobulin in cultured hepatocytes collected from rat fetuses (Lavoinne et al., 1998). α-2 Macroglobulin is the major positive acute-phase protein in adult rat, and its expression is under the control of interleukin-6.

Respiratory burst

The superoxide anion (Omath image) generated by NADPH oxidase serves as the starting point for the production of a number of reactive oxidants, including oxidized halogens, free radicals, and singlet oxygen (Babior, 1999). These oxidants are used by neutrophils to kill invading microorganisms, but they also cause damage to nearby tissues. Therefore, oxidant production has to be tightly regulated to ensure that they are only generated when and where required. Glutamine increases superoxide anion generation stimulated by PMA in rat neutrophils deprived of glutamine for 3 h (Pithon-Curi et al., 2002a). PMA markedly increased the expression of gp91phox, p22phox, and p47phox mRNAs. Glutamine at 2 mM increased the expression of these three proteins both in the absence and in the presence of PMA. Therefore, glutamine increased superoxide anion production in neutrophils, was partially due to regulation of the expression of the components of NADPH oxidase (Pithon-Curi et al., 2002).

Insulin secretion and action

Modulation of insulin secretion

Glutamine plays a “permissive” role in the pancreatic β-cell, enhancing glucose stimulated insulin secretion probably via metabolism involving the gamma-glutamyl cycle, glutathione synthesis, and subsequent enhancement of mitochondrial function (Brennan et al., 2003). The immediate product of glutamine metabolism, glutamate, is a powerful metabolic stimulus-secretion coupling factor in the pancreatic β-cell (Brennan et al., 2003). Recent unpublished work has demonstrated that addition of 10 mM glutamine differentially regulates the expression of approximately 140 genes by at least 1.8-fold (148 up-regulated and 18 down-regulated) related to signal transduction, metabolism, cell defense and repair and insulin secretion in a pancreatic β-cell line (Fig. 2). The metabolic genes regulated were related to fatty acid synthesis and electron transport.

Figure 2.

Glutamine-regulated genes in the pancreatic β-cell line, BRIN BD11, were related to signal transduction, metabolism, growth, channels/receptors, structural, apoptosis, immune response, and inflammatory response (only genes displaying a 1.8-fold change in expression or higher were included). Miscellaneous and expressed sequence tags (ESTs) are also displayed.

Modulation of insulin action

Traxinger and Marshall (1989) postulated that desensitization of the glucose transport system requires three components: glucose, insulin, and selected amino acids. Overall, these studies revealed that amino acids play an important role in modulating insulin action at the cellular level and provided new insights into the metabolic mechanisms mediating insulin resistance in the glucose transport system (Traxinger and Marshall, 1989). The primary amino acid modulating the glucose-induced loss of maximal insulin responsiveness was glutamine (Traxinger and Marshall, 1989, 1991, 1992; Marshall et al., 1991).

A later study demonstrated that hexosamine, a product of glucose and glutamine metabolism, was involved in the induction of insulin resistance. Azaserine and 6-diaxo-5-oxo-norleucine, the glutamine analogs that irreversibly inactivate glutamine-requiring enzymes, such as glutamine:fructose-6-amidotransferase (GFAT), the first and the rate-limiting enzyme of hexosamine biosynthesis, inhibit insulin-desensitization in cultured adipocytes. Glucosamine, an agent known to preferentially enter the hexosamine pathway at a point distal to enzymatic amidation by GFAT, effectively desensitizes the glucose transporter system in adipocytes in a dose-dependent manner. The authors also found that glucosamine was 40 times more potent than glucose in mediating desensitization, did not require glutamine for its desensitizing action and was able to induce desensitization in the presence of azaserine (Marshall et al., 1991).

Glutamine in association with glucose and insulin can increase activity and mRNA levels of pyruvate kinase (Traxinger and Marshall, 1992). Azaserine was able to prevent the increase in pyruvate kinase in a dose-dependent manner. However, azaserine was unable to prevent glucosamine-induced increase in pyruvate kinase activity, which is expected since glucosamine enters the hexosamine pathway at a point distal to the action of GFAT (Traxinger and Marshall, 1992).

