Scholarly article on topic 'Gastric digestion of protein through pancreozyme action optimizes intestinal forms for absorption, mucin formation and villus integrity'

Gastric digestion of protein through pancreozyme action optimizes intestinal forms for absorption, mucin formation and villus integrity Academic research paper on "Animal and dairy science"

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{"Intestinal proteolysis" / "Mucin formation" / "Nutrient absorption" / "Unstirred water layer" / "Villus maintenance"}

Abstract of research paper on Animal and dairy science, author of scientific article — Edwin T. Moran

Abstract Recovery of dietary protein proceeds through two phases of digestion before suitable forms exist for absorption. The first phase engages the gastric system where low pH weakens overall structures allowing pepsin to disrupt hydrophobic bonding and enhance aqueous compatibility. Secondly, trypsin, chymotrypsin and elastase proteolysis in conjunction with carboxypeptidases A and B cooperate to form a mixture of free amino acids and peptides that progressively arise in the small intestinal lumen. Free amino acids are dominated by the aromatic, aliphatic and basic ones while resulting peptides largely involve the nonessentials. Motility convectively transfers digestion products to the unstirred water layer of the upper villus where filtration limits entry to low molecular weight solutes that can be further digested by underlying enzymes to optimize membrane absorption. Mucin oligosaccharides are credited for creating a microenvironment having reduced pH’s ∼5.5–6.0 that favors enzymes finalizing digestion as well as absorption of two type peptides. This pH is speculated to optimize peptide forms having either a zwitterion-like charge for proton gradient transfer or be non-dissociated and passively diffused. A high frequency of peptides having either glycine or proline can be rationalized as providing particularly favorable electronic terms for peptide absorption by either approach while being non-competitive. The upper villus has first access to absorbed products where goblet cells assure continuance of mucin and continuity of the unstirred water layer. Glutamine is their dominant nutrient for synthesis of mucin oligosaccharides while also providing glutamic acid for either formation of its associated protein or is consumed for energy. Dietary cystine and threonine are frequently limiting, and their assurance is necessary for the mucin core while ready availability of glycine and/or serine together with proline also foster mucin formation. Unused absorbed products descend the villus in venules adjacent to the surface and provide nutrition as well as information about the lumen for adaptation of replacement cells. Sustaining the villus with absorbed nutrients takes priority for continuation of operational efficiency before their entry into the portal system and subsequent body use.

Academic research paper on topic "Gastric digestion of protein through pancreozyme action optimizes intestinal forms for absorption, mucin formation and villus integrity"

Animal Feed Science and Technology xxx (2016) xxx-xxx

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Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Gastric digestion of protein through pancreozyme action optimizes intestinal forms for absorption, mucin formation and villus integrity

Edwin T. Moran Jr.

Poultry Science Department, Auburn University, AL 36849, USA

ARTICLE INFO

ABSTRACT

Article history:

Received 2 September 2015

Received in revised form 19 May 2016

Accepted 26 May 2016

Available online xxx

Keywords:

Intestinal proteolysis Mucin formation Nutrient absorption Unstirred water layer Villus maintenance

Recovery of dietary protein proceeds through two phases of digestion before suitable forms exist for absorption. The first phase engages the gastric system where low pH weakens overall structures allowing pepsin to disrupt hydrophobic bonding and enhance aqueous compatibility. Secondly, trypsin, chymotrypsin and elastase proteolysis in conjunction with carboxypeptidases A and B cooperate to form a mixture of free amino acids and peptides that progressively arise in the small intestinal lumen. Free amino acids are dominated by the aromatic, aliphatic and basic ones while resulting peptides largely involve the nonessen-tials.

Motility convectively transfers digestion products to the unstirred water layer of the upper villus where filtration limits entry to low molecular weight solutes that can be further digested by underlying enzymes to optimize membrane absorption. Mucin oligosaccharides are credited for creating a microenvironment having reduced pH's ~5.5-6.0 that favors enzymes finalizing digestion as well as absorption of two type peptides. This pH is speculated to optimize peptide forms having either a zwitterion-like charge for proton gradient transfer or be non-dissociated and passively diffused. A high frequency of peptides having either glycine or proline can be rationalized as providing particularly favorable electronic terms for peptide absorption by either approach while being non-competitive.

The upper villus has first access to absorbed products where goblet cells assure continuance of mucin and continuity of the unstirred water layer. Glutamine is their dominant nutrient for synthesis of mucin oligosaccharides while also providing glutamic acid for either formation of its associated protein or is consumed for energy. Dietary cystine and threonine are frequently limiting, and their assurance is necessary for the mucin core while ready availability of glycine and/or serine together with proline also foster mucin formation. Unused absorbed products descend the villus in venules adjacent to the surface and provide nutrition as well as information about the lumen for adaptation of replacement cells. Sustaining the villus with absorbed nutrients takes priority for continuation of operational efficiency before their entry into the portal system and subsequent body use.

© 2016 Elsevier B.V. All rights reserved.

E-mail address: moranet@auburn.edu

http://dx.doi.Org/10.1016/j.anifeedsci.2016.05.015 0377-8401/© 2016 Elsevier B.V. All rights reserved.

2 E.T. Moran Jr. / Animal Feed Science and Technology xxx (2016) xxx-xxx

1. Introduction

Birds are known to maintain integrity of their alimentary tract at the expense of body growth as do mammals (Konarzewski et al., 1990). Increasing dietary protein with broilers has been shown to develop the small intestine, particularly duodenum-jejunum, before available amino acids are realized for growth (Wijtten et al., 2010). The requirements of amino acids for growth of individual tissues can be ascertained by differences in actual accumulation with time, whereas measurements conducted for maintenance were only valid on a collective basis for the body as a whole. Maintenance estimates for the growing bird have employed balanced amino acid diets (Baker et al., 1996). In these studies, the amino acid in question was progressively removed while determining the absolute decrease of all other amino acids in body protein (Baker et al., 1996). Leveille and Fisher (1958) employed a similar approach with adult roosters where nitrogen balance without body weight change was the objective. Maintenance requirements of the intestine perse apart from the body as a whole have not been attempted.

Measurement of digestible amino acids in dietary protein usually employs a correction based on ileal endogenous amino acids lost when feeding a N-free feed. The nature of such correction is prone to error by variation in amounts of mucosal sloughing as well as failed autolysis of digestive enzymes. Furthermore, these endogenous losses also vary with intestinal location used for collection, nature of the N-free feed, age of bird, and lumen microbial load (Butts et al., 1991; Adedokun et al., 2007a,b; Kamisoyama et al., 2010; Lee et al., 2011). Amino acids most consistently observed with endogenous loss indicate that mucin was a dominant contributor.

The small intestine has a particularly fast rate of growth during juvenile development while concurrently being confronted with an accentuated turnover of its mucosa (Bertalanaffy, 1960; Crompton and Walters, 1979). Protein nourishment of the villus appears to be prioritized and depend on absorbed amino acids before their release into the portal system. Extrusion of cells from the villus that occur with mucosal turnover can be rationalized as the primary reason for difference in amino acids between the lumen and their appearance in the portal system. Most protein associated with extruded cells is returned for reuse after its ensuing digestion; however, an exception to recovery is prominent with the surface mucins which defy digestion. Mucins arising from each part of the gastrointestinal tract during digesta transit co-mingle with other unsalvageable debris to represent endogenous N. Again, amino acids entering the ileum comprising endogenous N correlate well with those associated with mucin (Lien et al., 1997; Ravindran and Hendriks, 2004).

