EMERGING ROLE OF GLUTAMINE FOR CELL DEFENSE

In the past 20-30 years, many studies have reported the critical importance of glutamine to the body’s defenses, especially to cells of both the innate and adaptive immune system. However, based on results from both in vitro and in vivo studies no clear-cut trends were found with respect to cytokine secretory profiles, levels of activation, and physiological outcomes. For example, when incubated in vitro in the presence of lower levels of glutamine, identical to the lowest plasma glutamine concentration measured after some heavy sessions of exercise training (300-400 pM), lymphocytes will function normally (Newsholme 2001) as compared to lymphocytes incubated at the accepted physiological concentration (600 pM) (Hiscock and Pedersen 2002). Hence, glutamine-dependent deficiencies occur only in situations characterized by severely depressed glutamine availability, such as observed in a high-intensity sport environment (Hiscock and Pedersen 2002) or severe catabolic illnesses (Wernerman 2008).

In response to several forms of stress, cells rapidly increase glucose uptake and utilization, which may be related to the capacity to defend and respond appropriately (Moley and Mueckler 2000). Experimental evidence suggests that inhibition of both glycolysis and the hexosamine biosynthetic pathway (HBP) results in decreased cell survival (Zachara et al. 2004). HBP are vital components for maintaining integrity and function of mucosal surfaces, and under stress situations they appear to be part of an early cellular protective response. It appears that depletion of ATP is an important stimulus and signal to initiate a response to stress (Newsholme et al. 2014). However, low intracellular ATP levels do not necessarily explain dysregulation of cell function, as ATP turnover may increase in such situations, satisfying cellular demands. The source of fuel required to maintain elevated ATP turnover can be modulated to elevate glucose, amino acid, and fatty acid input, as required.

Glutamine and its metabolites are important for the production and optimal activity of HBP. Furthermore, glutamine stimulates the expression of argininosuccinate synthetase (ASS) gene via O-glycosylation of Sp1 (Hamiel et al. 2009; Zachara and Hart 2006), a key transcription factor required for the most basic mechanisms of cellular protection, namely, heat-shock proteins (HSP) response (Heck et al. 2011). Glutamine availability was also identified as a limiting step for the activation of the mammalian target of rapamycin (mTOR) (Nicklin et al. 2009). Many initiation factor complexes (e.g., eIF2, eIF4F) that are assembled from multiple subunits are sensitive to activation by the mTOR cascade (Sartorelli and Fulco 2004), which results in coordinated protein synthesis and degradation (Newsholme et al. 2014).

It is not only the key intracellular proteins and transcription factors, which are HBP O-glycosylated following stress or injury, such as Sp1 (Hamiel et al. 2009; Zachara and Hart 2006) but the phosphorylation of the eIF2 (Dokladny et al. 2013b) also promotes the activation of the main thermal shock eukaryotic factor (Heat Shock Factor [HSF-1]), leading to the expression of HSPs (Xue et al. 2012) (Figure 1.3). HSPs are a family of polypeptides clustered according to molecular weight, which are increased in amount in response to an increase in intracellular denatured proteins. Some examples of HSP include HSP110, HSP100, HSP90, HSP70, and HSP27. The HSP response can be triggered by variations in body temperature, inflammation, and oxidative stress. Early studies documented novel protein responses in the salivary gland cells of Drosophila buskii after heat shock by Ritossa in 1962 (Heck et al. 2011). At lower temperatures, less heat-shock protein expression occurs. Thus, these proteins were described as temperature sensitive; in other words, thermal or heat-shock proteins (Heck et al. 2011).

HSP can be considered as stress-sensitive proteins, as further studies by Ritossa demonstrated that a number of agents or metabolic stressors could stimulate HSP levels. Various events such as exposition to heavy metals, UV radiation, amino acid analogs, bacterial or viral infections, inflammation, cyclooxygenase inhibitors (including acetylsalicylic acid), oxidative stress, cytostatic drugs (anticancer), growth factors, and cell development and differentiation can induce the expression of HSP (Heck et al. 2011; Wischmeyer 2002). All these factors strongly activate HSF-1, leading to the expression of HSP (Xue et al. 2012).

Although the connection between glutamine availability and the HSP response is not clear and may need further investigation, the reduction of body’s glutamine concentration may contribute to cell death (Cruzat et al. 2014a; Lightfoot et al. 2009). Recent studies have demonstrated that glutamine-induced HSP70 response may modulate autophagy by regulating the mTOR/Akt pathway (Dokladny et al. 2013a) and block signaling pathways associated with protein degradation (Singleton and Wischmeyer 2007). However, it is unlikely that glutamine supplementation can be used as an enhancer of muscle performance or muscle mass gain and strength under normal situations (Candow et al. 2001; Gleeson 2008). Glutamine supplements may be useful in replenishing previously depleted body glutamine stores (e.g., by high-throughput exercise and inflammatory diseases).

The role of amino acids, especially glutamine, has been recognized in cell defense mechanisms, through impact on antioxidant properties. Made up of three amino acid residues—cysteine, glutamate, and glycine—glutathione (y-L-glutamyl-L-cysteinylglycine, GSH) is found in high concentrations in cells and can directly react with ROS in non-enzymatic reactions as well as acting as an electron donor in peroxide reduction, catalyzed by glutathione peroxidase enzyme (GPx) (Cruzat and Tirapegui 2009; Newsholme et al. 2011b). During the 1960s and 1970s, Sir Hans Krebs, Hems, and Vina in the Metabolic Research Laboratory, University of Oxford, made key findings in relation to GSH, especially in mammalian cells (Valencia et al. 2001). In 1978 they described the regulation of hepatic GSH and its maintenance in isolated hepatocytes (Vina et al. 1978). Subsequently, different groups around the world have demonstrated through studies that GSH levels are reduced in stress in animals and humans (Cruzat and Tirapegui 2009; Meister 1983; Newsholme et al. 2011b; Rodas et al. 2012). GSH is the most important and more concentrated non-enzymatic antioxidant in cells (Cruzat and Tirapegui 2009; Flaring et al. 2003). Decreased glutamine concentrations, especially in liver and skeletal muscles, may compromise de novo synthesis of GSH, since glutamine is the immediate precursor of glutamate, even if cysteine and glycine were maintained at relatively constant levels (Rutten et al. 2005).

During catabolic states, elevated oxidative stress can be observed associated with an increase in the intracellular redox state, as indicated by the ratio between the intracellular concentration of glutathione disulfide (GSSG) and GSH, that is, [GSSG]/[GSH], resulting in a reduction of GSH and an increase in the amounts of GSSG (Galley 2011). The redox state of cells is consequently related to GSH concentration, which is influenced by the availability of amino acids. A higher glutamine/ glutamate ratio reinforces substrate availability for GSH (Cruzat et al. 2014a).

Quantitatively, the liver is the main organ for de novo synthesis of GSH, being responsible for nearly 90% of the circulating GSH in physiological conditions. The elevated concentration of hepatic GSH is mainly due to the high activity of glutathione reductase in this tissue. Methods to directly increase the GSH concentration through supplementation, or even addition of glutamate, are not effective and can be toxic, sometimes accelerating the cell senescence process (Finkel and Holbrook 2000). A number of studies demonstrate that glutamine supplementation can be an effective option to increase the availability of GSH in the organism if required (Cruzat et al. 2007; Cruzat and Tirapegui 2009). Furthermore, it is important to note that gradual stimuli, such as physical activity, stimulates per se the hormesis and adaptation of the organism, stimulating antioxidant mechanisms (Finkel and Holbrook 2000; Lamb and Westerblad 2011).

 
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