Intestinal barrier function – A case to be studied in reduced protein diets

R. Barekatain, S. Gilani, S.M. Kitessa and R.J. Hughes

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Maintaining intestinal health and growth remains a concern as demands increase to limit the use of antibiotics in poultry production. There is also an interest to feed meat chickens with lower protein content diets using more synthetic amino acids to maintain productivity while reducing the environmental impact of poultry production. In that situation, closer attention needs to be given to those amino acids that maintain the intestinal barrier, decrease the variability of nutrient utilization and absorption, and make the birds less vulnerable to a physiological stress while not compromising performance. Optimum function of the epithelium of the intestine is important in controlling permeability. Poorly digested diets, fasting, endotoxins and several forms of stress have been shown to adversely affect intestinal barrier function. In addition to their usual role in the synthesis of proteins, amino acids are regarded as key regulators of fluxes through several major metabolic pathways with additional roles in maintaining gut health. Threonine, arginine (Arg), and glutamine (Gln) are regarded as the three most critical amino acids involved in metabolism, function, integrity and health of the intestinal tract across different species. There are therapeutic roles documented for specific amino acids including Gln, glutamate (Glu), Arg, glycine (Gly), Lys, Thr, and sulphur amino acids in gut- related disorders and disease.

Intestinal barrier function
The small intestine serves as an organ for nutrient absorption. The mucosa of the small intestine has finger like projections known as the villi. A single layer of epithelial cells (enterocytes) covers these villi. The enterocytes are linked with each other through complex proteins known as adherens junctions (AJ), tight junctions (TJ) and desmosomes. Adherens junctions and desmosomes create the mechanical link between enterocytes e.g. Zonula Adherens. Tight junctions including Claudin, Occludens and Jams play an important role in paracellular permeability as reviewed by Groschwitz and Hogan (2009). These junctions not only have a fundamental role in absorbing nutrients, but also prevent entry of microbes and toxins into the body. However, during stress or disease when these junctions are disrupted, the barrier function of the gut is compromised leading to increased intestinal permeability (IP).
Increased IP can lead to compromised health, bacterial and toxin translocation, lameness and compromised performance. Increased IP in chickens has not been studied in detail so far and needs further research. A few models have been studied in this regard e.g. dextran sodium sulphate and feed withdrawal for increasing IP. In addition to the limited knowledge of increased IP models in chickens, very little has been researched regarding the biomarkers to evaluate the changes in increased IP. Tight junctions mRNA expression and Ussing chamber have been used in the past, however, information regarding the biomarkers to assess increased IP in live birds is limited. Gilani et al. (2016) have concluded in their review that two-sugar methods can effectively be used to evaluate the changes in IP in live chickens. These sugar methods include lactulose, rhamnose, mannitol and fluorescein isothiocyanate dextran (FITC-d). The modulation of IP through nutrition warrants further research.

Key amino acids involved in intestinal function
Amino acids are involved in major metabolic pathways as regulators in addition to their normal roles as the building blocks of protein synthesis. Additional roles of amino acids in gut function and integrity has been reviewed by Wang et al. (2009). In this regard, only a few major amino acids are mentioned with specific focus on their role and their interactions on intestinal function.
There are also growing evidences that there are dietary requirements for non-essential amino acids (NEAA) including Gln to support optimum animal growth. Typical plant protein sources often do not contain adequate amount of NEAA. Glutamine has been traditionally considered as a NEAA being the most abundant amino acid in blood plasma. It is established, mainly in other species, that Gln is the main energy source facilitating proliferation of intestinal enterocytes and activated lymphocytes. Several studies have confirmed that Gln is vital in maintaining the functional integrity of the gut as it plays a nourishing role for rapidly dividing intestinal epithelial cells, enterocytes and lymphocytes. This maintenance role is directly related to tight junctions, mucosal cell proliferation and differentiation. Glutamine is involved in mucin synthesis. N-acetylglucosamine is a glycoprotein, a component of the mucin that protects mucosal surfaces and its formation is wholly dependent on Gln. Glutamine may be considered a conditionally essential amino acid when animal suffers from stress, injury or malnutrition. Under such circumstances the requirement may exceed the capacity for endogenous synthesis required to maintain gut integrity and reduce inflammation because during an immune response a marked increase in the uptake of plasma Gln by immunocytes occurs. Beneficial effects of Gln on growth performance and gut development in broiler chickens have been previously demonstrated. Nevertheless, its effectiveness in regards to enhancement of gut barrier function and more importantly its interaction with other nutrients with similar regulatory effects are still largely unknown for poultry.
Arginine has known roles in the urea cycle, in transport, storage, and excretion of nitrogen and eventually disposal of ammonia. Enterocytes can synthesize Arg from Gln and this is required to support optimum growth and intestinal function. Synthesis of nitric oxide (NO) is dependent on Arg as an essential substrate. Production of NO along with enterocyte migration is crucial for restoration of epithelial continuity. Arg is known to preserve intestinal barrier integrity and reduce bacterial translocation in mice. It has also been shown that Arg can attenuate inflammatory response in broiler chickens in response to lipopolysaccharide injection. Similar results have been reported for broilers subjected to a coccidial vaccine challenge.
From the arginine family of amino acids, Arg and Gln have some similar pivotal functions. It has been reported that dietary supplementation with these two amino acids can improve porcine intestinal immunity and growth performance. It appears that there is complementary mode of action. Gln and Arg have been shown to decrease gut permeability through regulation of TJ.
The role of Thr in synthesis of mucin and maintenance of intestinal function is well documented in literature. Deficiency of Thr impairs the mucosal integrity evidenced mainly by differences observed in intestinal morphology and mucin production. In regard to paracellular permeability, no data are available for poultry. However, research in piglets has shown that a moderate deficiency of Thr (30% reduction) increased paracellular permeability in the ileum assessed by Ussing chambers.

Dietary protein content
Dietary protein and therefore balance of amino acids plays a major role in animal health and performance. There is an interest to feed birds with low protein diets to reduce cost, environmental impact and wet litter. However, this practice has often resulted in impaired performance and unbalanced supply of amino acids. Supplementation of amino acids have been extensively researched in relation to the reduced protein diets and the order of limiting amino acids are very much known. Glycine and serine have been shown to improve the bird performance fed low protein diets. In the concept of intestinal gut health, the role of specific amino acids and their relationship together for intestinal integrity and development are still not fully understood when dietary protein is reduced. However, Chen et al. (2016) recently showed that reducing dietary protein exacerbated the effect of aflatoxin in broiler performance and nutrient utilisation along with a numeric tendency to increase intestinal permeability. These researchers found that increasing dietary protein to 26% completely restored the adverse effect of aflatoxin on bird performance.

Conclusion
There is a clear need to investigate the role of key amino acids including Gln, Arg and Thr for intestinal barrier function. Although some supporting evidence has emerged on positive effects of some amino acids, such as Gln, Gly and serine, traditionally regarded as non- essential, their inclusion in practical poultry diets may not be economically and practically feasible without reduction of the protein content of the diets; a practice that may potentially have some implications for the bird performance and intestinal function.
References are available on request
From the Proceedings of the 2017 Austrialian Poultry Science Symposium