The following is a holistic view of protein digestion and absorption of its products followed by their use to accommodate ever changing terms in the lumen. Such an overview seeks to better understand the sequence of events with digestion and a basis for the ultimate absorption of amino acids and peptides. Partial retention of these amino acids by the villus is employed to assure continuity of its function at nutrient retrieval and protection. An appreciation of this retention is critical in applying corrections used to measure amino acid digestibility as well as modifications in the dietary requirement that are intended to address intestinal maintenance.

2. Gastric improvement of protein recovery

An overall structural destabilization of feed components represents the primary task of gastric digestion. Muscular activity of the gizzard in combination with proventricular juices reduces particulate size while fostering aqueous compatibility of the composite before passage into the duodenum. Similar events occur with ruminants and simple stomached mammals though different anatomical locations and structures are involved. Invariably, plant proteins are encapsulated by fiber walls of differing strength as the primary obstacle to their access. The fowl's gastric system resorts to breaching these walls by employing a combination of very low pH and a unique proteolytic activity in concert with physical force exerted by the gizzard. Essentially, hemicelluloses are central to plant feedstuff fiber and provide extensive interconnections H-bonding among cellulose fibrils to convey wall structure (Henriksson and Gatenholm, 2001). Such bonding can be weakened by very low pH, particularly with neutral detergent type fiber which dominates seed endosperm cells. Plant cell walls also have small amounts of protein paralleling animal connective tissue that further contributes its structural stabilization (Ringli et al., 2001; Rhodes and Stone, 2002; Ryser et al., 2003). Pepsin is adept at hydrolyzing animal structural proteins such as collagen and elastin; in parallel, this protein in neutral detergent fiber appears equally structured and susceptible to digestion. Low pH aids pepsin action by enabling a structural dishevelment that avails cleavage sites in endosperm walls just as heat treatments improve access and digestibility with difficult animal source proteins. Robertson et al. (1997) were able to increase the solubility of barley non-starch polysaccharides from 23 to 52 and 83% by in vitro treatments with pepsin and its combination with pancreatic enzymes, respectively.

Considerable homology exists among animal pepsins and each has similar specificity in proteolytic action (Kagayama, 2002). Avian proventricular oxnyticopeptic cells produce both HCL and pepsinogen. Pepsin is formed from pepsinogen after intermolecular rearrangements and a subsequent peptide loss by autocatalysis once pH is less than four (Bohak, 1969, 1973; Horvath, 1974). Low pH also alters the secondary structure of dietary protein during proventricular-gizzard transit by decreasing hydrogen bonding that impairs overall structural stability when superimposed on salt linkage minimization. Pepsin focuses on peptide bonds between aromatic and large aliphatic amino acids, particularly where several other aliphatic-aromatic amino acids are in succession on either side to create a defined hydrophobic area on the chain (Fig. 1). Hydrophobic bonding can exist between the end groups of opposing chains having a similar array of amino acids. This affiliation in an aqueous environment substantially contributes to structural stabilization while also fostering insolubility. Peptide

E.T. Moran Jr. / Animal Feed Science and Technology xxx (2016) xxx-xxx

Fig. 1. Schematic representation of hydrophobic bonding that occurs between chains in protein where each has several aliphatic-aromatic amino acids in sequence. These amino acids each have hydrophobic ends on one chain that collaborates with the ends from a similar array on another chain. Exclusion of waterand mutual affinity creates bond strength while also lending to insolubility. Pepsin specificity favors cleaving of peptide linkages between hydrophobic amino acids at the center of each chain, thereby reducing size and integrity of the protein while improving water compatibility.

cleavage at the center of opposing chains creates smaller proteins having aromatic and large aliphatic amino acids assume a large proportion of their amino and carboxyl ends with the resulting proteins of reduced size. Concurrent improvement in water compatibility becomes particularly important to subsequent proteolytic actions by the pancreatic enzymes under neutral terms within the small intestine. Proteins that were already soluble usually have minimal hydrophobic bonding and negligible hydrolysis occurs by pepsin due to rapid passage through the gastric system.

3. Small intestine proteolytic actions

All proteolytic enzymes from the pancreas, like those of the pepsin family, have a common evolutionary background and homology of their own; however, specificities of action differ markedly (Hartely et al., 1965). Trypsinogen activation is central to the subsequent activations of all other pancreatic enzymes. Upon entry of digesta into a vacant lumen, trypsinogen initially released is first activated by enterokinase which involves a cleavage and peptide release; thereafter, resident trypsin acts on all subsequent trypsinogen but with greater expediency (Mayer et al., 1974; Jeno et al., 1987; Brunger et al., 1987; Varon et al., 1990). Enterokinase appears to be located below the unstirred water layer of mature enterocytes which means that its accessibility for trypsinogen activation can only occur after enterocyte extrusion from the villus and exposure of its disrupted surface to lumen contents (Nordstom and Dahlqvist, 1969; Louvard et al., 1973; Lebenthal and Morrissey, 1977). Trypsin acts as an endopeptidase cleaving at the carboxyl end of peptide bonds with amino acids expressing an amino or like basic group at its free end, i.e., lysine, arginine and histidine (Peterson, 1966). The net effect of trypsin is to further reduce the size of protein at locations wherever basic amino acids are located. In so doing, another population of smaller proteins is created in which basic amino acids are now appearing in greater frequency at the carboxyl end. Such action contributes to the previous action by pepsin with the aromatics and aliphatics that had arisen at both amino and carboxyl ends.

Chymotrypsin complements the action of pepsin. But first, trypsin must perform a cleavage on chymotrypsinogen appropriate to its specificity and create a-chymotrypsin. Both trypsin and chymotrypsin are remarkably similar in structure and purpose among mammals and fowls (Ryan, 1965). Like trypsin, a-chymotrypsin is an endopeptidase but peptide bond cleavage favors the carboxyl end of either aromatic or large alphatic amino acids that exist singly within the protein chain (Cohen, 1969; Keller et al., 1984; Schellenberger et al., 1991; Kallies and Mitzner, 1996). During subsequent autolysis, a-chymotrypsin together with trypsin act to create a series of P-, 7-, and 8-chymotrypsins which seem to have similar specificities for the hydrophobic amino acids. While pepsin previously led to any of these same aromatic- and aliphatic-amino acids at both amino and carboxyl ends after cleavage, chymotrypsin opens the protein chain to further numbers of aromatic/aliphatics that are now restricted to the carboxyl end. Resulting peptides from chymotrypsin action also leads to a random presentation of other amino acids residing at the point of cleavage but appearing at the amino end.

Elastase is a lesser defined pancreatic endopeptidase that bears considerable similarity in specificity to chymotrypsin and could be viewed as an autolysis product if not recoverable within the pancreas. Again, the carboxyl end of peptide bonds represents the point of attack, but the minor aliphatic amino acids, particularly alanine, are favored. Elastin has considerable

E.T. Moran Jr. / Animal Feed Science and Technology xxx (2016) xxx-xxx

Fig. 2. Combined action of gastric and pancreatic proteolysis creates a mixture of free amino acids and polypeptides dictated by their specificities. Such a mixture is continuously being formed and changes with ease of hydrolysis and distal progression of the luminal mass. Motility convectively optimizes concentration difference at the lumen interface with unstirred water layer enabling all amino acids and sufficiently small peptides to enterthis first phase of enterocyte absorption.

alanine and is notably vulnerable to elastase, hence its name (Gertler and Feinstein, 1971). Net effect of all chymotrypsin-type proteases when superimposed on the resultant products of pepsin and trypsin creates a population of polypeptides having aromatic, aliphatic, and basic amino acids predominate at the carboxyl end. The amino end still reflects pepsin influence by presenting some aromatic and aliphatic amino acids; however, a random array of the others is expected to assume dominant proportions, especially from readily soluble proteins. Such a mixture of free amino acids and peptides is not a fixed quantity but relates to the characteristics and amino acid composition that typify each dietary protein. Pancreatic enzymes can change in their proportional contributions in response to alterations in the amount of dietary protein being consumed. Given that aromatic and aliphatic amino acids collectively occupy a large part of most dietary proteins, chymotrypsin has been shown to be the greatest respondent to change. Increasing dietary protein for the pig from 0 to 40% was observed by Corring and Saucier (1972) to increase chymotrypsin by 250% but only a 20% gain in trypsin.

