24 Gastrointestinal protein and amino acidmetabolism in growing animals1

D. G. Burrin

USDA/ARS Children’s Nutrition Research Center,

Department of Pediatrics, Baylor College of Medicine,

1100 Bates Street, Houston, Texas 77030

The tissues of the gastrointestinal tract play a major role in the metabolism

of protein and amino acids in growing animals. Intestinal tissue has a high

rate of protein metabolism, which is directly linked to the high rates of pro-

liferation, protein secretion, cell death and desquamation of various epithe-

lial and lymphoid cells within the mucosa. Because the small intestine is the

first tissue exposed to the diet, it has a key regulatory role in the digestion,

absorption, metabolism and availability of dietary protein and amino acids

for growth. The major oxidative fuels for the intestine are glutamate, gluta-

mine, aspartate and glucose; however, some essential amino acids are also

oxidized, such as lysine, leucine and phenylalanine. Among the essential

amino acids, threonine utilization is particularly high, and may be critical

for normal intestinal function. Intestinal protein and amino acid metabolism

is primarily regulated by the modality (enteral versus parenteral) and quan-

tity of nutrient intake, and to a lesser extent by the composition of nutrients.

1This work is a publication of the USDA=ARS Children’s Nutrition Research Center, Department ofPediatrics, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX. The work was sup-

ported in part by federal funds from the U.S. Department of Agriculture Agricultural Research Service,

Cooperative Agreement No. 58-6258-6001, by the National Institutes of Health R01 HD33920. The con-

tents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agri-

culture, nor does mention of trade names, commercial products, or organizations imply endorsement by

the U.S. Government.

695 Biology of the Intestine in Growing Animals

R. Zabielski, P.C. Gregory and B. Westrom (Eds.)

# 2002 Elsevier Science. B.V. All rights reserved.

Intestinal metabolism has a significant influence on the quantity and pattern

of amino acids required for growth, particularly arginine and threonine.

1. INTRODUCTION

During the last 20 years, research studies established that the tissues of the

gastrointestinal tract have a substantial impact on whole-body protein and

amino acid metabolism in growing animals. The portal-drained visceral

(PDV) tissues, comprised largely of gastrointestinal tissues, contribute

between 3 and 6% of body weight, but they account for 20 to 35% of

whole-body protein turnover and energy expenditure (Burrin et al., 1989;

Yen et al., 1989; Nieto and Lobley, 1999; Stoll et al., 1999a). The dispro-

portionate impact of gastrointestinal tissues on whole-body metabolism is

a function of their relatively high fractional rates of protein synthesis and

oxygen consumption. Studies with domestic animals have demonstrated that

the fractional synthesis rate of protein in the intestinal tissues is several-fold

higher than that in peripheral tissues, such as muscle (Lobley et al., 1980,

1992; Attaix et al., 1986; Burrin et al., 1992; Davis et al., 1996). Although

we know much about the heterogeneous phenotypes and characteristic func-

tions of the cells that comprise the intestinal mucosa, the quantitative influ-

ence of these different cell types on mucosal protein metabolism as a whole

has yet to be fully delineated. Moreover, we have a limited knowledge of

how the spatial localization of cells within the mucosa determines the

source of substrate, its supply, and rate of metabolism.

Two critically important physiological functions of the intestine that are

necessary for maximal growth of the organism are: (1) to digest and

absorb dietary nutrients, and (2) to serve as a biological barrier in order

to prevent or minimize effects of environmental pathogens, toxins and

other antigenic molecules. Studies during the past decade have suggested

that ensuring an adequate nutrient supply to the intestine, mainly via the

oral or enteral route, is essential to maintain these functions. Currently,

efforts are increasing to quantify the extent to which the intestine uses

nutrients for its own purposes, and modifies the rate and pattern of amino

acid supply for peripheral tissue growth (Reeds et al., 1993; Reeds et al.,

1999; Burrin et al., 2001). Given the significance of the intestinal tissues

to whole-body metabolism, the investigation of those factors that substan-

tially alter intestinal mass has become increasingly relevant to the study of

whole-animal growth.

The intent of this review is to characterize gastrointestinal protein

and amino acid metabolism by examining (1) the underlying influence of

anatomy and cellular function, (2) the major metabolic fates of amino acids,

696 D. G. Burrin

and (3) the relative significance of nutrition, ontogeny, infection and inflam-

mation.

2. ANATOMICAL AND MORPHOLOGICAL BASIS

OF GUT METABOLISM

2.1. Regional metabolism within the gastrointestinal tract

There are notable differences in the rates of metabolism within the tissues of

the gastrointestinal tract. Studies in developing animals indicate that the

rates of protein synthesis are higher in the small intestine than in the sto-

mach and large intestine (Attaix and Arnal, 1987; Attaix et al., 1992; Burrin

et al., 1999a). Within the small intestine, the protein synthesis rate is highest

in the duodenum, and declines longitudinally (fig. 1) (Attaix et al., 1992;

Stoll et al., 2000a). There are numerous estimates of non-essential amino

acid metabolism (mainly glutamine, arginine, citrulline and ornithine) in

primary isolated enterocytes (Darcy-Vrillon et al., 1994; Quan et al.,

1998; Wu, 1998), colonocytes (Mouille et al., 1999), and colon carcinoma

cell-lines (Selamnia et al., 1998). One study comparing isolated cells

from the jejunum, caecum and colon found no difference in the glutamine

Fig. 1. Fractional rates of protein synthesis is small and large intestinal setments of milk-

fed and weaned lambs. (Adapted from Attaix et al., 1992).

Gastrointestinal protein and amino acid metabolism in growing animals 697

oxidation rate, despite regional differences in glucose and short-chain fatty

acid oxidation (Fleming et al., 1991).

