*New Acquisitions Regarding Structure and Function of Intestinal Mucosal Barrier DOI: http://dx.doi.org/10.5772/intechopen.105463*

from the lumen to the bloodstream and after its uptake from the bloodstream. This oxidative capacity coincides with a high energy demand of the epithelium, which is in rapid renewal and responsible for the nutrient absorption process. L-Glutamate is a precursor for glutathione and *N*-acetylglutamate in enterocytes. Glutathione is involved in the enterocyte redox state and in the detoxification process. *N*-acetylglutamate is an activator of carbamoyl phosphate synthetase 1, which is implicated in L-citrulline production by enterocytes. Furthermore, L-glutamate is a precursor in enterocytes for several other amino acids, including L-alanine, L-aspartate, L-ornithine, and L-proline. Thus, L-glutamate can serve both locally inside enterocytes and through the production of other amino acids in an inter-organ metabolic perspective. In colonocytes, L-glutamate also serves as a fuel but is provided from the bloodstream. Alimentary and endogenous proteins that escape digestion enter the large intestine and are broken down by colonic bacterial flora, which then release L-glutamate into the lumen. L-Glutamate can then serve in the colon lumen as a precursor for butyrate and acetate in bacteria. L-Glutamate, in addition to fiber and digestion-resistant starch, can thus serve as a luminally derived fuel precursor for colonocytes (**Figure 1**) [9].

Glutamine is the principal energy source for IECs, and during acute illnesses, patients experience nutritional depletion that is correlated to low plasma and low mucosal glutamine concentrations. Such deficiencies are common among hospitalized dogs and cats or human patients and are associated with an increased risk of developing infectious complications, organ failure, and death [10, 11]. A number of clinical studies reveal a significant benefit of glutamine use on mortality, length of hospital stay [12, 13], and infectious morbidity in critical illnesses, as well as in dog or cat parvovirus infection [11, 12, 14]. Patients receiving high-dose parenteral (rather than orally) glutamine presented the highest beneficial effects, and it is estimated that high doses of parenteral Gln (>0.50 g/kg/day) are the best treatment for humans and animals, demonstrating a greater potential to benefit [15]. However,

### **Figure 1.**

*Metabolic role of glutamine at the cellular level. In the catabolic phase, glutamine is transformed into glutamate and ammonium ions, thanks to the mitochondrial enzyme glutaminase (GA), while in the anabolic phase, at the level of most tissues, glutamine can be synthesized starting from glutamate and ammonia, in the presence of ATP, thanks to the enzyme glutamine synthetase (GS). Ammonia can be converted into Carbamoyl-phosphate, while Glutamate can form a-Ketoglutarate but also Glucose in the liver and kidney, while it is the basis for the synthesis of Glutathione in most cells, and of GABA (Gamma aminobutyric acid) at the neuronal level.*

the role of glutamine in the maintenance of normal gut and immune system function may be even more important for critically ill animals [16]. Glutamine is now considered by many investigators to be a conditionally essential nutrient during protein-calorie malnutrition, required in quantities that are greater than those that can be synthesized by the body. Based on this hypothesis and preclinical studies performed in dogs [17] the commercial veterinary critical care rations often recommended for cats and dogs with some severe enteropathies and cancer are routinely supplemented with glutamine. Glutamine supplementation has also been suggested as a way to promote more rapid resolution of acute side effects of the oral mucosa in dogs receiving oronasal radiotherapy and to maintain gut immunity and integrity in patients receiving radiotherapy or chemotherapy [18].