Free amino acids do not occur until exopeptidase cleavage is initiated. Both of the pancreatic procarboxypeptidases require activation by trypsin in a manner corresponding to its specificity as was done with all other enzymes. Carboxypeptidase A and B bear similarity to each other in structure and homology while using Zn++ as a co-factor (Zelikson et al., 1971; Coll et al., 1991; Villegas et al., 1995). However, A-carboxypeptidase devotes its attention to aromatic and aliphatics at the carboxyl end of polypeptides while the B-enzyme prefers releasing the basic ones, i.e. lysine, arginine and histidine in free form (Christianson and Lipscomb, 1989; Suh, 1990; Hendriks et al., 1993; Banci et al., 1994). In order for continued functioning of both endoproteases as well as carboxypeptidases within the lumen, resident polypeptides must remain sufficiently large or entry into the unstirred water would restrict their access.

The combined and concurrent actions of the endoproteases and exopeptidases within the lumen result in a progressive formation of free amino acids together with polypeptides that motility conveys to the unstirred water layer (Fig. 2). Aromatic, large aliphatic and basic amino acids dominated those free and are all essential. Resulting polypeptides overwhelmingly contain the nonessential amino acids with a small portion of aromatic-aliphatics residing at the amino end that arose from prior pepsin action. Dominance of the free amino acids as being essential while small peptides would be largely composed of nonessential ones suggests that this result is an evolutionary adaptation to improve attaining nutritional needs (Hull, 1991). Alternatively, this result may be a possible means to improve absorption of those units that would either be adverse to water or have particular difficulty in membrane transit (Bull et al., 1978). Again, amounts and proportions of free amino acids relative to peptides are unlikely to be fixed quantities as much as a function of the protein(s) being digested. Raghunath et al. (1987) fed different proteins to rats and noted that animal sources yielded more peptides than free amino acids being present in jejunal supernatants while the converse was true with plant sources.

Variation in free amino acids and peptides within the lumen is also likely to differ with duration of enzyme hydrolysis and distal progression of lumen contents. Complete feed comprises an array of proteins having extremes in character. Structurally labile protein likely precedes disappearance compared to resistant sources leading to an amino acid-peptide composite that is quantitatively diminishing and evolving in character within the lumen. Location of the duodenum in fowl can be defined as that portion of the small intestine surrounding the pancreas with jejunum progressing thereafter to yolk sac remnant then ileum continuing to the ceca. The largest part of lumen protein recovery generally occurs prior to the end of the jejunum with a distinctive decrease in relative amount once within the ileum. Subsequent reduction in lumen amounts for recovery leads to mucosal minimization of villi needed to absorb lesser amounts of nutrients and overhead cost to do so (Fig. 3).

Contributions of protein resulting from the addition of pepsin and pancreatic enzymes to the lumen when taken together are substantial and variable. The amount of protein contributed in the form of pancreatic enzymes approximates 5-8% of the pig's total amino acid requirement (Corring, 1975). Enzyme autolysis further complements the amino acid-peptide mix (Rovary, 1988; Harel et al., 1991). Pancreatic enzyme autolysis is not easily accomplished, thus, their contributions to absorbable products are likely deferred beyond the yolk sac remnant. Difficulty in digestion can be expected with connective

E.T. Moran Jr. / Animal Feed Science and Technology xxx (2016) xxx-xxx

Fig. 3. Illustration generalizing recovery oftotal lumen protein during progression of contents from duodenum through each part ofthe small intestine with broilers receiving a commercial feed (Moran, unpublished). Pepsin initiates protein hydrolysis but realization of absorbable products does not occur until addition of the pancreatic proteolytic enzymes in the duodenum. The digestive enzyme composite together with mucosal debris adds substantial protein to the lumen which becomes superimposed upon that from the feed. A cascade of amino acids and peptides occurs with rapid digestion of labile protein along with other readily digestible nutrients through to mid-jejunum where high profile villi recover absorbable units. Reduction of labile protein with continuance of enzyme resistant proteins leads to a decreasing rate of digestion, and villi minimize prominence to relieve overhead cost. Such resistance is not only encountered with structural proteins but also digestive enzymes because of their purposeful protection from autolysis. (Constructed and redrawn from Moran, 1982).

tissues that dominate animal meals and usually provide low proportions of most essential amino acids; however, delayed proteolysis of the digestive enzyme composite may well favor attaining a reasonable balance to the nonessential ones as the composite moves distally. All pancreatic enzymes have associated calcium which is credited with overall structural stabilization that delays loss in activity until digestion of dietary protein is largely fulfilled, particularly trypsin (Caldwell, 1992). Autolytic recovery of enzyme protein is a necessity to nutritional economics given their extensive contribution to the animal's total amino acid requirement. Souffrant et al. (1993) observed that the recovery of pancreatic enzymes and extruded cells from the mucosa based on levels existing within the pig's duodenum was 79% recovered once at the distal ileum and considered to be endogenous N loss.

4. Accessing the absorptive surface

Extent of protein digestion is expected to diminish as digesta progresses from duodenum through to ileum just as the free amino acid-peptide mixture can vary in amount and composition. Regardless of proportions, amino acid-peptide combinations have consistently been shown to be at advantage in the rate of recovery compared to equivalent amounts of free amino acids while employing less energy to do so (Leveille and Fisher, 1958; Kan, 1974; Silk et al., 1975; Rerat et al., 1992; Tanabe et al., 1993). Wilson et al. (1971) examined the uptake of L-methionine and L-methionyl-L-methionine using in situ intestinal loops with the rat and noted that the rate of free amino acid uptake was more rapid distally while dipeptide was at advantage toward the proximal end. Amino acids are actively transported and systems exist that can be generalized as having anionic, cationic, and neutral preferences such that any and all free forms can be retrieved (Ganapathy et al., 2001; Hyde et al., 2003). Tasaki and Takahashi (1966) placed an equimolar mixture of 18 amino acids into an intestinal loop of adult fowl in situ then measured net absorption that had occurred over 10 min. Individual absorptive rate of one amino acid to the other was generally more favorable with the essential ones that would have been released after pancreatic proteolysis of intact protein (Table 1). Of particular interest is the 30% decrease between methionine that had the fastest absorptive rate as compared to glutamic acid having the lowest rate of recovery while its associated variance escalated three fold.

Presenting large amounts of dietary free amino acids into the intestine is expected to be different than would occur with the progressive appearance of an amino acid-peptide combination resulting from whole protein digestion. The gradual appearance of amino acids and peptides within the lumen creates in a marginal advance in osmotic pressure while concurrently encountering broad exposure to the wall for absorption (McWhorter et al., 2009). Although an immediate and concentrated presence of free amino acids at one location would eventually lead to their absorption, an extensive competition and complicated absorption kinetics seems to impair overall rate of recovery (Gous et al., 1977). Active transport of aliphatic amino acids given orally was shown to be more rapid in their transfer through to the blood when given in low concentration whereas recovery was depressed when high (Szmelcman and Guggenhein, 1966).