2.2. Cellular basis of mucosal metabolism

The high rates of protein metabolism in the gut are directly linked to the

high rates of proliferation, protein secretion, cell death and desquamation

of various epithelial and lymphoid cells within the mucosa. In vivo labeling

studies in young pigs show that the epithelial cells have a life span of about

3 to 10 days, depending on the intestinal region and stage of development

(Smith and Jarvis, 1978; Fan et al., 2001). Within the small intestinal epithe-

lium, the proliferative crypt compartment contains pluripotent stem cells,

which undergo mitosis and differentiation into four cell lineages: absorptive

enterocytes, mucin-producing goblet cells, antibacterial peptide- producing

Paneth cells, and enteroendocrine cells. The differentiated phenotype of

these cells defines their main functions, which are to digest and absorb diet-

ary nutrients and provide an innate mucosal barrier to the luminal environ-

ment. Lymphoid cells are the other major class of cells located mainly in the

lamina propria region beneath the epithelial monolayer. These cells include

T- and B-lymphocytes, macrophages, neutrophils, mast cells, dendritic cells

and fibroblasts that are diffusely organized around a highly vascular and

neuronal network. Studies in vitro show that these epithelial (Higashiguchi

et al., 1993; Wu 1998) and lymphoid (Szondy and Newsholme, 1990;

Dugan et al., 1994) cell types exhibit high rates of protein synthesis and

glutamine metabolism. The protein secretory capacity of goblet cells is also

substantial, and mucin protein represents a significant component of endo-

genous secretions that pass undigested to the terminal ileum (Lien et al.,

1997; Montagne et al., 2000).

2.3. Luminal vs arterial substrate input

Another important anatomical consideration with respect to the metabolism

of epithelial cells is that they derive nutrients from two separate sources: the

intestinal lumen, via the apical=brush-border membrane, and the blood, viathe basolateral membrane. Studies in piglets with isotopically labeled sub-

strates have shown that there is significant utilization of both luminal and

arterial amino acids. It is of interest to note that, in pigs fed a milk-based

formula, the input of amino acids from the luminal route (i.e. diet) is far

greater than that from the arterial circulation (fig. 2). With respect to the

uptake of amino acids, particularly glutamate and glutamine, there is mark-

edly greater uptake from the luminal (67–96%) than the arterial (11–21%)

698 D. G. Burrin

input. Interestingly, the uptake of luminal glucose, both on a relative and

absolute basis, is considerably less than that of amino acids (� 6% of

input). It is possible that this combined phenomenon is a function of the

inherent amino acid and glucose transport capacities of the apical (luminal)

and basolateral (arterial) surfaces of intestinal enterocytes. However, in

absolute molar terms, the uptake of some amino acids from the arterial

and luminal routes is nearly equal, because the rate of arterial amino acid

input is more than five times greater than that from the diet. Thus, some

amino acids used by the gut are derived equally from the diet and arterial

supply. In contrast, intestinal glucose is supplied largely by the arterial cir-

culation (85% total), with only a small contribution from the diet (15%).

The relatively high rates of first-pass metabolism of dietary amino acids

may be a phenomenon that is largely confined to the small intestine rather

than the stomach or large intestine. That is because when intact protein is

consumed, the level of free amino acids is normally low in the stomach

as well as the large intestine due to bacterial fermentation. If the digestive

environment in the stomach and large intestine limits the luminal amino

acid availability, then the arterial circulation must meet the amino acid

needs of these tissues. This idea is supported by the evidence that during

total parenteral nutrition, atrophy occurs primarily in the proximal region

Fig. 2. Contribution of dietary and arterial amino acid input into the portal-drained visceral

tissue in young pigs fed cow’s milk formula. (Adapted from Van Goudoever et al., 2000;Stoll et al., 1999b; Stoll et al., 1998; Burrin et al., 1999b).

Gastrointestinal protein and amino acid metabolism in growing animals 699

of the small intestine, while the mass of the distal small intestine, stomach

and large intestine is preserved (Goldstein et al., 1985; Morgan et al., 1987;

Ganessunker et al., 1999).

2.4. Crypt vs villus cell metabolism

The extent to which epithelial cells derive their nutrients from the luminal

or vascular input is further influenced by their stage of differentiation and

physical location along the crypt-villus axis. Early studies (Alpers, 1972)

showed that crypt cells are more highly labeled with isotopic tracers derived

from the blood, whereas villus cells are more highly labeled with tracers

given luminally (fig. 3). Although these findings seem logical, in a general

sense, we still know very little about how the stage of differentiation and

amino acid transporter expression on the apical and basolateral surface of

enterocytes affect the rate and pattern of nutrient utilization. Studies with

cultured enterocytes indicate that the mitogenic effect of glutamine appears

to be dependent on its metabolism, but interestingly, it cannot be reproduced

with glutamate. This apparent specificity of glutamine as a crypt cell mito-

gen may be due to the fact that the crypt cell lines used in these studies had a

limited capacity to transport glutamate across the cell membrane. Yet, this

contradicts the findings of extensive intestinal glutamate metabolism docu-

mented in vivo (Windmueller and Spaeth, 1975; Reeds et al., 1996). On the

Fig. 3. Relative incorporation of luminally (�-3H-leucine) and intravenously (*-14C-leucine) administered leucine into enterocytes isolated from the crypt to villus axis of the

intestinal mucosa (adapted from Alpers, 1972).

700 D. G. Burrin

other hand, it may be that crypt cells are capable of basolateral transport of

blood-borne glutamine (but not glutamate) for proliferative processes, but as

the cells differentiate and mature, they acquire the ability to transport lumi-

nal glutamate for use as a metabolic fuel and as a biosynthetic precursor

(fig. 4). This idea is supported by evidence that shows that glutamine trans-

port is high in undifferentiated Caco-2 cells, but in confluent differentiated

cells glutamine transport is decreased, whereas glutamate transport is sub-

stantially increased (Mordrelle et al., 2000).

