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.
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.
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.