Glutamine and intestinal barrier function
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The intestinal barrier integrity is essential for the absorption of nutrients and health in humans and animals. Dysfunction of the mucosal barrier is associated with increased gut permeability and development of multiple gastrointestinal diseases. Recent studies highlighted a critical role for glutamine, which had been traditionally considered as a nutritionally non-essential amino acid, in activating the mammalian target of rapamycin cell signaling in enterocytes. In addition, glutamine has been reported to enhance intestinal and whole-body growth, to promote enterocyte proliferation and survival, and to regulate intestinal barrier function in injury, infection, weaning stress, and other catabolic conditions. Mechanistically, these effects were mediated by maintaining the intracellular redox status and regulating expression of genes associated with various signaling pathways. Furthermore, glutamine stimulates growth of the small intestinal mucosa in young animals and also enhances ion transport by the gut in neonates and adults. Growing evidence supports the notion that glutamine is a nutritionally essential amino acid for neonates and a conditionally essential amino acid for adults. Thus, as a functional amino acid with multiple key physiological roles, glutamine holds great promise in protecting the gut from atrophy and injury under various stress conditions in mammals and other animals.
KeywordsGlutamine Intestinal barrier function Nutrition
Neutral amino acid transporter type 2
Essential amino acids
Eukaryotic translation initiation factor 4E-binding protein-1
Eukaryotic translation initiation factor 4E
Forkhead box O transcription factor
GlutaminaseGALT gut-associated lymphatic tissue
Glutamate cysteine ligase
Inhibitor of NF-κB
Intrauterine growth restriction
Mitogen-activated protein kinases
Mammalian target of rapamycin
Reduced form of nicotinamide adenine dinucleotide
Reduced form of nicotinamide adenine dinucleotide phosphate
p70 Ribosomal protein S6 kinase
Protein kinase B
Pentose phosphate pathway
Ras homologue enriched in brain
Reactive oxygen species
Receptor tyrosine kinase
Sodium coupled neutral amino acid transporter 2
Tricarboxylic acid cycle
Tumor necrosis factor-alpha
Total parenteral nutrition
Tuberous sclerosis complex
Zonula occludens protein 1
Gln and intestinal development
Both enterocytes and intestinal luminal bacteria degrade Gln (Dai et al. 2010, 2012; Wu and Knabe 1994). Approximately 70 % of Gln in the enteral diet is catabolized by the absorptive cells of the small intestine during the first pass (Wu 1998). However, Gln is abundant in plasma, skeletal muscle, fetal fluid and milk, due to endogenous synthesis from branched-chain amino acids and glucose (Wu et al. 2011). The concentration of free Gln in porcine milk markedly increases from <0.1 mM at day 1 of lactation to 4 mM on day 29 of lactation, becoming the most abundant amino acid in sow’s milk (Wu and Knabe 1994). The predominance of Gln in sow’s milk is consistent with the notion that Gln plays a vital role in the growth and development of the neonatal gastrointestinal tract (Sheard and Walker 1988). In addition, dietary Gln supplementation to early-weaned piglets prevents jejunal atrophy during the first week postweaning (Wu et al. 1996). Moreover, oral administration of Gln (0.5 g/kg of body weight, twice daily) to 7- to 21-day-old suckling piglets enhances intestinal and whole-body growth (Haynes et al. 2009). Thus, milk-borne Gln is insufficient for maximal growth of the neonate. Likewise, supplementing Gln to Gln-free total parenteral nutrition (TPN) solution prevents intestinal atrophy in humans and rats under catabolic conditions (Buchman et al. 1995; Schroder et al. 1995). Similarly, dietary supplementation with Gln or glycyl-Gln dipeptide improves growth performance, small intestinal morphology and immunity response in endotoxin-challenged weaning piglets (Jiang et al. 2009; Yi et al. 2005).