3. METABOLIC FATE OF AMINO ACIDS

Once taken up by the intestinal tissues, amino acids can be utilized for three

major metabolic purposes: (1) incorporation into protein; (2) conversion via

Fig. 4. Model of mucosal glutamate, glutamine, and aspartate metabolism derived fromdietary intake and the arterial circulation.

Gastrointestinal protein and amino acid metabolism in growing animals 701

transamination into other amino acids, metabolic substrates and biosynthetic

intermediates; and (3) complete oxidation to CO2. In the first two pathways,

amino acids can be deposited and recycled by the body for purposes of

growth or other biological functions. In the case of some amino acids,

namely threonine and cysteine, incorporation into endogenous secretions

that are fermented in the large intestine represents a net nutritional

loss. Likewise, if the amino acids are completely oxidized to CO2 by the

mucosal cells, this is also a nutritional loss, especially in the case of essen-

tial amino acids.

3.1. Protein synthesis and degradation

The major metabolic fate of amino acids in the gut is considered to be their

incorporation into cellular proteins. Although this is true of essential amino

acids, there is extensive metabolism of several non-essential amino acids.

Numerous studies have measured the rates of protein synthesis in various

tissues of the gastrointestinal tract. In general, these studies show that the

fractional rates of protein synthesis are relatively high compared to other

major tissues such as muscle, due to the high rates of cell turnover. More-

over, gut protein synthesis rates are modulated by several nutritional and

hormonal factors, the details of which are described below. However, there

is a lack of fundamental knowledge about the cellular and molecular

mechanisms that regulate protein synthesis in gut tissues in comparison with

our knowledge of other tissues, such as liver and muscle. Recent evidence

has shown that the stimulation of muscle protein synthesis by food intake,

insulin and amino acids is mediated by activation of intracellular signal

pathways which include IRS-1, PI-3 kinase, MAP-kinase, mTOR and

p70-S6-kinase (Kimball et al., 1998; Davis et al., 2000; Kimball et al.,

2000; Anthony et al., 2001). These signaling pathways play a critical role

in the proliferation and differentiation of intestinal epithelial cells (Taupin

and Podolsky, 1999; Wang et al., 2001) and can be activated by amino acids

(Rhoads et al., 2000); yet, their role in the regulation of protein synthesis is

largely unknown. We know even less about the regulation of protein degra-

dation in gut tissues, whether at the molecular and cellular level, or with

respect to nutritional and hormonal regulation (Baracos et al., 2000).

3.2. Nonessential amino acid metabolism

Of the amino acids ingested in the diet, the intestinal metabolism of gluta-

mine and glutamate has been studied most extensively (fig. 5). It has

been known for more than 25 years that there is substantial utilization of

702 D. G. Burrin

glutamine and glutamate by the intestine, and much of the glutamine is

derived from the arterial circulation (Windmueller and Spaeth, 1975;

1980). More important, however, is the fact that in situ studies (Windmuel-

ler and Spaeth, 1980) have shown that at least 50% of the dietary glutamate

and glutamine taken up by the gut is completely oxidized to CO2. These

results were recently confirmed by in vivo studies of 13C-labeled glutamate

and glutamine metabolism by the PDV tissues in piglets indicating that as

much as 95% of enteral glutamate is metabolized by the gut, largely to

CO2 (Stoll et al., 1999a) (fig. 6). Consistent with this finding, studies in pre-

mature infants and adult humans, based on estimates of whole-body kinetics

of enterally fed 13C- and 15N-labeled glutamate and glutamine, indicate that

approximately 50 to 95% of the dietary glutamine and glutamate is

extracted by splanchnic tissues, and most of this is oxidized to CO2 (Battez-

zati et al., 1995; Darmaun et al., 1997; Haisch et al., 2000).

In addition to the role of glutamate as metabolic fuel, studies in rats

(Horvath et al., 1996) have shown that adding 4% glutamate to an elemental

diet deficient in both glutamine and glutamate significantly increases intest-

inal growth, crypt cell mitosis, and lactase activity, without affecting body

weight gain. These results serve to challenge the long-standing notion that

glutamine is the primary oxidative fuel for the gut, and suggest that gluta-

mate and perhaps aspartate ingested in the diet are equally important intest-

inal fuels. Moreover, studies with short-term cultures of isolated enterocytes

have shown that, along with glutamine, glucose is a major intestinal oxida-

tive fuel (Darcy-Vrillon et al., 1994; Kight and Fleming, 1995; Wu et al.,

1995). Although our results indicate that significant quantities of glucose

Fig. 5. Synthesis and interconversion of glutamate and glutamine. (1) Glutamate dehy-drogenase; (2) Alanine aminotransferase, aspartate aminotransferase, or branched-chain

aminotransferase; (3) Glutaminase; (4) Glutamine synthetase.

Gastrointestinal protein and amino acid metabolism in growing animals 703

are indeed oxidized by the gut, they demonstrate a preferential utilization of

arterial rather than dietary glucose. It is important to note that, although

glucose represents a major oxidative fuel (29%) in terms of total PDV

CO2 production, the proportion of glucose oxidized completely to CO2 is

substantially less than that of either glutamate or glutamine. This finding

is entirely consistent with results from cultured enterocytes showing that

glutamine can potently suppress glucose oxidation, whereas glucose has lit-

tle impact on glutamine oxidation (Kight and Fleming, 1995; Wu et al.,

1995). The implication is that glutamine and glutamate are preferentially

channeled towards mitochondrial oxidation, while most of the glucose is

utilized for other metabolic or biosynthetic purposes. A small percentage

of the portal glucose utilization is metabolized to alanine (7.5%) and lactate

(5.9%), which leaves approximately 60% unaccounted for. The biosynthetic

products potentially derived from gut glucose utilization are numerous;

however, incorporation into mucin glycoproteins, fatty acids and lipids

presents a variety of intriguing possibilities.

