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U.S. Department of Agriculture/Agricultural Research Service, Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONApproaching the study of...Contribution of glutamate to...Biosynthetic role of dietary...SUMMARYREFERENCES |
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KEY WORDS: • glutamate • small intestine • metabolism • pigs • stable isotopes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONApproaching the study of...Contribution of glutamate to...Biosynthetic role of dietary...SUMMARYREFERENCES |
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More than 40 years ago, Neame and Wiseman (1957)
showed
in dogs that only a small proportion of a dose of enteral glutamate
appeared in the circulation and that the lumenal infusion of glutamate
led to considerable production of alanine by the intestine. After these
observations, there was relatively little interest in this subject
until the influential series of papers published by Windmueller and
Spaeth during the 1970s and early 1980s (Windmueller 1982
, Windmueller and Spaeth 1974
, 1975
, 1976
, 1978
and
1980
). Much subsequent work has been stimulated by their
finding that in rats, intestinal mucosal metabolism accounts for 30%
of total body glutamine turnover. This has been confirmed in a number
of mammalian species, and a large literature attests to continuing
interest in the role of systemic glutamine as a metabolic substrate
(Wu 1998
), and as a trophic (Wilmore 1997
) and functional factor in enterocytes (Burrin and Reeds 1997
).
Although there seems little doubt that glutamine plays an important,
but remarkably poorly characterized role in the metabolism of many
proliferating cells, much of the more recent literature on intestinal
metabolism has in effect ignored two other observations made by
Windmueller and Spaeth (1975)
, i.e., first, that the
metabolism of lumenal glutamate was even more extensive than that of
arterial glutamine; and second, that the presence of high
concentrations of glutamate in the intestinal lumen had only a small
(~25%) effect on intestinal utilization of glutamine. This suggests
that these two closely related amino acids may have different
functional roles in the multicellular system termed the intestinal
mucosa. The first of these observations in the rat was confirmed by
stable isotopic studies in humans (Battezzati et al. 1995
) and by our recent studies in piglets (see below).
From a metabolic perspective, the intestinal mucosa is unique. First,
the absorptive enterocytes are specialized for the unidirectional
movement of nutrients from the lumen to the basal lamina. As a result,
the proteins expressed in the apical and basolateral membranes,
including the transporter types, are quite different. The consequence
of this heterogeneous expression is that the rate of uptake of
different substrates across these two surfaces probably differs, both
qualitatively and quantitatively. Second, from a strictly metabolic
perspective, the mucosal cells are presented with high quantities of
substrates from both the intestinal lumen and the mesenteric arterial
circulation. A third observation, admittedly not unique to the mucosa,
is that on the basis of studies in vitro, enterocytes display
substantial metabolic plasticity (Darcy-Vrillon et al. 1994
, Fleming et al. 1991
and 1997
, Kight and Fleming 1993
, Wu et al. 1995
, Wu 1998
), and there is "metabolic competition" among different
substrates (Fleming et al. 1997
, Kight and Fleming 1995
).
With the exception of the work of Windmueller and Spaeth, there is very
little direct in vivo information on substrate metabolism in the gut
mucosa [although, see Yu et al. (1990)
], and much of
our current understanding is based on in vitro studies. For justifiable
experimental reasons, the majority of these experiments have
investigated the metabolism of single carbon-labeled substrates.
However, as pointed out above, under normal circumstances, the mucosa
is exposed to many potential precursors. Furthermore, there are
indications that the metabolic capabilities of enterocytes alter once
they are isolated. For example, as observed by Windmueller and Spaeth
and by ourselves in both pigs (see below) and mice (Pascual et al., 1998
), mucosal first-pass oxidation of dietary glucose
is very low in vivo, yet isolated enterocytes will readily metabolize
glucose via the Krebs cycle. It seemed to us, therefore, that given the
polarized nature of the enterocytes and the multiplicity of substrates
that are presented to them in vivo, there is little alternative but to
carry out in vivo studies, and to perform these studies under as
physiologically and nutritionally normal circumstances as possible.