Neonates with intrauterine growth restriction (IUGR) have higher perinatal mortality and morbidity in both humans and animals, including pigs (D’Inca et al. 2011; Rezaei et al. 2011, 2013b). IUGR neonates are characterized by reduced concentrations of Gln in plasma and a relatively thin intestine in both preterm and term neonates of many species (D’Inca et al. 2011; Wu et al. 2011). Alterations in intestinal development impair nutrient absorption and utilization and contribute to IUGR-related high morbidity and mortality (D’Inca et al. 2010, 2011; Wang et al. 2008). Interestingly, oral administration of Gln (1.0 g/kg of body weight per day) between day 0 and day 21 of age enhances the growth of IUGR piglets and reduces post-weaning mortality (Wu et al. 2011). Similar observations have been reported in low-birth-weight infants (Neu et al. 1999), suggesting Gln provision as an effective nutritional strategy to enhance intestinal development in IUGR neonates. All these results indicate a beneficial effect of Gln on postnatal intestinal growth and development.
Gln and intestinal cell proliferation
The epithelium of the mammalian intestine undergoes rapid renewing and turnover every 3–5 days (Camilleri et al. 2012). This process is fulfilled by the fine-tune balance between cell proliferation and loss, thus constituting a homeostasis which is required for the physiological function of the intestinal mucosal barrier (Bach et al. 2000). The underlying regulatory mechanisms may involve multiple signaling and growth factors. It is generally accepted that intestinal stem cells located near the base of the crypts divide rapidly and migrate along the crypt/villus axis and differentiate into absorptive enterocytes, enteroendocrine cells, mucous-secreting goblet cells, tuft cells, and Paneth cells (Barker et al. 2008; Shaker and Rubin 2010).
Gln enhances intestinal cell proliferation through a number of mechanisms. First, oxidation of Gln provides ATP to support intestinal ion transport, cell growth and migration and to maintain intestinal integrity (Curi et al. 2005). Second, Gln is a precursor for the synthesis of purine and pyrimidine nucleotides, which are essential for DNA synthesis and the proliferation of cells (Wu 1998). Third, Gln is a major substrate used to produce glutathione, an antioxidant in cellular environment (Reeds et al. 1997). Fourth, Gln up-regulates the expression of ornithine decarboxylase, a key enzyme for converting ornithine into polyamines, which are required for DNA and protein synthesis (Wu 2013). Fifth, Gln stimulates expression of heat shock proteins (Marc Rhoads and Wu 2009) to promote cell survival. Sixth, Gln regulates cellular signaling pathways for cell proliferation. For example, addition of physiological levels of Gln (0.5 and 2 mM) increases protein synthesis and inhibits protein degradation in enterocytes, resulting in enhanced cell proliferation (Xi et al. 2012). Similarly, Gln has been reported to stimulate protein synthesis in the small intestinal mucosa of humans and rats (Deniel et al. 2007; Rhoads et al. 1997; Ziegler 1994). Finally, Gln enhances expression of genes for mitogen-activated protein kinases, including both ERK1/2 and JNK, resulting in activation of AP1-dependent gene transcription, thereby contributing to cell proliferation (Rhoads et al. 1997).
Growing evidence shows interactions between Gln and growth factors in intestinal cells. Of note, several growth factors, such as epidermal growth factor (EGF), transforming growth factor, and insulin-like growth factor 1 (IGF-1), may regulate proliferation and differentiation in various cell types, including enterocytes (Booth et al. 1995). It is known that the intestinal epithelium contains receptors for IGFs, including IGF-1 (Rhoads et al. 1997). Gln supplementation may increase protein abundances of these growth factors by modulating rates of protein turnover, and consequently promotes cell proliferation by activating downstream signaling cascades. It has also been reported that EGF up-regulates the activity of Gln transporter B0/ASCT2 and system B0,+ and ASCT2 expression in a multiple kinases (MAPK, PI3K, Rho)-dependent manner in human enterocytes (Avissar et al. 2008), indicating a feedback loop between growth factors and Gln-induced cell signaling. These signaling pathways contribute to Gln-mediated enhancement of intestinal cell proliferation (Rhoads 1999).