The importance of glutamine and glutamate to gut metabolism goes

beyond their roles as oxidative fuel for rapidly proliferating cells, such as

enterocytes and lymphocytes; they also function as important precursors

Fig. 6. Relative rates of arterial and dietary substrate uptake and oxidation by the portal-drained visceral tissues in young pigs fed cow’s milk formula (GLN, glutamine; GLU,

glutamate; GLUC, glucose; ASP, aspartate; Adapted from stoll et al., 1999a).

704 D. G. Burrin

of nucleic acids, nucleotides, amino sugars, amino acids, and glutathione

(Smith, 1990; Souba, et al., 1990; Rouse et al., 1995; Klimberg, 1996; Gate

et al., 1999). A number of studies with adult rats, neonatal pigs and porcine

enterocytes have demonstrated that glutamine=glutamate are precursorsfor the synthesis of several non-essential amino acids, including alanine,

aspartate, proline, citrulline and arginine (Windmueller and Spaeth, 1980;

Blaicher et al., 1993; Wu et al., 1995; Wu and Knabe, 1995; Stoll et al.,

1999a). In addition, Reeds et al. (1997) reported that luminal glutamate,

rather than glutamine-derived glutamate, is the preferential source of glu-

tathione synthesis in the intestinal mucosa of the infant pig, suggesting a

highly compartmentalized catabolism of intestinal glutamate and glutamine.

It is also noteworthy that the enzymes necessary for de novo synthesis of

both glutamate and glutamine are present in gut tissues, and their expression

is up-regulated during the suckling-to-weanling transition (Shenoy et al.,

1996; Madej et al., 1999).

Studies with cultured intestinal epithelial cells have demonstrated that

glutamine has pluripotent actions, including stimulation of cell prolifera-

tion, differentiation, ornithine decarboxylase (ODC) and immediate early

gene (c-jun) expression, and polyamine synthesis (Kandil et al., 1995; Wang

et al., 1996). Glutamine also suppresses apoptosis, which suggests that it

may be an important survival factor (Papaconstantinou et al., 1998). In a

recent review, Rhoads (1999) postulated that glutamine stimulates cell pro-

liferation by a signaling mechanism which involves the activation of two

related, but distinct, classes of mitogen-activated protein kinases. The bio-

chemical mechanism whereby glutamine affects intestinal function may

be related to its conversion to glucosamine, which reduces the cellular

NADPH and suppresses nitric oxide synthesis (Wu et al., 2001).

3.3. Essential amino acid metabolism

A number of essential amino acids play a key role in gut function and thus

their metabolism by the gut has a critical influence on essential amino acid

requirements. Dietary threonine and cysteine are utilized by the intestinal

mucosa for mucin and glutathione synthesis. From a nutritional perspective,

although threonine is considered essential, cysteine is not. However,

cysteine can be synthesized from the essential amino acid, methionine.

Therefore, increased metabolism of methionine to meet cysteine needs

may become limiting for growth, and nutritionally significant. The secretory

mucins play a key role in the innate immune defense of the mucosa, and the

core protein of the major intestinal mucins (van Klinkenet al., 1997)

contains a large amount of threonine and cysteine. Likewise, cysteine is a

Gastrointestinal protein and amino acid metabolism in growing animals 705

component of the tripeptide antioxidant glutathione, which is critical for the

maintenance of the structural integrity and barrier function of the intestinal

mucosa (Martenssonet al., 1991). Given that the rates of mucosal synthesis

and secretion of glutathione and mucins are likely significant, it follows

that their production by the gut could have a quantitatively significant

impact on the animal’s dietary requirement for threonine, cysteine, and per-

haps methionine. For threonine, but not cysteine, there is evidence to sup-

port this hypothesis. First, based on net portal utilization, as much as 60

to 85% of dietary threonine is utilized by the gut (Stoll et al., 1998; Van

Goudoever et al., 2000). More conclusive, however, was a recent study

(Bertolo et al., 1998) demonstrating that the threonine requirement of pig-

lets maintained by parenteral nutrition was only 40% of that observed in

piglets receiving enteral feedings. Moreover, a subsequent report found

that feeding threonine-deficient diets to piglets significantly reduces intest-

inal mass and goblet cell numbers, and this suppression of intestinal growth

cannot be fully restored by providing threonine parenterally (Ball et al.,

1999). Another study demonstrated that the methionine requirement of

piglets maintained by parenteral nutrition was 35% lower than that of ent-

erally fed piglets (Shoveller et al., 2000; 2001). This value is lower than

the value of 46% based on our measurements of net portal utilization of

dietary methionine. Nevertheless, both findings suggest that the gut utilizes

substantial amounts of dietary methionine, and raise the question of

whether methionine is being metabolized to cysteine and used for intestinal

mucin synthesis.