Presence of the bolus in the lumen by itself together with a post-absorptive sensing of nutrients in the lamina propria represents the basis for generating intestinal motility. The enteric nervous system is envisaged as coordinating motility such that absorbable nutrients arising during digestion do not concentrate nor stay in same location but move distally over a broad area commensurate with their formation in the lumen (Shuttleworth and Keef, 1995; McWhorter et al., 2009). Fowl

Table 1

E.T. Moran Jr. / Animal Feed Science and Technology xxx (2016) xxx-xxx

Absorption of individual amino acids from an eqimolar mixture administered to an in situ loop of rooster small intestine through a 10 min duration3.

Order AA %Absorb SEMb Order AA %Absorb SEMb

1 Methionine 89.6 3.3 10 Serine 78.5 4.1

2 Isoleucine 86.7 4.2 11 Threonine 78.4 5.7

3 Valine 86.0 3.6 12 Tyrosine 78.2 4.8

4 Leucine 84.9 4.1 13 Cystine 77.7 5.7

5 Tryptophan 83.3 4.4 14 Proline 77.7 6.4

6 Phenylalanine 82.9 4.2 15 Arginine 75.3 6.1

7 Histidine 79.8 4.3 16 Glycine 73.5 8.1

8 Lysine 79.6 4.9 17 Aspartate 69.2 8.5

9 Alanine 78.7 5.1 18 Glutamate 61.9 9.9

a Data taken from Tasaki and Takahashi (1966). Each amino acid was calculated to provide 12.5 ^moles/5 ml with the composite injected into a loop from 10cm above to 10cm below Meckel's Diverticulum. b Values represent 3 measurements ± standard error of the mean.

Fig. 4. Diagrammatic illustration of the primary components of the unstirred water layer. Membrane associated mucin (glycocalyx) linearly projects from the apex of enterocyte microvilli as repetitive O-glycosylated areas along its protein core. Length of the glycocalyx approximates depth of the unstirred water layer. Secretory mucins differ by being released in free form from near-by goblet cells and have similar repetitive glycosylated areas, but their arrangement one to the other resembles a net because of cystine inter-linkages. Once released, secretory mucin is envisaged as entangling with membrane associated mucin fixed at the surface to form the unstirred water layer where pore dimensions dictate solute size for entry. Microvilli also have contractile fibers within their core that convectively engage amino acids and peptides occurring within the unstirred network to finalize absorption. (Constructed and redrawn from Moran, 1982).

generally employ a refluxive type of motility as opposed to progressive peristalsis and segmentation with mammals. In both situations the extent of resulting surface convection is primarily driven by the major circular muscles while minor contractile fibers of the muscularis mucosa rotate villi to create a secondary motion further complementing wall exposure (Hodgkiss, 1982; Moran, 1982; Csonkya et al., 1990; Suzuki et al., 1996; Olssenand Holmgren, 2001). Mature surface cells and ability to formally conduct absorption reside on the upper villus where the greatest advantage from overall lumen convection occurs.

Enterocytes dominate the mature absorptive surface with goblet cells dispersed among them in a mosaic fashion (Michael and Hodges, 1973a; Humphrey and Turk, 1974a; Kurosumi et al., 1981). Nutrient absorption is the only defined activity conducted by mature enterocytes in place, whereas goblet cells are engaged in the continual formation and release of mucin. Intestinal motility beyond the intention of absorption also acts to move mucin upon its release across the microvilli surface of adjacent enterocytes thereby assuring continuance of the unstirred water layer. Depth of the unstirred water layer is largely determined by the length of membrane associated mucin extending from the microvilli apex or referred to as glycocalyx (Atuma et al., 2001). This co-operative effort between membrane associated mucin (glycocalyx) and secretory mucin from goblet cells results in what has been considered as the primary and purposeful barrier to formal absorption (Nimmerfall and Rosenthaler, 1980; Smithson et al., 1981; Smithson, 1983) (Fig. 4).

Fundamental differences exist between membrane associated and secretory mucins that are central to establishing the unstirred water layer and separation of lumen contents from absorptive membrane (Fig. 5). The glycocalyx corresponds to mucin fibers that are linear and anchored to the membrane by a short hydrophobic amino acid tail (Maury et al., 1995). Core protein extends into the lumen with many repetetive areas involving serine, threonine, proline, and alanine that are separated by intervals of hydrophobic amino acids. The hydroxyl ends of threonine and serine provide lateral extensions having 6-8 saccharides that sterically hinder destruction of the core from proteolytic action at the lumen interface (Bloomfield, 1983). Goblet cell mucins have similar bottle-brush like repeating units; however, these repeats appear to be farther apart between these intervals and largely interconnected by cystines to create a fishnet-like arrangement (Flood, 1981; Bansil et al., 1995;

E.T. Moran Jr. / Animal Feed Science and Technology xxx (2016) xxx-xxx

Fig. 5. Representation of absorptive activities of protein digesta within the unstirred water layer. O-linked saccharides are speculated to act as a buffer and create a microenvironment having a low pH. Such pH is envisaged to balance peptides between isocharged units for proton gradient uptake while the uncharged alternatives are thought to diffuse through the membrane. Taken together with the concurrent active transport of free amino acids unfavorable for peptide absorption, overall protein recovery by enterocytes would be minimally competitive, have an optimal rate, and employ less energy to do so.

Table 2

Dipeptides having an absorption rate more favorable as free amino acidsa.

Glycyl-L-Tryptophan

Glycyl-L-Tyrosine L-Phenylalanine-L-Phenylalanine Glycyl-L-Histidine L-Prolyl-L-Hydroxylproline a-L-Glutamyl-L-Glutamic Acid L-Arginyl-L-Aspartic Acid

a Taken from a list in Matthews (1971) of 36 different peptide combinations that were compared in their rate of absorption with an equivalent mixture of free amino acids. Peptides shown are those having a disadvantage in the rate of absorption when compared to presence as free amino acids.

Gendler and Spicer, 1995; Bansil and Turner, 2006; Perez-Vilar and Maboto, 2007). Essentially, this floating net is envisaged as becoming entangled with the fixed glycocalyx once released to create the surface composite referred to as the unstirred water layer. Pore dimensions of the soluble mucin now act as a molecular filter by restricting passage to small water compatible molecules such as sugars, amino acids, small peptides and lipid micelles while excluding molecules of greater size, particulates, and microflora.

5. Conducting absorption

Microvilli projecting from each enterocyte surface not only have an attached glycocalyx, but also expose immobilized enzymes that finalize digestion (Maroux et al., 1979; Kushak et al., 1981). While free amino acids passing through the unstirred water layer are ready for absorption, most polypeptides likely require further reduction to di- and tripeptides not to mention reduction of the dextrins, maltotriose, maltose from starch to glucose. Several aminopeptidases exist to reduce polypeptides, but two having broad specificities have been generalized (Danielson and Hansen, 2006). Aminopeptidase A can be credited for hydrolyzing the acidic amino acids, glutamic and aspartic acids as well as the large aliphatic and aromatic ones from the amino end of peptides while aminopeptidase N is credited with preference for the neutral ones (Arvanitakis et al., 1976; Benajiba and Maroux, 1981; Feracci et al., 1981; Matsushima et al., 1991). Such actions seem particularly favorable for release of the aliphatic-aromatic amino acids previously availed at the amino end by pepsin. The numerous di- and tri-peptides subsequently evolving have a propensity to contain either proline or glycine (Winkler et al., 1999; Aito-Inoue et al., 2007). Matthews (1971) compared the rate of intestinal uptake of di- and tripeptides having many different amino acid combinations versus the equivalent amounts when amino acids were in free form. Advantage in the rate of absorption between the two forms was particularly apparent when either glycine or proline shared the peptide, whereas the presence of aromatic, large aliphatic and basic amino acids usually led to peptides having a poorer rate of uptake than if the corresponding units had existed free (Table 2).