Wu et al. (2001) have shown that GFAT is present in endothelial cells and that the hexosamine synthesis increased with the extracellular concentration of glucose and glutamine. In addition, high concentrations of glucose and glutamine increased GFAT activity. These findings may have implications for poorly controlled patients with diabetes mellitus as these patients display elevated plasma concentrations of both glucose and glutamine. Glucosamine, the main bioproduct of the hexosamine biosynthetic pathway, inhibits nitric oxide synthesis in endothelial cells. Moreover, increasing extracellular concentrations of both glutamine and glucose resulted in decreased nitric oxide production by endothelial cells (Giugliano et al., 1997; Okuda et al., 1997; Cosentino and Luscher, 1998; Mitchell et al., 2000), suggesting that the glucosamine biosynthetic pathway mediates the inhibition of endothelial nitric oxide synthesis induced by hyperglycemia and high plasma glutamine levels (Wu et al., 2001).

We have previously reported that glutamine increased mouse mesangial cell proliferation (Lagranha et al., 2001). Our data suggested that glutamine potentiated the level of glucose-induced mesangial cell proliferation via activation of the GFAT metabolic and MAPK signaling pathways. As several complications of diabetes mellitus including those leading to diabetic glomerulosclerosis appear to be mediated via the GFAT and MAPK pathways, we suggest that glutamine acting synergistically with elevated glucose may contribute to the development and progression of diabetic nephropathy (Lagranha et al., 2002). Accordingly, Schleicher and Weigert (2000) have shown that the increase in transforming growth factor-β (TGF-β1) production in mesangial cells induced by high glucose levels was abolished following inhibition of the GFAT pathway. This suggests that the GFAT pathway at least in part mediates the hyperglycemia-induced production of TGF-β, a prosclerotic cytokine involved in the development of diabetic nephropathy.

Recently, Weigert et al. (2003) reported that the hexosamine pathway-mediated induction of TGF-β1 synthesis in mesangial cells is dependent on GFAT enzyme activity. This study suggested that the hexosamine pathway increases transcriptional activity of nuclear proteins leading to an enhancement of cytokine biosynthesis. It has been observed that a stable overexpression of GFAT increased the levels of TGF-β1 protein, mRNA, and promoter activity and that these effects appear to be transduced by PKC. Involvement of the hexosamine pathway in hyperglycemia-induced production of cytokines (TGF-α and basic fibroblast growth factor-bFGF) has also been demonstrated in vascular smooth muscle cells (Schleicher and Weigert, 2000). These studies revealed a rapid increase in GFAT activity following treatment with agents that elevate the levels of cyclic adenosine 3′,5′ monophosphate (cAMP), thus indicating that GFAT activity is tightly regulated by cAMP-dependent phosphorylation. Using immuno-histochemistry and in situ hybridization techniques, high expression of GFAT was detected in human adipocytes, skeletal muscle, vascular smooth muscle cells, and renal tubular epithelial cells (Schleicher and Weigert, 2000). Significant immunostaining for GFAT was found in glomerular cells of patients with diabetic nephropathy. These findings support the proposition that an increased flow through the hexosamine pathway, regulated by GFAT, may be causally involved in the development of diabetic vascular disease, and in particular diabetic nephropathy (Schleicher and Weigert, 2000).

CONCLUDING REMARKS

Evidence is presented herein that glutamine is involved in many processes vital to cell function and integrity. The molecular mechanisms of glutamine action remain to be elucidated but undoubtedly involve changes in gene and protein expression, protein activity, and changes in intracellular metabolite concentrations. This is best illustrated by recent published and unpublished studies by some of the authors of this review who assessed the effect of glutamine on pancreatic β-cell metabolism and function. Glutamine metabolism resulted in the generation of key stimulus secretion coupling factors including glutamate and glutathione, which indirectly stimulate ATP production and enhance insulin secretion (Brennan et al., 2003). Glutamine additionally differentially regulated the expression of genes involved in the regulation of insulin secretion including ion-channels, metabolic enzymes, and protein kinases and phosphatases. Thus, glutamine may have both acute and chronic effects on cell metabolism and function. A note of caution should be considered, however, when interpreting the described results. as with most of the cited in vitro studies very high concentrations of L-glutamine were used in cell incubations or culture. Concentrations of L-glutamine added to cell incubations generally vary between 2 and 10 mM, concentrations well in excess of physiological L-glutamine (0.7 mM) but in vitro the higher concentrations will result in optimal glutamine transport into target cells.

Therapeutically the parenteral or enteral administration of glutamine has been recommended for critically ill patients where it is known to have beneficial effects on recovery. However, this amino acid and protein hydrolyzates enriched with glutamine have been widely used by healthy individuals, in particular by athletes, to maintain immune function. Glutamine regulates the synthesis and activation of important proteins including those of the mesangial ECM, a key element in the development of glomerulosclerosis. Thus although glutamine supplementation brings about clear benefits in many situations, problematic adverse effects of the use of high concentrations cannot be fully ruled out.

Ancillary