“IMPROVED MILBOND-TX® PROMOTES INTESTINAL HEALTH” “UNDERSTANDING THE MECHANISM OF ACTION”
In the previous issue of Milwhite’s Journal (September, 2016) the research conducted by Almeida et. al., (2014) at the University of Illinois (USA) was discussed and documented the fact that of two smectite clays and a zeolite that were tested only one smectite clay was able to promote intestinal health. That clay was IMTX. These authors provided data that showed all three clays restored the lost performance in chick body weight and feed intake resulting from a Salmonella challenge. However, of the two smectite clays tested only IMTX was reported to initiate changes in goblet cell function by stimulating mucus synthesis and release which strengthened the integrity of the intestinal mucus barrier. The purpose of this issue of Milwhite’s Journal is to explain, in an easy to understand fashion, the reason why these investigators decided to select out and study specific components of the mucosal immune system when testing these clays. These components of the immune system, which are proteins, are considered to be among the most important proteins that are synthesized when the animal’s intestinal tract is challenged by pathogenic microbes A full explanation of the details of their in-vivo and In-vitro experiments can be found in their publication cited below.
GOBLET CELLS AND ITS MUCIN PROTEINS
At this point, in order to facilitate a better understanding of the data collected and reported by these authors which explains how IMTX is promoting intestinal health it is essential to appreciate the difference between mucus and mucin. Mucus is the viscous gel-like fluid that is actually secreted onto the surface of the intestinal epithelium by the goblet cells associated with the intestinal villi. The gel-like nature of mucus is a property resulting from the numerous sugar molecules in its structure. Simply, mucus is a composite composed of a protein core (backbone) to which individual sugars are attached. This carbohydrate-rich protein complex also has inorganic salts associated with its structure. Thus, this complex is commonly referred to as a “glycosylated protein” (i.e., glycoprotein) known as “mucin” and there are several different glycoproteins (mucins) associated with mucus. It is the sugars that are coating (surrounding) the protein core that are responsible for the high water holding capacity of mucus since the mucins attract an enormous quantity of water molecules. In fact, some mucin proteins are composed of over 85% of these sugars and are responsible for mucus being composed of approximately 95% water. So, mucus is composed of mucins which are glycoproteins associated with inorganic salts that becomes very viscous after this composite attracts water.
GOBLET CELL SYNTHESIS OF SPECIFIC MUCINS
In order for goblet cells to synthesize their many mucin proteins there must be a mucin gene that encodes each protein. Each individual mucin protein has a specific defined function. Specifically, one of the most important mucin proteins is referred to as the MUC2 protein (i.e., MUCIN 2 protein) and the coding gene for this protein is the MUC2 gene. Even though there are different types of mucins produced in various locations within an animal’s body, MUC2 is quantitatively the most important protein in intestinal mucus associated with epithelial barrier protection and this is one reason that Almeida et. al., (2014) were interested in this goblet cell protein. It has been estimated by numerous researchers that as much as 80% of the mucins produced by goblet cells are MUC2. This MUC2 protein is responsible for the viscus “net-like” structural property of the mucus secreted by goblet cells. Not only did Almeida and coworkers report on the goblet cell production of MUC2 protein, but they also reported data on how two other essential goblet cell proteins were influenced by different clays. These two proteins were TREFOIL FACTOR 3 (TFF3) and RESISTIN-LIKE MOLECULE BETA (RELM-ß). As with MUC2, being only one of several in the family of mucin proteins, TFF3 and RELM-ß are only one in a family of different TFF and RELM proteins produced by goblet cells. The reason why these two families of proteins were also a main focus of this study was because they have extremely important functions in maintaining integrity of the intestinal mucosal surface associated with “certain situations”.