Dietary arginine is essential for young piglets. Studies have shown that

the small intestine is an important site of arginine and proline synthesis

(fig. 7) (Murphy et al., 1996; Wu, 1998; Stoll et al., 1999a). Similarly, in

adult rats and weanling pigs (Windmueller and Spaeth, 1980; Dugan et al.,

1995), intestinal citrulline synthesis from glutamine, glutamate and proline

is the main source for circulating citrulline, which plays a critical role in

arginine homeostasis. In many adult animals and humans, arginine is

synthesized by the kidney from intestinally derived citrulline at rates that

are inadequate to support growth in neonatal animals. As the milk of

humans, pigs, rats and many other mammals is deficient in arginine (Davis

et al., 1994), it is considered an essential amino acid for growing animals. In

the healthy suckling pig, the intestinal synthesis of arginine provides only

about half of the animal’s needs for growth; thus, arginine is required in

the diet. Moreover, the net intestinal synthesis of arginine declines substan-

tially during the late suckling period (Wu and Morris, 1998). These obser-

vations raise the possibility that both the endogenous (via gut synthesis) and

dietary arginine supply may be limiting for maximal growth of suckling

piglets. Studies with enterocytes from newborn pigs (Blaicher et al.,

706 D. G. Burrin

1993; Wu and Knabe, 1995) and developing mouse and rat small intestine

(Hurwitz and Kretchmer, 1986; DeJonge et al., 1998) have shown develop-

mental changes and metabolic zonation along the crypt-villus axis of argi-

nine-metabolizing enzymes. At birth, the enterocytes of the upper villus

(DeJonge et al., 1998) are the major site of arginine synthesis, but gradually

become the major site of net citrulline production as intestinal arginase

expression increases via a glucocorticoid-dependent mechanism. This tran-

sition is compensated by the gradually increasing capacity of the kidney to

use citrulline for arginine synthesis. Thus, following the transition from

suckling to weanling, the intestine appears to become a site of arginine

degradation rather than synthesis (see review, Wu and Morris, 1998).

The limited arginine degradation by enterocytes from newborn pigs

ensures a maximum output of arginine (synthesized from glutamine or

derived from milk) into the portal circulation for utilization by extraintest-

inal tissues. Type II arginase, a mitochondrial enzyme that is expressed at

lower levels in kidney, brain, small intestine, mammary gland, and macro-

phages but not in liver, is distinct from type I arginase, a cytosolic enzyme

that is highly expressed in liver as a component of the urea cycle. The

induction of type II arginase in enterocytes after weaning possibly regulates

the availability of arginine for the synthesis of nitric oxide (NO), ornithine

and thus, polyamines, proline, and glutamate, as well as ureagenesis in the

small intestinal mucosa (fig. 7). lthough considerably less than in the liver,

Fig. 7. Metabolic pathways and interconversion of arginine, ornithine, glutamate, and proline.

(1) arginase; (2) ornithine aminotransferase; (3) glutamate-g-semi-aldehyde dehydrogenase;(4) g-glutamyl kinase; (5) g-glutamyl dehydrogenase; (6) spontaneous, non-enzymatic;(7) pyrroline-5-carboxylase reductase; (8) proline oxidase; (9) ornithine transcarbamoylase;

(10) NO synthase; (11) arginine decarboxylase.

Gastrointestinal protein and amino acid metabolism in growing animals 707

gut ureagenesis may be a first-line defense in detoxification of ammonia

derived from tissue metabolism and luminal microorganisms (Wu, 1995).

However, the major end-products of arginine metabolism by intestinal

enterocytes from weaned pigs are proline and ornithine. Proline, required

for collagen synthesis, is one of the most abundant amino acids in human

milk (Davis et al., 1994). Arginine-derived proline is not detectable in enter-

ocytes of newborn or suckling pigs. However, providing proline in the diet

can ameliorate the hyperammonemia associated with dietary arginine defi-

ciency in neonatal pigs (Brunton et al., 1999). Thus, while proline is not

considered an absolute dietary essential nutrient, it may be conditionally

essential for maintaining arginine synthesis in neonates. It is apparent from

a number of studies that a normally functioning gut is important for main-

tenance of whole-body arginine and proline status, especially in neonates.

Furthermore, these amino acids become conditionally essential under con-

ditions that markedly reduce gut mass or compromise function, such as

massive small bowel resection (Wakabayashi et al., 1995).

Recent studies in cultured intestinal cells (Blaicher et al., 1995) have

shown that ornithine derived from arginine metabolism is converted to poly-

amines (fig. 8). Polyamines (putrescine, spermidine, spermine, cadaverine)

Fig. 8. Polyamine synthesis. (1) ornithine decarboxylase; (2) spermidine synthetase;(3) spermidine synthase; (4) polyamine oxidase; (5) methionine adenosyl transferase;

(6) S-adenosymethionine decarboxylase.

708 D. G. Burrin

are ubiquitous cationic amines involved in cell proliferation and differentia-

tion in many tissues, including the gastrointestinal tract. Ornithine decar-

boxylase (ODC) and S-adenosyl-methionine decarboxylase (SAMDC),

converting ornithine to putrescine and putrescine to spermine, respectively,

are the rate-limiting enzymes in polyamine synthesis. The synthesis of

polyamines from arginine is negligible in enterocytes of newborn and suck-

ling animals (Blaicher et al., 1991; Blaicher et al., 1992), but increases in

enterocytes of postweaning animals, concurrent with the induction of both

arginase and ODC (Wu and Morris, 1998). Luminal administration of poly-

amines increases intestinal growth in adult rats (Seidel et al., 1985) and has

been shown to enhance intestinal maturation (Grant et al., 1990) and cell

proliferation in developing rats (Dufour et al., 1988). Polyamines are pre-

sent in human milk in micromolar concentrations for up to 4 months of lac-

tation (Pollack et al., 1992; Buts et al., 1995). Results of a recent study in

cultured intestinal cells suggested that both human and rat milk, but neither

bovine milk nor infant formula, contain sufficient amounts of polyamines to

sustain cell growth during inhibition of polyamine synthesis with difluoro-

methylornithine (DFMO), a specific and irreversible inhibitor of ODC

(Capano et al., 1998). Thus, when the ingestion of milkborne polyamines

by the neonate ceases after weaning, the induction of intestinal polyamine

synthesis from ornithine, arginine and proline may become physiologically

significant for the maintenance of normal intestinal growth and function

(Wu et al., 2000a; 2000b). Furthermore, the induction of intestinal poly-

amine synthesis is dependent on the weaning-induced cortisol surge.