'Release of the aromatic and aliphatic amino acids in free form by the pepsin-chymotrysin-carboxypeptidase A sequence along with basic ones by trypsin-carboxypeptidase B pancreatic enzyme combinations is viewed as purposeful to favor the remainder to be within peptides. A difference exists in the solution characteristics between amino acids arising in free form and those likely to be as peptides. Those free have either an ionizable group in addition to the a-amino and a-carboxyl lending to an isoelectric point distant from an average pH of six or provide a dominant hydrophobic end to create poor aqueous solubility (Table 3). Aminopeptidase from chicken intestine has an optimal activity at pH 6 (Jamadar et al., 2003). The broad range of intestinal proton gradient peptide transporters optimize at pH 5.5-6.0 (Ganapathy and Leibach, 1985).

8 E.T. Moran Jr. / Animal Feed Science and Technology xxx (2016) xxx-xxx

Table 3

General values for amino acid solubility and their isoelectric points3.

AA g/100 ml pI AA g/100 ml pI

Alanine 16.7 6.00 Isoleucine 4.12 6.02

Arginine 14.9 10.76 Leucine 2.16 5.95

Asparagine 2.51 5.41 Lysine 9.47 9.74

Aspartate 0.54 2.77 Methionine 5.95 5.74

Cystine 1.12 4.60 Phenylalanine 2.80 5.44

Glutamate 0.86 3.22 Proline 5.00 6.30

Glutamine 4.30 5.65 Serine 25.0 5.65

Glycine 25.1 5.97 Threonine 9.80 5.54

Diglycine 23.3 - Tryptophan 1.38 5.89

Triglycine 6.30 - Tyrosine 0.045 5.63

Histidine 4.33 7.54 Valine 2.50 5.96

a Based on values in water approximating 25 °C.

Peptide combinations created during final digestion within the unstirred water layer would be dominated by amino acids that create a favorable solubility while also exhibiting a moderately acid isoelectric point to optimize rate of membrane transfer.

Preservation of glycine and proline content seems to be especially important to peptide absorption. Glycine is capable forming electrically neutral molecules as well as zwitterion types when at a moderately low pH while also being particularly soluble (Imamura et al., 1969; Shipman and Christoffersen, 1973). Proline having anomalous hydrophilic character can be rationalized as providing somewhat similar terms that would also advantage membrane transit of existing peptides (DeTar andLuthra, 1977; Gibbset al., 1991; Prajapati et al., 2007). Using frequent combinations of either glycine or proline with other amino acids can be rationalized as creating peptides that present two electronic forms permissible for ready absorption. Such terms are speculated to allow peptides having no apparent charge to be passively permeable through lipid bi-layers (Temple et al., 1998; Nagle et al., 2007) while enabling those being equivalently charged to employ a peptide membrane transporter (Ganapathy et al., 2001). Both peptide forms are visualized as being in a balance and coexisting one to the other within the unstirred water layer, thereby, allowing their recovery to be non-competitive concurrent with active transport of free amino acids (Fig. 5).

An array of peptide transporters having generalized preferences exist in parallel with those actively transferring amino acids. Peptide transporters are not absolute in requiring the existence of a peptide bond because many non-amino acid nutrients can be absorbed in this manner. Carboxylic acids, such as fumarate and citrate, are favored at low pH when dissociated as well as passive diffusion if non-dissociated (Wolffram et al., 1992). Absence of dissociation and/or having an isocharged molecule by 2-hydroxy-4-methylthiobutanoic acid also seems to agree with these same terms and relative absence of active transport (Maenz and Engele-Schaan, 1996a,b). While many peptides enter the enterocyte once absorbed, most are hydrolyzed to amino acids within the cytosol before basolateral exit to the vascular system (Josefsson and Sjostrom, 1966; Peters, 1970). Exceptions exist and peptides have been shown to be released from enterocytes when using gelatin hydrolysates; in turn, peptides having a preponderance of glycine and proline appear in the blood (Ohara et al., 2007).

The unstirred water layer not only filters digesta but protects underlying enzymes from demise by the lumen pancreatic enzymes. Once the products from microvillus enzymic action appear at the membrane surface, the unstirred water layer also prevents their being swept away with motility and readily consumed by lumen microbes. Microvilli submerged in the unstirred water layer can move to create convection within the surface mucin gel in parallel to villi with lumen contents. Thus, the internal contractile filaments of microvilli not only create convective favor for enzymes in finding substrate, but concurrently improve product access to absorptive sites (Mooseker and Tilney, 1975; Drenckhahn et al., 1983; Maroux et al., 1988).

6. Unstirred water layer microenvironment

The unstirred water layer can be rationalized as the basis for maintaining a consistently moderate and low pH as its microenvironment (Daniel et al., 1985; Shiau et al., 1985). Such an interface becomes central to effective nutrient recovery. This narrow range in pH optimizes operation of overall enzymes finalizing digestion on the microvilli (Mizuno et al., 1982; Jamadar et al., 2003) while supporting maximal proportions of peptides having electrical terms favorable for their transport. Glycine and proline become favorable participants in fostering absorption when combined with many other amino acids (States and Segal, 1968; Bojesen, 1987; Wolffram et al., 1992; Munck and Munck, 1994; Brandsch, 2006) as well as eventually being significant mucin components. Mediation of peptide transport by creation of a H+ gradient between the enterocyte surface and cytoplasm may not be the gradient perse as much as basis for presenting neutral and zwitterionic electrical peptides via a favorable microenvironment. Surface membrane "export" of H+ would concurrently balance the inward movement of Na+ associated with active transport (Ganapathy and Leibach, 1985; Ganapathy et al., 2001) while fortifying mucin oligosaccharide buffering from lumen influence.

Mucin oligosaccharides are negatively charged and act to buffer the unstirred water layer by virtue of constituent saccharides selected by the goblet cell during synthesis. Hansson et al. (1991) observed at least 28 structures involving modified,

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Fig. 6. Sequential development of cells on the villus that occurs during their movement from crypt to apex. Enterocyte maturation can be defined by its progressive prominence of microvilli and glycocalyx whereas goblet cells express increased content and release of secretory mucin granules. Proportions of one type cell to the other seem to arise during reproductive activity in the lower villus after provision of a common progenitor from the crypt.

sulfated, and sialyated oligosaccharides with swine small intestinal mucin. Distribution of fucose, galactose, mannose, N-acetyl-glucosamine, N-acetylgalactosamine, and sialic acid represents the basis of ionic character and whether the cell histologically stains either acid or neutral (Wesley et al., 1983; Apparecida et al., 1985; More et al., 1987; Pastor et al., 1988). Goblet cells represent the greatest contributor of mucin to the unstirred water layer as a whole. These cells turnover their contents several fold to maintain surface continuity whereas the glycocalyx from enterocytes is of a lesser quantity established during prior development (Lehr et al., 1991; Forstner, 1995).

Secretory mucins are synthesized within goblet cells then packaged by their golgi apparatus into granules for storage. In order to condense their size and minimize space, Ca++ appears to be layered within the granule to relieve the high charge density created by silationand sulfation (Black and Smith, 1989; Karlsson et al., 1996; Paz et al., 2003; Koga and Ushiki, 2006; Perez-Vilar, 2007). Subsequent granule release into the lumen is followed by Ca++ dissipation allowing the resulting sol to flow across the villus surface (Humbert et al., 1989; Specian and Oliver, 1991). Released mucins from all goblet cells can be envisaged as converging on the surface thereby providing an acidic buffer as a function of their continual contributions.