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Approaching the study of intestinal glutamate metabolism in vivo |
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TOPABSTRACTINTRODUCTIONApproaching the study of...Contribution of glutamate to...Biosynthetic role of dietary...SUMMARYREFERENCES |
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To answer these questions in a model that was of relevance to our
central interest in pediatric nutrition and metabolism, we developed
the necessary surgical and isotopic techniques in the piglet. This is a
particularly useful experimental animal, because not only are its
intestinal function and metabolism analogous to that of the human [see
Ball et al. (1996)
, Moughan et al. (1992)
, Reeds et al. (1997b)
for discussion),
but it is sufficiently robust that it can survive and prosper after
quite extensive surgery.
The experiments that we summarize in this paper required the
combination of two techniques. The first was the surgical placement of
catheters that allowed the measurement of the portal appearance, and
hence the net absorption or utilization of dietary organic substrates
by the portal-drained viscera to be studied directly (Ebner et al. 1994
). The application of this technique to dietary
glutamate utilization (Reeds et al. 1996
, Table 1
), confirmed the preceding literature by showing that in piglets
receiving dietary protein at a rate of 12 g/(kg · d), essentially no
dietary glutamate (and aspartate) appeared in the portal circulation.
In contrast, >85% of the dietary carbohydrate and 50% of the dietary
essential amino acids were absorbed into the body.
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, Yu et al. 1990
). The interpretation of
data on tracee (mass) and tracer balances is relatively simple when
applied to compounds such as essential amino acids, which organisms
cannot synthesize (Stoll et al. 1997
, 1998a
, and 1998b
).
However, when the substrate of interest can be synthesized by the
animal, its carbon is recycled extensively both between and within
organs. The end result is that with substrates such as glutamate or
glucose, simple measurements of isotope balance can give a grossly
misleading impression of true absorption.
This problem can be solved by the use of tracers in which all of the
carbons are 13C [so-called uniformly labeled
(U)13C-tracers] in combination with mass
isotopomer distribution analysis (Berthold et al. 1991
,
Brunengraber et al. 1997
, Reeds et al. 1997b
). Data on the portal appearance of the
[U-13C]tracer allow the unequivocal measurement
of its true rate of absorption [see Reeds et al. (1996)
], whereas measurements of the production of lower mass
isotopomers quantify the degree to which
13C-label is recycled in mucosal intermediary
metabolism (Pascual et al. 1998
, Reeds et al. 1997a
). Finally, and importantly from the perspective of these
studies, measurements of the isotopomer distribution in compounds
generated from the mucosal metabolism of the
[U-13C]tracer can be used to identify the
relative importance of enteral and systemic substrates to mucosal
metabolism (Reeds et al. 1997a
).
Table 2
shows data on the tracer (e.g., [U-13C])
balances of enterally infused [U-13C]glutamate
and glucose or [U-13C]glucose, glutamine and
[2H3]glutamate given intravenously.
These results indicate that >95% of the enteral glutamate but only
5% of the enteral glucose was utilized by the mucosa. At the same
time, the visceral tissues extracted 6% of the glucose and 11% of the
glutamate arriving in the mesenteric artery. It was of particular
interest that the total extraction of arterial glutamine (22% of
arterial flux) was higher than the net extraction (11% flux, Table 1
).
This provides further evidence of the absorption of significant
quantities of dietary glutamine, an observation that has also been made
in rats (Windmueller 1982
), adult humans
(Matthews et al. 1993
) and, recently, in infants
(Darmaun et al. 1997
).
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Contribution of glutamate to visceral intermediary metabolism |
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TOPABSTRACTINTRODUCTIONApproaching the study of...Contribution of glutamate to...Biosynthetic role of dietary...SUMMARYREFERENCES |
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). The former is represented by the partial
catabolism of a substrate to 3-carbon products (i.e., pyruvate, lactate
and alanine). Of course, it is obvious that when glucose is the
metabolic substrate, its complete oxidation necessarily involves the
movement of carbon through the pyruvate pool. However, when glutamate
is the oxidative substrate, its carbon enters intermediary metabolism
via 
-ketoglutarate, synthesized either via glutamate transamination
or via glutamate dehydrogenase. This leads to the direct synthesis of
oxaloacetate and (subsequently) citrate.