SYNTHESIS OF TREFOIL FACTOR 3
A situation where TFF3 protein is synthesized from its specific gene (TFF3 gene) in goblet cells occurs when there is a need for mucosal defense or a healing function in the intestine as a result of some insult. Also, there is evidence that TFF3 protein assists in stabilizing the entire mucus layer along with providing protection against ulcerative conditions in the intestinal tract which are common during pathogen colonization. For instance, whenever there is an injury or an ulcerative condition in the intestinal epithelial surface the TFF proteins act as “hormone-like messengers” and promote the migration of healthy cells in the region of the injury without promoting cell division. Thus, a mode of action of these TFF proteins is related to their ability to stimulate the migration of surviving cells from the edge of any damaged region of the intestine over the uncovered (denuded) area promoting the healing process. One might wonder why this family of intestinal proteins is referred to as the “Trefoil peptide family”. The Latin word “Trifolium” (tri-“three” and folium-“leaf) refers to the genus of the legume family of plants that have leaves divided into three distinct areas called leaflets (i.e., common clovers). Since the proteins belonging to the Trefoil family have three distinct and identifiable “loops” in their structure this name reflects these three distinct structural loops or more commonly referred to as a protein having three distinct structural “domains”. The TFF family of peptides is characterized by having six molecules of the sulfur containing amino acid, cysteine, in their structure.
This is of importance because as part of the protein structure these six cysteine molecules form three distinct intra-chain disulfide bonds which are responsible for forming and maintaining the unique three-loop structural domain configuration of the trefoil peptide family.
SYNTHESIS OF RESISTIN-LIKE MOLECULE ß
The third cysteine-rich goblet cell protein, RELMß, was of importance to these investigators since the chicks were challenged with Salmonella and RELMß is synthesized and released from goblet cells in situations where bacterial colonization occurs. Maintaining and stabilizing the mucosal defense barrier as well as helping to regulate and control intestinal inflammation following mucosal injury are also considered main responsibilities of this protein. RELMß has also been shown to upregulate the MUC2 gene so more mucus will be formed during infections and released from goblet cells and as a result goblet cells decrease in size. This hormone-like protein derives its name from another cysteine-rich protein called “Resistin” which is known to be produced by fat cells and was only discovered in 2001 at the University of Pennsylvania Medical School (USA). It was called Resistin (Resist-“Resistance” and in-“insulin”) because it promoted insulin resistance when it was injected into mice. Insulin resistance occurs when muscle, fat, and liver cells do not respond properly to insulin and results in the absence of glucose absorption into the cells of these tissues and the beta-cells in the pancreas become hyperactive attempting to produce more insulin. Over time insulin resistance leads to Type-2 diabetes. Because this goblet cell-derived protein has also been shown to impair insulin function just like Resistin, it was named “Resistin-Like Molecule Beta” (RELMß).
The importance of the data reported by these researchers investigating the smectite clay, IMTX, should be obvious. Their data, related to goblet cell number and size, supported the concept that of the three clays tested only IMTX was able to promote effects consistent with strengthening the intestinal mucus barrier. In their in-vitro experiment data indicated that no clay showed an ability to alter the gene expression of TFF3. However, only IMTX increased expression of the RELMß gene in the human colorectal cell-line, LS174T, selected for use in this study. Data collected on gene expression in these cells indicated that IMTX promoted more synthesis and release of the RELMß protein. In this study, all three clays were shown to have a beneficial effect in restoring the loss in chick body weight and feed consumption which resulted from the Salmonella challenge. However, these researchers mentioned that in certain cases only IMTX was the smectite clay that promoted positive benefits compared to the other clays tested. Importantly, they emphasized that just because a clay is a smectite does not mean that it will promote similar positive benefits as those observed with IMTX in this study.
The information presented in this issue of Milwhite’s Journal was compiled by Dr. Orlando Osuna, Director of Health Science at Milwhite, Inc. and Dr. Richard Miles, Professor Emeritus, Univ.of Florida, Gainesville, FL, USA.
Almeida J.A.S., N.P. Ponnuraj, J.J. Lee, P. Utterback, H.R. Gaskins, R.N. Dilger, and J.E. Pettigrew. 2014. Effects of dietary clays on performance and intestinal mucus barrier of broiler chicks challenged with Salmonelle enterica serovar Typhimurium and on goblet cell function in vtiro. Poultry Science 93:839-847.