7. Absorptive surface replacement

A mucosal cell's half-life averages 3.5 days enabling the surface to continually be replaced and adapt to ever changing conditions in the lumen. The upper villus faces perpetual hazards from lumen microflora, particularly reduction of its oligosaccharides at the unstirred water layer surface (Fernandez et al., 2000; Cheld-Shoval et al., 2014). Lumen viscosity can occur with many feedstuffs, particularly wheat and barley that readily limits oxygen transfer from mucosa to lumen and foster anaerobe activity (Hillman et al., 1993; Moran, 2014). Such microbes are known to have an array of fucosidase and neuraminidase enzymes capable of hydrolyzing mucin polysaccharides, thereby availing core protein to lumen prote-olytic enzymes (Wold et al., 1974; MacFarlane et al., 1989; Forder et al., 2012). Cumulative damages to the absorptive area accentuate the rate of villus turnover (Cook and Bird, 1973; Turk, 1982).

Surface regeneration is initiated by multiplication of stem cells which leave the crypt then progressively mature commensurate with anticipated needs as they ascend the villus. Crypt multiplication not only relates to enterocyte-goblet cell progenitors which dominate the surface, but minor proportions of other cells dedicated to defensive activities appear as well (Muller et al., 2005; Barker et al., 2008; Salzman et al., 2007). While cell definition is lacking in the crypt, subsequent elevation into lower villus together with further multiplications lead to eventual commitment as being either an enterocyte or goblet cell (Cheng, 1974; Cheng and Leblond, 1974a,b; Paulus et al., 1993; Van den Brink et al., 2001). Uni et al. (1998) using PCNA staining illustrated proliferating cells on the lower villus with chicken intestine apart from those multiplying in the crypt. Differences in the dynamics between cells in the crypt and villus suggest that each population is being influenced to do so in a separate manner (Kaur and Potten, 1986b).

Cells on the lower villus once committed to their objective develop to meet assigned tasks with maturity (Fig. 6). Ente-rocytes during development form surface enzymes, transport systems, and glycocalyx that become associated with the concurrent presentation of microvilli (Brunner et al., 1979; Chambers and Grey, 1979; Shehata et al., 1984; Hoffman and Chang, 1993; Naim et al., 1999). Goblet cells increase in their ability to accrue and store mucin as they differentiate while yielding structurally distinct granules during the progression (Chambraud et al., 1989; Oliver and Specian, 1991). Presumably, granule modifications involve selection of saccharides during assembly of oligosaccharides that best support microenvironment. Enzymes associated with the synthesis of membrane and gel-forming mucin oligosaccharides are apparent during

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Fig. 7. Representation of the villus vascular system emphasizing arteriole and venule locations. One arteriole from the mucosa directly ascends to the villus apex with oxygen then venules subsequently carry absorbed nutrients to the base. Nutrients and other information about the lumen are conveyed via vascular contents to enable accommodation by developing cells to expected conditions. Another arteriole communicates with crypt about body information and cell needs for of the villus before returning to form a confluence with descending venules from the villus and portal entry. (Adapted and redrawn from Aharinejad et al., 1991; Courtesy of Scanning Electron Microscopy Inc.).

development with both enterocyte and goblet cells, particularly after microbial encounters in the lumen (Weiser, 1973a,b; Umesaki et al., 1982; Wilson et al., 1984).

8. Villus vascular system

Villus microvessels accomplish many objectives: supply oxygen, remove absorbed nutrients, and provide logistical support for all facets of epithelial operation (Fig. 7). Arterioles in the submucosa divide with a branch directly accessing and devoted to the crypt while another ascends to the villus apex (Kumoro and Hashimoto, 1990; Aharinejad et al., 1991). Once at the apex, a multitude of venules subsequently cascade down the villus immediately adjacent to mature, developing, and eventually multiplying epithelia at the base. Not only is oxygen provided during vascular transit, but nutrients that had been absorbed are now accessible for use by all villus cells. Immediate to absorption, a portion of these nutrients appear to be removed by adjacent goblet cells to support actively forming and secreting mucin before their subsequent descent to cells in the midst of development and multiplication at the villus base (Fig. 8).

Absorbed nutrients are expected to influence the cells of each population and foster activities that optimize their operation. Cells multiplying at the base are envisaged as either lengthening or shorting of their synthetic phase during mitosis commensurate with nutrient concentration. In turn, this alteration in regeneration rate leads to either a lengthening or shortening of the villus (Rose et al., 1971; Michael and Hodges, 1973b; Rijke et al., 1974; Fasina et al., 2007). Beyond multiplying cells on the villus, a confluence now occurs below the base with the vessel separately returning from crypt. This villus-crypt vascular composite now enters the portal system to carry contents for the body at-large. All vessels throughout the epithelial area are of the fenestration type to allow ready molecular exchanges as opposed to the continuous type that follow the confluence. Unlike mammals, fowl have no central lacteal nor associated lymphatic system (Perry and Granger, 1981; Bohlen, 1984; Ohtami, 1987). Total mesenteric arterial flow is dominated in use by the mucosa and submucosa vessels in a manner that decreases from duodenum to ileum to accommodate overall reduction of digestive intensity and villi dimension as nutrients absorbed from the lumen dissipate. In a converse manner, returning blood within the portal system is low distally and increases toward the duodenum in order to accommodate the progressive amounts of absorbed nutrients to the liver (Mailman, 1982; Shepard, 1982; Dinda and Beck, 1986). Overall control of blood flow within the small intestinal system revolves around the array of gastrointestinal hormones, extent of motility, and neural integration (Fondacaro, 1984; Premen et al., 1985; Holzer, 2006).

The mature surface of the upper villus appears to influence lower cells beyond nutrients provided from absorption. Ornithine decarboxylase is central to controlling polyamine formation and continuance-stabilization of RNA-DNA such that protein synthesis and development are supported accordingly. Substantial activity of this enzyme is located at the upper villus and crypt but lacking within cells between these areas (Raina and Janne, 1975; Iwami et al., 1990; McCormack and Johnson, 1991). A short half life of ornithine decarboxylase approximating 10-30 min is key to having cells respond when

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MATURE SURFACE

Lumen Digestion

VILLUS VASCULAR SYSTEM

Fig. 8. Illustration of epithelial cell interrelationships at the upper villus surface. Enterocyte absorption of protein digesta is transferred to the underlying lamina propria for venule removal as its primary activity. Goblet cells at the mature surface are concurrently forming mucin using immediately absorbed nutrients commensurate with need to maintain integrity of the unstirred water layer. Secretory mucins are continuously formed and released to maintain the unstirred water layer whereas membrane associated mucins had been fixed in place during earlier development. (Adapted and redrawn from Freeman and Geer, 1965; Courtesy Springer International Pub. AG).

opportunity and need exist as well as minimize synthetic activity in the absence of nutritional support. Its presence high on the villus occurs when no developmental activity is apparent other than mucin synthesis. Feeding and fasting alter the activity of this enzyme, particularly the presence of glycine, cystine, glutamine, and asparagine (Minami et al., 1985; Dagostino et al., 1987; Kandil et al., 1995). Emphasis in stimulation of ornithine decarboxylase by the aforementioned amino acids and their high content in mucin infer that maintenance of the unstirred water layer is the key motive. Thus, polyamine appearance can largely be attributed to goblet cell response as nutrients become available. Enterocytes in the upper villus have matured and nutrient absorption perse represents their near exclusive activity. Polyamines generated at the upper villus do not appear to be restricted to this location but purposely released into circulation to foster cell development at a lower level concurrent with the presentation of nutrients that had just been absorbed (Kobayashi et al., 1992; Johnson et al., 1995; McCormick and Johnson, 2001).