Gln and tight junction
Intestinal epithelial cells are tightly bound together by intercellular junctional complexes (Fig. 1) that regulate the paracellular permeability and are crucial for the integrity of the epithelial barrier (de Santa Barbara et al. 2003). To date, four types of junctional complexes have been identified, including tight junctions, adherens junctions, gap junctions, and desmosomes. Tight junctions are the most apical structure of the apical complex demarcating the border between apical and basolateral membrane domains, selectively regulate the passage of molecules and ions via the paracellular pathway, and also restrict the lateral movement of molecules across the cell membrane (Prasad et al. 2005). Adherens junctions are located beneath the tight junctions and are involved in cell–cell adhesion and intracellular signaling (Farhadi et al. 2003; Matter and Balda 2003). Desmosomes and gap junctions contribute to cell–cell adhesion and intracellular communication, respectively. Among the junctional complexes, tight junctions are extensively studied due to its critical role in regulating barrier function and preventing relocation of toxins and pathogens from the gut lumen into mucosal tissue and circulation (Chen et al. 2014; Zhang et al. 2013).
Three types of structural transmembrane components that are enriched at tight junctions include the IgG-like family of junctional adhesion molecules, the claudin, and occludin families of transmembrane proteins (Furuse and Tsukita 2006; Schneeberger and Lynch 2004). Regulation of the assembly, disassembly, and maintenance of tight junction structure are influenced by various physiological and pathological stimuli that activate several kinases, including protein kinase C, mitogen-activated protein kinases (MAPK), myosin light chain kinase, and the Rho family of small GTPases (Ulluwishewa et al. 2011). In general, the activation of protein kinase phosphorylates tight junction protein, leading to alterations of barrier integrity and permeability. It should be noted that the results from different research groups are not always consistent and need further clarification (Findley and Koval 2009). Although the beneficial effect of Gln on intestinal health was documented in the 1990s (Deniel et al. 2007), the regulation of tight junction protein by Gln was reported only in recent years. Gln deprivation or inhibition of Gln synthetase led to significant decreases in transepithelial resistance, an increased permeability, as well as reduced tight junction proteins, claudin-1 and occludin in Caco-2 cells, a classic human cell line model for gut barrier function (DeMarco et al. 2003; Li et al. 2004). Importantly, the dysfunction of gut barrier function can be rescued by Gln addition (DeMarco et al. 2003). Mechanistically, Gln deprivation up-regulates the PI3K/AKT pathway, which, in turn, reduces the abundance of tight junction protein claudin-1, resulting in barrier function breakdown (Li and Neu 2009). It is unknown whether rates of claudin-1 synthesis, degradation, or both processes are affected in the gut. These observations suggest that Gln can directly or indirectly function as regulator of signal pathways associated with gut function. In addition, the protective effect of Gln on tight junction protein is also observed under stress response. For example, it has been reported that Gln prevented the acetaldehyde (a carcinogenic metabolite of ethanol)-induced increase in permeability to endotoxin by ameliorating the disruption of tight junction, adherence junction, and redistributing the tight junction proteins, ZO-1, occludin, E-cadherin, and β-catenin from the intracellular junction complex in Caco-2 cell monolayer (Basuroy et al. 2005; Seth et al. 2004), indicating a therapeutic potential of Gln on carcinoma progression. It should be noted that several lines of studies indicated that endogenous Gln synthesis by Gln synthetase is critical for the beneficial effect on protein expression of tight junction in human Caco-2 monolayer (DeMarco et al. 2003; Li and Neu 2009). However, in our recent study, we found that deprivation of Gln decreased monolayer transepithelial resistance which can be reversed by the provision of Gln to the culture medium in the absence of the Gln synthetase inhibitor in intestinal porcine epithelial cells. This suggests that the amount of endogenous Gln synthesized by Gln synthetase is much less than that of the Caco-2 cells used in the previous study (DeMarco et al. 2003; Li and Neu 2009). Indeed, Gln synthetase activity is negligible in enterocytes of neonatal pigs (Haynes et al. 2009) and lactating sows (Li et al. 2009). Another reason for this discrepancy might be due to metabolic, genetic, or epigenetic differences between cancer cells and normal intestinal epithelial cells (Meadows et al. 2008). In a recent study, Noth and his colleague demonstrated that oral Gln supplementation ameliorated intestinal permeability dysfunction as shown by increased occludin expression, reduced gastrointestinal permeability and reduced apoptotic cells in the crypt (Noth et al. 2013). All these in vivo and in vitro data suggest an important role for tight junction in the maintenance of gut function. Regulation of tight junction proteins by Gln is responsible, at least partially, for the beneficial effect of Gln on gut barrier function in humans and animals.