3.4. Essential amino acid oxidation

The catabolism of essential amino acids by the gut has received little recent

attention, in part because early studies failed to demonstrate the presence of

the catabolic enzymes in the small intestinal mucosa (Wu, 1998). However,

studies based on isotopic tracer kinetics in PDV tissues suggest that dietary

essential amino acids may indeed be oxidized by the intestinal mucosa. Stu-

dies in sheep (Pell et al., 1986; Cappelli et al., 1997; Yu et al., 2000), pigs

(Burrin et al., 1999b) and dogs (Yu et al., 1992) have found that approxi-

mately 5–10% of whole-body leucine flux is oxidized by the portal-drained

viscera. Recent studies in piglets enterally fed [U-13C] threonine, leucine,

lysine and phenylalanine suggested significant first-pass metabolism by

the intestinal mucosa (Stoll et al., 1998; Stoll et al., 1999; Van Goudoever

et al., 2000; Van der Schoor et al., 2001). In growing pigs, lysine is consid-

ered to be the first limiting dietary amino acid. Studies in young piglets

showed that intestinal oxidation of dietary lysine accounted for about

Gastrointestinal protein and amino acid metabolism in growing animals 709

one-third of whole-body lysine oxidation. Interestingly, although about 10%

of the arterial flux of lysine was taken up by the portal-drained viscera, none

of this was oxidized, suggesting a preferential oxidation of dietary lysine.

Studies in young pigs suggest that approximately 15 to 30% of the whole-

body leucine and phenylalanine flux is oxidized by the PDV tissues (Van

der Schoor et al., 2001; Bush et al., 2001). The oxidation of phenylalanine

implies that phenylalanine hydroxylation occurs in the gut and is consistent

with previous observations suggesting de novo gut tyrosine production.

However, threonine seems to be unique among the essential amino acids

in that there is negligible 13C-threonine oxidation to CO2 when given either

enterally or systemically to young piglets. This may be due to the fact that it

is preferentially used for synthesis of mucins.

4. ROLE OF ONTOGENY

The fractional rates of whole-body protein metabolism are highest

during foetal life, and decline progressively with advancing post-conceptual

age, commensurate with fractional growth rates (Waterlow et al., 1978;

Goldspink and Kelly, 1984). In rats, the intestinal protein mass increases

approximately 750-fold (2.5 to 1893 mg between 18 days gestational age

and 105 weeks postpartum age (Goldsplink et al., 1984). During this life

span, however, the fractional protein synthesis rate (FSR) in the small

and large intestine declines by 43%. After weaning, the decline in intestinal

FSR with age is largely due to a decreased synthesis in the muscularis and

serosal layers, whereas the mucosa remains constant (Merry et al., 1992).

Despite the overall age-related decline, studies in neonatal rats and mice

indicate that the FSRs of the stomach, small intestine and pancreas increase

significantly after weaning (Burrin et al., 1991; Burrin et al., 1999a). In

domestic animals, there are few, if any, estimates of gastrointestinal FSRs

before birth and beyond pubertal ages, yet the changes between birth and

weaning in pigs and sheep tend to parallel those in rodents (Seve et al.,

1986; Attaix et al., 1992; Davis et al., 1996). In milk-fed animals, the intest-

inal FSR declines during the neonatal period, but increases markedly (40–

50%) after weaning. Even more striking is the relative contribution of the

small and large intestine to whole-body FSR, which was found to be

approximately 10% in milk-fed lambs and 20% in weaned lambs. The

explanation for this sharp increase in gut protein synthesis after weaning

is likely the substantial change in the composition diet and resultant stimu-

lation of mucosal cellularity and proliferation (Attaix and Meslin, 1991;

Pluske, 1997; Jiang et al., 2000). The extent to which these changes are

related to alterations in the gut microflora is also a probable factor.

710 D. G. Burrin

With respect to the ontogenic changes in the metabolism of specific

amino acids, most studies have focused on glutamine, arginine, citrulline,

ornithine and proline. The rates of glutamine and glucose oxidation in iso-

lated enterocytes decrease by roughly 90% in the first 3 weeks of life

(Darcy-Vrillon et al., 1994; Wu et al., 1995). The percentage of glutamine

and glucose oxidized to CO2 at birth is 36 and 21%, respectively, and

declines at 3 weeks of age to 4 and 2%, respectively. After weaning, in

intraepithelial lymphocytes, glutamine is mainly metabolized to glutamate

and ammonia (92%), with minimal oxidation (4%). The decline in gluta-

mine oxidation with age is paralleled by increased activities of glutamine

synthetase, glutaminase, and glutamate dehydrogenase (Hahn et al., 1988;

Shenoy et al., 1996; Madej et al., 1999). Other studies in isolated entero-

cytes show that the synthesis of citrulline and ornithine increases with

age, particularly after weaning, and proline is a major precursor for their

synthesis (Wu, 1997). Recent studies indicate that this shift in ornithine pro-

duction is associated with increased ODC activity and is dependent on

increased circulating cortisol (Wu et al., 2000a; Wu et al., 2000b).