Multiplying crypt cells have also been found to express ornithine decarboxylase activity. The apparent driving force for its synthesis at this location seems largely involved with peripheral trophic hormones in conjunction with basal nutrient levels in blood conveyed by its separate arteriole (Fitzpatrick et al., 1986; Johnson et al., 1989; Ginty et al., 1990). Blood descending the villus will have established a confluence with blood returning from the crypt then both contributions proceed to the portal system. This separation would avoid mutual sensitivity from polyamines generated at either source. Thus, multiplication and kinetics of cells in lower villus versus those in the crypt would essentially be performing as separate populations (Kaur and Potten, 1986a,b,c). Conceptually, the crypt directly receives data about body condition at-large then accommodates villi with new cell support based on these conditions (Loeffler and Grossman, 1991; Slupecka et al., 2010). Should overall body reserves be at risk then a corresponding decrease of cells from the crypt would reduce overhead cost of villi prominence; conversely, fission and budding could initiate additional villi for mucosal expansion if abundance is perceived.

9. Villi adaptation to lumen conditions

Extent and nature of intestinal protein is in a continual flux; in turn, all sympathetic modifications incorporated with cells on the lower villus should be developing in concert with lumen conditions. Amounts of amino acids, peptides, sugars, and fat absorbed high on the villus provide information needed for subsequent response with cells below (Hirst, 1993). Microvillus enzymes and transfer sites for dissacharides (Siddons, 1972; Cezard et al., 1983; Ferraris et al., 1992; Wetzel et al., 2009; Moran et al., 2010) as well as peptides (Feracci et al., 1982; Raul et al., 1987; Gilbert et al., 2010) have been shown to be modified with developing enterocytes for subsequent tasks. Such changes are not expected to be uniform with the intestine-at-large but responsive to lumen contents from anterior to distal locations commensurate with the nature and extent of nutrient exposure (Ozols and Sheshukova, 1984; Rouanet et al., 1990; Gilbert et al., 2010). Given that digestion progresses along the intestine with varying amounts of nutrients and difficulties in their hydrolysis, then villus optimization likely becomes location specific. In turn, nutrients ultimately released into the portal system at any one location can be expected to vary accordingly. However, the sum of contributions at each location from beginning to end of the small intestine can be expected reflect total feed nutrients ultimately entering the portal system for hepatic manipulation and subsequent use by the body-at-large.

As suggested earlier, cells exiting the crypt continue to multiply but do so in a different manner once on the villus to create a separate dynamics. Commitment to being either enterocyte or goblet cell does not seem to be established for several generations on the lower villus (Kaur and Potten, 1986a,b). Ornithine decarboxylase generating polyamines released at the

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GLUCOSE <

Fructose-6-P Glucosamine-6-P

"Grain " Starch-Prolamines

GLUTAMINE

lutamic Acid

N-CHO's....^ MUCIN ï FORMATION

Fig. 9. Nourishment of enterocytes and goblet cells to form their respective mucins depend heavily on absorbed glutamine and glucose. Both nutrients can be readily provided by prolamines and starch in grain. This combination of substrates is the primary path to N-glucosamine and all related saccharides for oligosaccharide construction. Subsequent glutamic acid can further contribute to the protein core while an alternative transamination of pyruvate forms alanine while resultant a-keto glutaric acid provides substantial energy.

mature surface is positioned to alter the objective of these lower villus cells in agreement with previously absorbed nutrients. Polyamines would not only support mitotic intensity but likely influence commitment to being either an enterocyte or goblet cell (Sepulveda et al., 1982; Buwjoom et al., 2008; Laudadio et al., 2012). The ratio of enterocyte to goblet cells is not a fixed quantity but appears to vary with cell invasion at the surface and/or need for mucin protection. Microbial threats seem particularly effective at stimulating enhanced goblet cell number and mucin (Humphrey and Turk, 1974b; Kudweis et al., 1989; Meslin et al., 1999; Yi et al., 2005; Walk et al., 2011). Escalating microbial load also accentuates the proportion of sulfated and sialyated mucins contributed to the surface (Forder et al., 2007; Cheld-Shoval et al., 2014). As inferred earlier, lumen nutrients in quantity and quality would be continued after absorption in villus blood. These terms could also direct saccharide modifications to accommodate pH appropriateness within the unstirred water layer (Uni et al., 2003; Thompson and Applegate, 2006; Sharma et al., 1997).

10. Operational energy

Research on the metabolic needs of intestinal epithelia has generally been conducted on a cell composite without distinction as to either cell type or location on the villus. Results have been based either on metabolite differences between lumen presentation and serosal/portal appearance or changes associated with cell collections isolated from the brush-border. Duee et al. (1995) approximates that the GI is responsible for 20-25% of whole body oxygen consumption that can be attributed to energy expenditure in sustaining its high fractional rate of protein synthesis and epithelial cell turnover. Overall results have established that glutamine is the primary respiratory fuel, particularly when provided in conjunction with glucose which itself is only partially combusted (Windmueller and Spaeth, 1978, 1980; Porteus, 1980; Windmueller, 1980; Kight and Fleming, 1995a; Cremin and Fleming, 1997; Fleming et al., 1997; James et al., 1998). In reality, substantial differences in the nature of energy consumption can be expected between enterocytes and goblet cells from absorbing surface to crypt. Once mature, enterocytes are primarily devoted to transferring nutrients from lumen through to basolateral membrane; thus, glucose would be readily available and uncomplicated as an energy source (Reisenfeld et al., 1982). Although goblet cells in the same area are usually far fewer, their relative needs would be more extensive and complicated because of continuous synthesis and release of mucin.

Exclusive devotion to mucin formation and release is suggested as the basis for a dominant consumption of glutamine and considerable other amino acids by goblet cells. Glutamine is intricately involved in many facets of mucin formation (Fig. 9). Glutamine together with fructose-6-phosphate from glycolysis is the only means to form glucosamine-6-phosphate representing the first and rate limiting step in the formation of all hexosamines (Li et al., 2007; Buschiazzo and Alzari, 2008; Durand et al., 2008; Floquet et al., 2008). Selective polymerization of resulting sialic and neuraminic acids leads to short chain negatively charged oligosaccharides that are attached to the mucin protein core. Glutamic acid resulting after amine removal not only substantially contributes to protein core but can readily be used to transaminate pyruvate from glycolysis and form alanine which is another meaningful mucin contributor. Remaining a-ketoglutarate can now become the dominant and direct source of energy via the Krebs Cycle (Volman-Mitchell and Parsons, 1974; Watford, 1994; Kight and Fleming, 1995b; Wu, 1998; Lambert et al., 2002; Blanchier et al., 2009). The benefits of dietary glutamine to gut integrity and health are well established (Van der Hulst et al., 1993; Bartell and Batal, 2007).

The importance of glutamine in sustaining the intestine is illustrated by its continuing supply from the body when dietary sources diminish. Glutamine is the most abundant amino acid in circulation and maintained by degradation of muscle proteins which can be substantial by virtue of available mass (Change and Goldberg 1978; Teleni, 1993). Such release by muscle exclusively involves branched chain amino acid transamination, particularly leucine. Donor a-ketoglutaric acid from

E.T. Moran Jr. / Animal Feed Science and Technology xxx (2016)xxx-xxx 13

the Kreb's cycle forms glutamic acid which then leads to glutamine by via glutamine synthetase (Wu and Thompson, 1987a,b; Wu et al., 1989; Thompson and Wu, 1991). The by-product ketone body (2-oxoisocaproate, KIC) is also released to serve as an energy source for the mucosa and complement glutamine. Muscle from mammals seems more adept at glutamine formation than birds probably because of their prominence of mitochondria, which relates to differences in proportions of dark as opposed to white fibers (Wu et al., 1991). Chick embryos in the midst of hatching preferentially use the yolk sac membrane as a means of supplying glutamine for the developing intestine (Pons et al., 1986). Mucin formation is not isolated with the small intestine but all other mucosal surfaces where the vascular system represents the only access to nutrients. On a practical basis, considerable glutamine can be provided by the grain prolamines to exceed intestinal needs and enter portal circulation. Superfluous amounts of glutamine that increase blood level are now available to muscle fostering a repletion of myofibers appearing as enhanced growth (Wu and Thompson, 1990; Moundras et al., 1993).