Gln and stress response
Preventive effects of Gln against apoptosis
Gln, bacterial translocation and intestinal immunity
In addition to its role in digestion and absorption of nutrients, the small intestine is also viewed as the largest immune organ in the body to protect the internal milieu from potentially hostile pathogens which is required for the maintenance of normal intestinal epithelial barrier function (Menard et al. 2010; Veldhoen and Brucklacher-Waldert 2012). The normal intestinal barrier to bacteria invasion mainly depends on specific IgA antibody secreted from the gut-associated lymphatic tissue (GALT) with which several types of specialized cells, such as macrophages, natural killer cells, mast cells, and intraepithelial lymphocytes, are involved (Ruth and Field 2013). Under physiological conditions, IgA is secreted into the intestinal tract and has an ability to prevent the adherence of bacteria to the mucosal cell, which is the initiating and prerequisite step for colonization and invasion of bacteria to the deeper layers of the intestine (Artis 2008; Jacobi and Odle 2012). Abnormal regulation of secretory lgA (s-IgA) production due to pathologic invasion, stress exposure, or perturbation of the components of GALT, will impair the mucosal immune system, while leading to bacterial translocation and defective barrier integrity (Alverdy et al. 1988; Mestecky et al. 1986). The beneficial effect of Gln addition on mucosal during provision of standard total parenteral nutrition (Grant and Snyder 1988) promoted Burke and colleagues to elucidate immune function of Gln on the gastrointestine (Burke et al. 1989). These authors reported that addition of Gln to TPN reversed the decrease of s-IgA production, bacterial adherence to the intestinal mucosa, and bacterial translocation, suggesting a critical role of Gln in gut immune function (Alverdy 1990; Li et al. 2007). Experimental studies support the role of intestinal s-IgA as a significant component of the intestinal barrier function and that impaired production of s-IgA is associated with increased adherence of bacteria to the intestinal mucosa and breakdown of barrier function (Spitz et al. 1995). To demonstrate the role for Gln in bacterial translocation, rats exposed to a single dose of abdominal radiation were supplemented with Gln or isonitrogenous control in their drinking water for 4 days. The results indicated that provision of Gln blunts mucosal injury and the incidence of bacterial translocation (Souba et al. 1990). It has also been demonstrated that administration of Gln improves gut barrier function and reduces bacterial translocation in both in vivo (Chen et al. 1994; Souba et al. 1990) and in vitro (Scheppach et al. 1996) models of gut disorder. Furthermore, Gln deprivation facilitates TNF-α-induced bacterial translocation in Caco-2 cells, and the sensitization of TNF-α-induced bacterial translocation was blocked by an inhibition of the conversion of Gln to α-ketoglutarate (α-KG), a key step in oxidative metabolism of Gln (Clark et al. 2003). α-KG can activate mTOR and stimulate protein synthesis in enterocytes (Yao et al. 2012), thereby improving intestinal function (Hou et al. 2011). ATP depletion following Gln deprivation may be responsible, in part, for the sensitization effect of Gln on bacterial translocation (Clark et al. 2003). This view is supported by the observation showing that Gln is the major fuel energy for fast-dividing cells, including intestinal epithelial cell and lymphoma cells (Mates et al. 2006; Wu 1998).