5. ROLE OF NUTRITION

5.1. Enteral vs parenteral

Oral feeding is a potent stimulus of intestinal protein and amino acid meta-

bolism in growing animals (McNurlan et al., 1979; Patureau Mirand et al.,

1990; Burrin et al., 1991; Burrin et al., 1995). Prolonged fasting leads to

markedly reduced protein mass in gut tissues via suppressed protein synth-

esis and increased protein degradation, especially in the small intestine

(Burrin et al., 1991; Samuels et al., 1996). Recent studies have shown that

the stimulatory effect of nutrient intake on gut protein metabolism is highly

dependent on the administration of nutrients via the enteral route (Dudley

et al., 1998; Burrin et al., 2000; Stoll et al., 2000a). These piglet studies

showed that net loss of intestinal protein occurs during 7 days of TPN,

and that intestinal protein accretion is highly correlated with the level of

enteral intake; intestinal protein balance occurred at 20% enteral intake,

and maximal intestinal protein accretion at 60% enteral intake (fig. 9).

Numerous studies have shown positive effects of glutamine-supplemen-

ted TPN in preventing atrophy, stimulating protein anabolism and maintain-

ing intestinal permeability (Tamada et al., 1992; Platell et al., 1993; Inoue

et al., 1993; Haque et al., 1996; Naka et al., 1997; Khan et al., 1999). In

contrast, other studies have shown no effect of glutamine on gut growth

or protein metabolism in animals receiving glutamine supplementation

Gastrointestinal protein and amino acid metabolism in growing animals 711

parenterally (Spaeth et al., 1993; Burrin et al., 1994; Marchini et al., 1999;

Humbert et al., 2001). Furthermore, parenteral infusion of lipid has been

shown to stimulate intestinal protein synthesis more than either glucose

or glutamine (Stein et al., 1994). Thus, the intestinal trophic effects of glu-

tamine-supplemented TPN are evident under conditions of compromised

gut function, such as sepsis, inflammation, and small-bowel resection, while

in healthy animals, glutamine provides limited benefit.

Although the provision of nutrition via the enteral route is critical for

intestinal growth, the effects of first-pass metabolism can significantly

increase whole-body requirements for arginine, threonine, and methionine

(Brunton et al., 2000). Moreover, studies in piglets have shown that when

first-pass intestinal metabolism is bypassed during TPN, arginine and pro-

line become essential amino acids because their biosynthesis requires the

enteral input of precursors (Bertolo et al., 1999a). In contrast, the conse-

quences of reduced gut mass following TPN may enhance the absorption

of nutrients when enteral feeding is resumed. Our preliminary studies indi-

cated that the 52% reduction in intestinal mass observed after 7 days of

TPN was associated with a 30% increase in the net portal absorption of ent-

erally administered leucine (Burrin et al., 1999b). Moreover, the increased

absorption was largely due to reduced portal utilization of arterial leucine,

Fig. 9. Small intestinal protein balance in neonatal pigs fed varying proportions of enteraland parenteral nutrition (Adapted from Stoll et al., 2000a).

712 D. G. Burrin

secondary to the reduced intestinal mass. This finding indicates that a 50%

reduction in gut mass actually increased the net systemic amino acid avail-

ability for growth. Interestingly, these results provide a metabolic explana-

tion for the phenomenon of compensatory growth, which occurs when

animals are fed ad libitum following periods of restricted food intake. Pre-

vious studies in ruminants have shown that the reduction in the mass and

energy expenditure of the visceral organs during nutrient restriction results

in increased dietary energy availability for compensatory growth (Ferrell,

1988). It would appear equally plausible that the increased systemic amino

acid availability resulting from a reduction in intestinal mass and amino acid

metabolism also could lead to enhanced efficiency of dietary protein use and

growth rate, typical of compensatory growth.

5.2. Nutrient Composition

In addition to the quantity of dietary intake, the composition of dietary

nutrients can also substantially affect gut protein and amino acid meta-

bolism. There is an extensive literature describing the impact of dietary

composition on gut growth and function (Spector et al., 1977; Weser

et al., 1986; Buts et al., 1990; Bragg et al., 1991; Jenkins and Thompson,

1994). However, considerably less is known about how the composition

of the diet alters gut protein metabolism. Some studies have shown that

restriction of dietary protein has limited effects on intestinal protein synth-

esis and growth (Seve et al., 1986; Ebner et al., 1994), whereas others have

reported a decrease in protein synthesis (McNurlan and Garlick, 1981;

Wykes et al., 1996). The study with neonatal pigs demonstrated that,

although protein malnutrition significantly reduced whole-body growth

and amino acid absorption, this was associated with reduced carcass growth,

since gut tissue growth was maintained (Ebner et al., 1994). A recent study

based on metabolism of enterally fed 13C-lysine showed that in pigs fed

low-protein diets, the PDV tissues utilized more than 75% of the lysine

intake, compared to 45% in high- protein-fed pigs (Van Goudoever et al.,

2000). Another important finding of this study was that on the high-protein

diet, all of the PDV lysine utilization was derived from the arterial circula-

tion, whereas on the low-protein diet, the gut used lysine equally from the

enteral and arterial input. This study highlights the preferential use of diet-

ary amino acids by the gut and how this limits the systemic availability of

lysine for lean tissue growth during protein malnutrition.

Studies focusing on the impact of dietary macronutrients found that ent-

eral amino acids, but not carbohydrate or lipid, stimulated intestinal protein

synthesis, whereas each of these stimulated gut protein accretion, suggesting

Gastrointestinal protein and amino acid metabolism in growing animals 713

that carbohydrate and lipid suppressed gut proteolysis (Stoll et al., 2000).

In contrast, in other studies, enteral amino acids rapidly decreased intestinal

protein synthesis and proteolysis in young pigs (Adegoke et al., 1999).