Recently, glutamine parenteral supplementation evidenced restoration of interdigestive migrating contraction in an experimental canine model of postoperative ileus [19]; in this research is hypothesized that the benefit derives from glutamine's ability to maintain glutathione concentration and thereby counteract the deleterious effects from surgical injury, inflammation, and oxidative stress. Similarly, parenteral administration of L-alanyl-L-glutamine [20] in dogs prevented the immune suppression induced by high-dose methylprednisolone sodium succinate, and experimental studies in the current literature indicate that glutamine use may prevent the occurrence of lung injury, tissue metabolic dysfunction, and reduce mortality after injury [21]. Glutamine's beneficial effects on critical illnesses or during IBD, may result from two principal ways: (a) the direct effect on IECs metabolism that helps to maintain the integrity of the epithelial barrier, preventing bacteria translocation; and (b) enhanced heat shock proteins (HSP) expression [22, 23] by enterocytes, and leucocytes [10, 24]. Heat shock proteins are a group of proteins essential to cellular survival under stressful conditions. The stress-inducible HSP60, HSP70 and HSP72 are inducible forms of the stress protein, which may confer cellular protection [10]. The cellular functions of intracellular HSP70 and HSP72 are responsible for limiting protein aggregation, facilitating protein refolding, and chaperoning proteins; an intra-mitochondrial concentration of these proteins is associated with an increase of mitochondria wellness, metabolic activity and ATP production for IECs (see below). IECs-specific deletion of the mitochondrial chaperone protein heat-shock protein 60 (HSP60) led to mitochondrial dysfunction, impairment of cell proliferation and loss of stemness of intestinal stem cells [25]. Additionally, mitochondrial dysfunction impaired the ability of the CBCSs to produce ATP, leading to altered CBCSs self-renewal and differentiation. Furthermore, L-glutamine potentiation of HSP72 is associated with increased gut epithelial resistance to apoptotic injury, and reduced HSP72 may be associated with increased caspase activity in glutamine-deficient [26]. In fact, glutamine induces autophagy under stressed conditions, and prevents apoptosis under heat stress through its regulation of the mTOR and p38 MAP kinase pathways [27]. Glutathione (GSH) metabolism is also closely related to the apoptotic processes of epithelial and immune cells. The increase of intracellular GSH is sufficient to reduce Fas-triggered increase in apoptotic cells. Over expression of Bcl-2, an anti-apoptotic protein, causes redistribution of glutathione to the nucleus, thereby altering nuclear redox and blocking caspase activity [28, 29].

Also, the amount and type of dietary fiber influence the end-products of fermentation and thus fuel availability to intestinal tissue in a *specie* depending manner. The metabolic fuel usage was studied in intestinal cells isolated from dogs consuming a commercial diet to examine preferential fuel usage and the effect of diet on canine enterocytes and canine colonocytes, respectively, indicating that glutamate/glutamine

### *New Acquisitions Regarding Structure and Function of Intestinal Mucosal Barrier DOI: http://dx.doi.org/10.5772/intechopen.105463*

is preferentially used by enterocytes, while butyrate (found in food and produced as an intestinal fermentation by-product of dietary fiber by gut bacteria) followed by glutamine is preferentially used by isolated canine colonocytes [30].

IBD has been suggested to involve a state of energy-deficiency, whereby oxidative metabolism is altered within IECs [31, 32]. Butyrate undergoes catabolic degradation through β-oxidation in the mitochondrial matrix of colonocytes, providing over 70% of the energy demand of the colonic epithelium [33]. Butyrate metabolism was demonstrated to be impaired in an animal model of colitis [34], and numerous studies have reported impaired metabolism in the intestinal mucosa of patients with IBD [35, 36]. Similarly, intestinal mucosal inflammation results when butyrate oxidation is inhibited in experimental animals [33]. Santhanam et al. [37] showed that the mitochondrial acetoacetyl CoA thiolase, which catalyzes the critical last step in butyrate oxidation, was significantly impaired in the colonic mucosa of patients with ulcerative colitis. Furthermore, they conclude that an increase in mitochondrial ROS may trigger this enzymatic defect [37]. Thus, defective β-oxidation in the mitochondria has deleterious effects beyond energy requirements. Likewise, a dysfunctional gut microbiome or a poor diet may also result in a decrease of butyrate metabolism in the colonic epithelium. Enhanced production of butyrate may potentially benefit the colonic epithelial cells by stimulating an enhancement in cellular homeostasis, including antioxidant and anti-inflammatory roles as well as protective gut-barrier functions.

### **1.2 Role of mitochondria in IECs homeostasis and barrier integrity**

The integrity of the intestinal epithelium, tight junction maintenance, and β-oxidation are key cellular processes within the intestinal epithelium that are not only dependent upon properly functioning mitochondria but are also known to be altered in animal models of intestinal inflammation and in humans with IBD.

Control of intestinal epithelial stemness is crucial for tissue homeostasis. Disturbances in epithelial function are implicated in inflammatory and neoplastic diseases of the gastrointestinal tract. Mitochondrial function plays a critical role in maintaining intestinal stemness and homeostasis. Using murine IECs, Berger et al. [25] demonstrated that loss of mitochondrial chaperone HSP60, activates the mitochondrial unfolded protein response (MT-UPR) and results in mitochondrial dysfunction [25].