Growing evidence shows that Gln inhibits intestinal expression and activation of nuclear factor-κB (NF-κB) (Haynes et al. 2009; Mondello et al. 2010) which is a pleiotropic transcription factor present in almost all cell types (Pasparakis 2012). NF-κB has been recognized as a critical regulator of immune responses and implications in the pathogenesis of diverse inflammatory diseases (Pasparakis 2009). While early studies mainly focused on the role of NF-κB in the development and function of immune cells, accumulating experimental evidence indicates that NF-κB signaling in epithelial cells is important for the maintenance of immune homeostasis in barrier tissues such as the skin and the intestine (Pasparakis 2012). Under normal conditions, NF-κB is located in the cytosol and complexed with the inhibitory protein IκB-α, and thus exists in an inactive state. Extracellular signals, such as cell stress and inflammatory signal, can activate the enzyme IκB kinase (IKK). IKK, in turn, phosphorylates the IκBα protein, which results in ubiquitination, dissociation of IκB-α from NF-κB, and eventual degradation of IκB-α by the proteasome. The activated NF-κB is then translocated into the nucleus where it binds to the promoter region of specific genes and results in their functional change (Li and Verma 2002). It becomes clear that the immune homeostasis of intestine depends on the interactions of the bacteria with the mucosal immune system. Activation of NF-κB has been observed in stress-induced or bacteria-induced disruption of barrier integrity (Banan et al. 2007; Han et al. 2009), suggesting that NF-κB signaling might contribute to disease pathogenesis. Pharmacological inhibition of NF-κB had protective effects in gut disease, including colitis, supporting a pathogenic role for NF-κB in intestinal inflammation. Commensal bacteria are believed to activate NF-κB in epithelial cells by stimulating pattern recognition receptors including Toll-like receptors and NOD-like receptors, which are expressed to allow sensing of commensal bacteria in intestinal epithelial cells (Abreu 2010). In additional to the classic activation of NF-κB signaling, including IκB degradation and direct modifications on NF-κB protein (Li and Verma 2002), the activity of NF-κB can also be regulated by Gln under stress condition or critical illness. Experimental in vitro studies show that Gln deprivation modulates endotoxin-induced IL-8 production via decreasing IκB protein in both human fetal and adult enterocytes, with the immature intestine showing the greatest response (Liboni et al. 2005). A recent study also demonstrated that, in addition to modulating IKK activity, Gln can induce nuclear degradation of the NF-κB p65 subunit via the ubiquitin–proteasome pathway, thus diminishing inflammatory response (Lesueur et al. 2012). These results clearly indicate a repressing effect of Gln on intestinal inflammation, which might be another mechanism responsible for its beneficial effect on barrier function as observed from the in vivo studies (Wu et al. 1996).
Conclusion and perspectives
Gln is a truly functional amino acid in nutrition (Wu et al. 2013). Dietary Gln has been shown to be important for maintenance of the intestinal mucosal barrier by regulating expression of genes and proteins involved in cell proliferation, differentiation and apoptosis, protein turnover, anti-oxidative property, and immunity responses. The critical dependence of gut function on the provision of dietary Gln is observed under physiological and stress conditions, such as weaning, lactation, gestation, and various gastrointestinal disorders (Wu 2014). This beneficial effect of Gln continues to stimulate research on basic and clinical studies involving animal models and humans. Compelling evidence showed that Gln is a nutritionally essential amino acid for neonates and a conditionally essential nutrient for adults (Rezaei et al. 2013a; Wu et al. 2014). Despite much progress in this research area, the underlying mechanisms for the actions of Gln remain largely unknown and should be addressed in future studies involving in vitro and in vivo experiments. It should be borne in mind that in vitro studies can provide useful information about the biochemistry, nutrition and physiology of Gln, but cannot fully mimic conditions in intact animals which require coordination of multiple systems for maintenance of homeostasis. For example, Gln regulates metabolism of intestinal bacteria (Dai et al. 2013), which is expected to have profound impacts on the health of the gut and the whole body. Cautions should be taken to interpret and extrapolate in vitro data to in vivo models, particularly with regard to concentrations of Gln and other amino acids in cell culture medium. Integration of results from all studies is necessary to better understand how Gln functions in animals and humans and how to develop new means to improve Gln nutrition in the organisms.
This work was supported by National Key Basic Research Program (2013CB127302), the Natural Science Foundation of China (31172217, 31272450, 31301979, and 31272451), the Chinese Universities Scientific Fund (2013RC002), the Program for New Century Excellent Talents in University (NCET-12-0522), and the Program for Beijing Municipal Excellent Talents, and Texas A&M AgriLife Research (H-8200).
Conflict of interest
The authors declare no conflicts of interest.
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