Feeding a high- fat versus high- carbohydrate diet to young pigs stimulated

intestinal protein synthesis (Ponter et al., 1994). Numerous studies have

examined the effect of dietary supplementation with specific amino acids

on intestinal growth and metabolism. Supplementing glutamine, glutamate,

and arginine has been shown to be stimulatory to the gut in some cases,

but not in others (Michail et al., 1995; Wu et al., 1996; Horvath et al.,

1996; Vanderhoof et al., 1997; Hasebe et al., 1999; Bertolo et al., 1999b;

Ewtushik et al., 2000). Dietary factors that stimulate mucosal proliferation

and cell turnover also tend to increase protein synthesis. Feeding fibre and

lectins have been shown to stimulate intestinal protein synthesis in some

cases (Southern et al., 1985; Palmer et al., 1987), but not in others (Nyachoti

et al., 2000). Studies show that neonatal pigs fed elemental diets have higher

rates of protein synthesis and cell proliferation than those fed cow’s milk

formula, even though intestinal mucosal growth and villus morphology

are similar (Stoll et al., 2000c).

6. INFECTION AND INFLAMMATION

Infestation with pathogenic microbial and viral organisms, or exposure to

the toxins they produce, is known to adversely affect both intestinal struc-

ture and function. Studies with growing animals indicate that exposure to

pathogenic and non-pathogenic organisms impart a protein metabolic cost

to the animal associated with stimulation and maintenance of the pro-

inflammatory, acute-phase response (Von Allmen et al., 1992; MacRae,

1993; Higashiguchi et al., 1994; Johnson, 1997; Breuille et al., 1998; Wang

et al., 1998; Breuille et al., 1999; Mack et al., 1999). The studies demon-

strate that treatment with pro-inflammatory stimuli such as bacteria, enteric

parasites, endotoxin and cytokines significantly increases protein synthesis

in visceral tissues, especially the liver, gut and spleen, while increasing cat-

abolism and net loss of muscle protein body weight. Recent work in sheep

demonstrated that parasitic infection increases the rate of leucine utilization

and oxidation by PDV tissues, thereby reducing the systemic availability of

dietary amino acids by 20–30% (Yu et al., 2000). In addition, most of the

increased PDV leucine utilization is either oxidized or lost as endogenous

protein secretion; together, these losses account for most of the reduced

nitrogen retention associated with infection (Yu et al., 2000).

These results illustrate a mechanism whereby the gastrointestinal micro-

flora activate the immune system and suppress the growth rate in domestic

714 D. G. Burrin

animals. Antimicrobial compounds are fed to domestic animals in order to

suppress the activity of the gut microflora and enhance growth; however, the

exact mechanism for this effect is unknown. It has been shown that by sup-

pressing microbial activity, antimicrobials reduce the luminal concentration

and associated toxic insult of ammonia, and thereby diminish the thickness

and mass of the intestinal mucosa and associated lymphoid tissue (Visek,

1978). Studies in pigs and chickens show that feeding antimicrobial com-

pounds significantly reduces small intestinal mass, cell proliferation and

intestinal ammonia absorption (Yen et al., 1987; Yen and Pond, 1990;

Krinke and Jamroz, 1996). Additional evidence indicates that much of the

luminal ammonia originates from bacterial hydrolysis of urea and deamina-

tion of dietary amino acids. Thus, given the recent evidence that the gut may

be an important site of dietary amino acid catabolism, the question arises as

to whether this activity is associated with the luminal microbes or the cell

populations of the mucosa. Although it is likely that both possibilities exist,

the end result is the catabolism and loss of dietary amino acids, especially

those that are essential, that otherwise would be used for growth.

7. FUTURE PERSPECTIVES

It is evident from past studies that the tissues of the gastrointestinal tract

play an integral role in the whole-body protein and amino acid metabolism

of growing animals. It has also become increasingly evident that the gut not

only consumes a substantial fraction of the dietary intake, but is critical for

the metabolism of key amino acids, such as arginine and threonine, that are

essential for the growth of developing animals. There has been considerable

progress in our understanding of intestinal amino acid metabolism, espe-

cially nonessential amino acids such as glutamate, glutamine, citrulline,

ornithine, and proline. However, with the exception of arginine and, to some

extent, threonine, there is only a very limited understanding of the cellular

basis for how other essential amino acids are metabolized within the gut tis-

sues. With the expanding range of analytical tools, from the molecular to the

whole-organ level, there is a clear rationale and opportunity to explore these

issues in growing animals. For example, many of the biochemical pathways

responsible for oxidation of essential amino acids have either not been well

characterized or even shown to exist in intestinal tissues. Although it is well

established that glutamine and glutamate are major oxidative fuels for the

gut, the metabolic fate of nitrogen from these substrates is only vaguely

understood. There is also a compelling need to identify the cellular signal-

ing mechanisms whereby nutrients and other extracellular molecules affect

the rates of protein and amino acid metabolism of intestinal cells. Many of

Gastrointestinal protein and amino acid metabolism in growing animals 715

the pathways and cellular targets that have been shown to be important in

other cell types are obvious candidates (e.g., MAP kinases, PI3 kinase,

mTOR, proteasome, NF-kB). As we begin to establish the underlying cellu-

lar basis of protein metabolism, it should become increasingly evident as to

which specific nutrients are important for intestinal growth and function;

this will then lead to more rational diet formulation. In addition, continued

efforts should be aimed at establishing how regulatory elements, such as

nutrients, hormones and environmental factors, modulate the growth and

metabolism of the gut tissues, and then quantifying their impact on nutrient

availability and growth of the animal. Further studies that couple the use of

trans-organ substrate balance and isotopic kinetic measurements will be par-

ticularly useful in attempting to answer these questions. An especially cru-

cial area will be the impact of the gut microflora, and the extent to which it

modifies gut amino acid utilization and the nitrogen economy of the animal.

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