During IBD, a destruction of the intestinal epithelial barrier, an increased gut permeability, and an influx of immune cells through the intestinal mucosa are observed. Given that, most cellular functions as well as maintenance of the epithelial barrier are energy-dependent, it is logical to assume that mitochondrial dysfunction may play a key role in both the onset and recurrence of disease. Indeed, several studies have demonstrated evidence of mitochondrial stress and impaired functions, such as oxidative stress and impaired ATP production, within the intestinal epithelium of patients with IBD and mice undergoing experimental colitis [38].

Recently, we have observed that mitochondria dysfunction has a central role in human detrimental intestinal barrier effects of chronic HIV infection [39].

Mitochondria are membrane-bound organelles that maintain cellular energy production through oxidative phosphorylation [40], and contain a circular small genome that encodes only 13 proteins [41]. Despite the limited coding-capacity of the mtDNA, mitochondria regulate vital cellular functions aside from energy production, such as the generation of ROS and reactive nitrogen species (RNS), the induction of programmed cell death, and the transduction of stress and metabolic signals [42]. The current literature would support a key correlation between mitochondrial function and intestinal barrier dysfunction/inflammation. Nonetheless, it is important to understand how any alteration in the multifaceted functionality of the mitochondrion may contribute to the initiation and propagation of an inflammatory insult (**Figure 2**).

Supporting the importance of mitochondrial form and function, enterocytes isolated from patients with IBD have been reported to exhibit swollen mitochondria with irregular cristae [43, 44]. Abnormal mitochondrial structure is also seen in IECs from mice subjected to experimental models of colitis [45]. Similar observations are made on canine IECs during IBD or lymphangiectasia [46].

These morphological changes are suggestive of cellular stress and bioenergetic failure. Indeed, patients with IBD have reduced ATP levels within the intestine [33, 47]. As would be expected, morphological changes in mitochondria have been shown to result in deficiencies in the β-oxidation of short-chain fatty acids (SCFA) [48]. The intestinal mucosa of IBD patients has been demonstrated to be in a state of energy deficiency characterized by low ATP levels and low energy charge potential [33, 49], calling into question the functionality of this organelle during disease. To further prove this, in a recent study, it was demonstrated that mtDNA released into the serum in IBD patients was recognized as a damage-associated molecular pattern (DAMP) potentially by toll-like receptor 9 (TRL9), and could provide a biomarker of inflammation [50].

Thus, defects in intestinal epithelial homeostasis result in an inadequate intestinal barrier defense, which may allow luminal antigens and/or microbes to interact with or violate the intestinal epithelium and consequently cause inflammation [51]. However, the role of mitochondrial dysfunction during IECs differentiation needs to be further

### **Figure 2.**

*A condition of eubiosis involves the correct synthesis/absorption of glutamine and glutathione by the enterocytes. Furthermore, the presence of "healthy" bacterial species producing NEFAs in the correct proportion, with an excess of butyrate, preserves the mitochondria from oxidative damage from ROS. A condition of dysbiosis increases mitochondrial damage, critically reducing the number of mitochondria but above all modifying their morphology and permeability. A critical reduction in mitochondria leads to a decrease in the production of ATP by the enterocyte (due to a reduction in the Krebs cycle and beta-oxidation). A reduction in energy leads to a lower "hold" of the intercellular junctional complexes and an increase in bacterial translocation through the intestinal epithelium, which becomes more permeable. At the submucosal level, this condition increases inflammation and the recall of leukocytes, further worsening the condition of the mucosal barrier.*

*New Acquisitions Regarding Structure and Function of Intestinal Mucosal Barrier DOI: http://dx.doi.org/10.5772/intechopen.105463*

evaluated in order to understand the role it may play in the development of intestinal inflammation. Recently, Bär et al. [52] demonstrated that altered mitochondrial oxidative phosphorylation activity influences intestinal inflammation in mice models of experimental colitis. The study suggests that increased regeneration of the intestinal epithelium (by means of increased mitochondrial function) is a key factor in combating intestinal inflammation. Mucosal healing also results in improved mitochondrial structure in the IECs of patients with ulcerative colitis [53].