11. Mucin and limiting amino acids

All amino acids comprising mucin are important; however, only those limiting in the diet to the extent that villus operation would suffer are critical. Amino acids associated with endogenous N are expected to reflect replacement for mucin; again, the extent of this replacement would not be fixed but largely differ in amount by virtue of feed characteristics and its microbial load (Ravindran et al., 2004, 2009). Invariably, the sulfur amino acids are first limiting in commercial feeds, particularly cystine when corn-soybean meal combinations are employed. The sulfur amino acids are especially crucial to the intestine where over 25% of dietary methionine is consumed for transmethylations and transulfurations en route to cystine as well as polyamine and nucleic acid support (Webel and Baker, 1999; Burrin and Stoll, 2007). Cystine is central to formation of secretory mucin by goblet cells (Park et al., 2009). A sulfur amino acid deficiency is known to up regulate methionine cycle activity while suppressing villus epithelial cell growth (Bauchart-Thevret et al., 2009).

More often than not, supplemental methionine is employed to rectify a dietary cystine deficiency. In turn, substantial ability exists by the intestine to convert D-, L-methionine, and DL-2-hydroxy-4-methylthiobutyrate into cystine. An absolute deficiency of all sulfur amino acids in the feed creates a more severe repercussion on live performance than absence of any other amino acid (Kino and Okumura, 1986; Martin-Venegas et al., 2006). As a result of prerequisite need for cystine in mucin and use of methionine for its synthesis, the amount reaching portal system represents a net loss with swine (Fang et al., 2010). Essentially, use of methionine to replace cystine leads to an accentuated appearance of cystine in ileal loss as mucin to imply that cytine is poorly digested from the feed being examined (Ravindran et al., 2002). Indirect support of this metabolic redirection within the mucosa appears as a 30% greater enteral need for methionine in swine than when parenterally given (Shoveller et al., 2005).

Like cystine, low dietary threonine would preferentially be used for mucin formation, thereby expressing an array of secondary problems in live performance when marginal (Kidd, 2000; Azzam et al., 2011). Low dietary threonine with broilers can distinctly reduce energy and N retention to reveal an impaired efficiency of nutrient recovery while also decreasing live performance (Dozier et al., 2000, 2001). Subclinical Clostridium infections that increase mucin need, in turn, accentuate threonine requirement (Star et al., 2012). Like methionine, threonine also suffers a loss during first-pass intestinal metabolism with pigs as an apparent retention in the mucosa (Stoll et al., 1998; Shaart et al., 2005). Again in parallel to methionine, threonine requirement can be attained at a lower level when given parenterally as opposed to enteral access (Shoveller et al., 2003). Other potentially limiting essential amino acids that could influence mucin formation involve valine and isoleucine given their core protein incorporation between bottle-brush units, particularly when using corn-soybean formulations.

Glycine, serine, and proline are substantial nonessential amino acid contributors to mucin's core protein as is glutamic acid. Although serine and glycine can be readily converted to one the other (Featherston, 1975,1976a), their de novo synthesis is not easily performed (Walsh and Sallach, 1966). Glycine has been shown to be of consistent advantage for broilers receiving minimal crude protein in conjunction with marginal levels of either threonine and/or sulfur amino acids (Dean et al., 2006; Corzo et al., 2009; Waguespack et al., 2009; Powell et al., 2011; Ospina-Rojas et al., 2013). Similarly, proline can be formed from glutamic acid, but numerous steps are also encountered to create a delay in appearance for use (Ross et al., 1978). Evidence for proline being of dietary advantage by itself is similar to glycine and occurs when a substantial immediate need exists. On the other hand, glutamic acid can be easily synthesized for cell use from diverse sources and has not been shown to be an issue (Sugahara and Arioshi, 1967; Parsons and Volman-Mitchell, 1974; Featherston, 1976b; Maruyama et al., 1976).

In practical feeds, glycine, serine and proline have to be considered together when viewing mucin formation. Delays in ready proline and glycine-serine synthesis result in reduced live performance when these amino acids are substantially deficient (Bhargava et al., 1971; Graber and Baker, 1973). Edwards et al. (1997) measured the chick's total maintenance requirement in terms of its whole body change in amino acids. As dietary threonine increases from inadequate to sufficient levels with chicks, the whole body content of glycine, proline, serine, and cystine concurrently decrease to indirectly suggest significant diversion for intestinal maintenance. Proline and glycine are known to cooperate in structural proteins such as collagen (Ananthanarayanan et al., 1985; Okuyama, 2008) and also appear together within the mucin core; but in this situation, use avoids structural stability. Digestion of structural proteins is known to provide various glycine-proline peptides that pass through the absorptive membrane then continue as such and enter circulation (Liu et al., 2009). Immediate access to such forms concurrent with other crucial amino acids appears especially favorable for goblet cells on a post-absorptive

14 E.T. Moran Jr. / Animal Feed Science and Technology xxx (2016) xxx-xxx

basis as ready support for mucin formation. Marginal feed levels of glycine and proline also become important when directly supplemented for broilers confronted with a coccidial vaccination challenge (Lehman et al., 2009).

12. Overview

The travail of dietary protein through the GI and absorptive recovery subsequently escalates in complexity by virtue of an array of uses by the villus to perpetuate its optimal functioning. Many voids in established understanding exist. In order to provide continuity in a holistic manner, considerable indirect evidence provided by extensive cited literature was employed to support speculation. Digestion of protein through selective hydrolysis by lumen enzymes from the pancreas and those at the absorptive membrane provide a mixture of amino acids and peptides that are favorable for maximizing rate of absorption with minimal expenditure in energy. The free amino acids have characteristics of being either highly ionized and/or especially hydrophobic, thus, active transport is employed to access the enterocyte. Peptides are usually no more than three amino acids and a high proportion of these contain either glycine or proline. Negatively charged oligosaccharides associated with mucin have been rationalized as buffering the unstirred water layer. An acidic value is speculated to favor generation of peptides having electrical arrangements which enable noncompetitive means for their absorption. Presumably, proton gradient transport uses peptides having electro-neutral peptides while diffusion is an alternative when charges are absent. Incorporation of either glycine or proline appears to play a significant role to this end for both type peptides. An array of digestion products that are largely not competitive with each other during membrane transport translates into an overall advantage in rate of recovery and energy expenditure to do so.

Maintenance of the unstirred water layer is considered central to effective nutrient recovery. A substantial commitment devoted to this end makes glutamine especially important, particularly for oligosaccharide synthesis while providing energy in the process. Goblet cells and enterocytes progressively develop as they ascend the villus to mature. During ascent, digestion products in descending blood vessels convey broad-based information for cells to functionally adjust their absorptive efficiency and surface protection. Threonine and cystine are critical to mucin synthesis because of their marginal levels in feed. Considerable amounts of glycine, serine, and proline are also needed; although being nonessential, their difficult de novo syntheses make dietary presence an advantage. Amino acids used for mucosa maintenance take priority before portal access and body support; thus, marginal inadequacies are more likely to impair feed conversion from increased consumption in order to satisfy growth potential. Relative impact of these inadequacies probably varies with mucosal threat to influence intestinal maintenance apart from amino acid needs of the body at-large. The practice of assuming that amino acid requirements are a fixed ratio of one to other seems inappropriate given that variation in specific ones for mucin formation often differ in their relative amounts needed for body weight gain.

Conflict of interest

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