However, it is critical to recognize that it is only the acetyl-CoA
portion of the citrate molecule that is oxidized net in the Krebs
cycle. It follows that for a compound such as glutamate (or for that
matter, any substrate entering the Krebs cycle beyond the level of
citrate) to be oxidized completely, it must lead to the synthesis of
acetyl-CoA. Thisin turn demands that pyruvate be synthesized from
the oxaloacetate. Table 1
shows that in the fed state, the
portal-drained viscera clearly synthesize and release considerable
quantities of alanine and lactate. Indeed, in our studies of gut
metabolism in fed piglets, the carbon outflow from the
gut as alanine and lactate (2700 µatoms carbon) is slightly more than
half the production of CO2 by the viscera. Thus, the
identification of the sources of both lactate/alanine and
CO2 is of equal importance to our understanding of
substrate flow in the mucosa.
A further advantage of the use of [U-13C]tracers is that
the introduction of [U-13C]glutamate, glutamine or
glucose into the cell can lead to the production of
[U-13C]pyruvate and hence lactate and alanine. This
allows us to make unequivocal calculations of the direct contribution
of any of these substrates to the lactate and alanine released to the
portal blood. Our measurements (Table 3
) show that enteral glutamate, enteral glucose and arterial glutamine
contribute 36, 6 and 15%, respectively, of the CO2
production by the portal-drained viscera. However, they also reveal
that the partitioning of glucose and glutamate metabolism between
incomplete and complete oxidation is quite different. Thus, with
arterial glutamine or dietary glutamate as substrates, ~10% of the
carbon is released as alanine and lactate and 90% as CO2,
whereas with glucose, 68% of the carbon appears as lactate and alanine
and only 34% as CO2. Under fed conditions, therefore,
enteral (dietary) glutamate is a far more important
oxidative substrate than either dietary glucose or
arterial glutamine.
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Biosynthetic role of dietary glutamate in the mucosa |
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TOPABSTRACTINTRODUCTIONApproaching the study of...Contribution of glutamate to...Biosynthetic role of dietary...SUMMARYREFERENCES |
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, Wu 1996
).
Accordingly, in our experiments with
[U-13C]glutamate infusions, we examined the
incorporation of glutamate carbon into arginine, proline and
glutathione. We were also interested in the general question of whether
dietary glutamate played a specific role in these metabolic activities,
as has been suggested by recent radioisotopic studies in pigs
(Murphy et al. 1996
) and
[U-13C]protein studies in humans
(Berthold et al. 1995
). Arginine was of interest because
mixed milk proteins contain insufficient arginine to support normal
rates of growth (Davis et al. 1994
) in the neonate and
work with isolated porcine enterocytes suggested that the mucosa of the
neonatal pig is capable of carrying out complete arginine synthesis
(Blachier et al. 1993
, Wu and Knabe 1995
,
Wu et al. 1997
). Glutathione was of interest because its
rate of synthesis in the mucosa is very high (Jahoor et al. 1996
) and it clearly plays an important role in the protection
of the mucosa from peroxidative damage and from dietary toxins
(Aw and Williams 1992
).
The objectives of both quantifying the pathways of glutamate metabolism
and identifying the role of enteral glutamate were aided substantially
by the use of mass isotopomer distribution analysis. This revealed
[Table
4; see also Reeds et al. (1996
and 1997a
)] that there was
extensive intracellular cycling of glutamate carbon within the mucosal
cells. Furthermore, the marked difference in isotopomer distribution
between the tracer and intracellular glutamate allowed us to
differentiate between end products synthesized directly from the
enteral tracer (M + 5 isotopomer) and from the bulked intracellular
pool (M + 1 to M + 3 isotopomers). The results were clear cut and
revealed that all three metabolic end products that we studied were
derived almost exclusively from the dietary tracer and not from the
intermediary metabolic pool of glutamate. Subsequent studies (data not
shown) showed also that arterial glutamine was a poor substrate for all
three end products.
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SUMMARY |
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TOPABSTRACTINTRODUCTIONApproaching the study of...Contribution of glutamate to...Biosynthetic role of dietary...SUMMARYREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported 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
(RO1-HD35679) and by the International Glutamate Technical Committee.
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TOPABSTRACTINTRODUCTIONApproaching the study of...Contribution of glutamate to...Biosynthetic role of dietary...SUMMARYREFERENCES |
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