SPECIAL COMMUNICATION
Glucose and glutamine provide similar proportions of energy to
mucosal cells of rat small intestine
Sharon E.
Fleming,
Kirsten L.
Zambell, and
Mark D.
Fitch
Department of Nutritional Sciences, University of California,
Berkeley, California 94720-3104
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ABSTRACT |
The objectives of
this study were to establish a reliable method for quantifying
glycolytic flux in intestinal epithelial cells, to determine the
proportion of energy provided to small intestine epithelial cells by
glucose vs. glutamine, and to determine whether there was an energetic
advantage to having both substrates present simultaneously. There was
substantial retention of 3H in
alanine and lactate when
[2-3H]glucose was used
as tracer for quantifying glycolysis, and the magnitude of the
3H retention was influenced by the
presence of other substrates and metabolites. Detritiation was at least
99% complete, however, when
[3-3H]glucose was used
as tracer in this system and the tritium was recovered as
3H2O.
Glycolytic flux was six- to sevenfold higher in cells of the proximal
than distal small intestine but was not significantly different for
young adult (4 mo) vs. aged adult (24 mo) rats. Net ATP production from
exogenous substrates was higher when both glucose and glutamine were
present simultaneously than when either substrate was present alone,
and glucose was calculated to provide 50-60% of the net ATP
produced from these two substrates. Most of the energy produced from
glucose was produced via the anaerobic metabolic pathways (78% for
glucose alone, 95% with glucose and glutamine). Net energy production
was calculated to be 10% lower in cells from aged animals than in
those from young animals, since CO2 production from these major
substrates was lower in cells from aged animals.
detritiation; method
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INTRODUCTION |
GLUCOSE IS UTILIZED BY epithelial cells of the mucosa
of the small intestine, and the major products include lactate, carbon dioxide, alanine, and other amino and organic acids (26, 46, 49, 50).
Through 13C nuclear magnetic
resonance (NMR) techniques (7), it has been shown that glucose
metabolism proceeds primarily via the glycolytic pathway. Other
experiments have shown that <5% of the glucose utilization occurs
via the pentose phosphate pathway in epithelial cells of both the
proximal and distal small intestine (21, 26).
It has generally been well recognized that epithelial cells of the
proximal small intestine exhibit a high rate of glucose utilization and
lactate production, yet glutamine has been increasingly considered to
be the major fuel for these cells (2, 45). The major objective of this
work was to determine the proportion of energy provided to small
intestine epithelial cells by glucose vs. glutamine. To do this, we
needed to measure both glycolytic flux and conversion of substrates to
metabolites that result in either a net production or utilization of
energy. These oxidation and metabolite data could then be used to
calculate the relative energetic contributions of glucose and glutamine
for cells of the proximal vs. distal small intestine. Also, this
approach could be used to assess the influence of senescence on
glycolytic flux, since the oxidation of some substrates was reported to
be significantly lower in epithelial cells isolated from the proximal
small intestine of aged vs. young rats (12). If the metabolic functions
of these cells were generally downregulated in senescence, we
hypothesized that glycolysis and thus the energy status would be
notably lower in cells from aged animals than from young animals.
Data that quantify flux of glucose through glycolysis in intestinal
cells were not previously available, although glycolytic activity of
the rat jejunum was first reported many years ago (39, 41). In those
and other studies (3, 6, 8, 35, 36, 39, 41), it was established that
glycolysis proceeded at a higher rate in the proximal small intestine
than in the distal small intestine. This was determined by
observing a decreasing distal gradient in oxygen consumption (3, 35,
39), decreased glycolytic enzyme activity (6, 8, 36), and a decreased rate of lactate accumulation (3, 39).
Net lactate production has been used in most cases to estimate
glycolysis in intestinal epithelial cells, but this approach is unable
to distinguish glucose-derived lactate from lactate produced by other
nutrients, such as alanine and glutamine, or from lactate produced
after efflux of tricarboxylic acid cycle intermediates. This technique
also does not consider the energy contributed by glucose-derived
pyruvate when it is channeled into other products, such as alanine.
These limitations can be overcome by quantifying the flux of glucose
through the glycolytic cycle by using the glucose detritiation
technique. This was the approach taken in the experiments that are
reported, since this technique allows assessment of the influence of
other lactate-producing nutrients on glycolysis. In addition,
tritium-labeled glucose can be used to quantify glycolysis under
conditions when there are no appreciable changes in glucose
concentration, and also when glycolysis and gluconeogenesis occur
simultaneously. Although tritiated glucose had been used previously in
studies of intestinal cell metabolism (37, 38), data were not available
to ensure that these techniques would provide an accurate quantitative
analysis of glycolytic flux in these cells. Thus it was necessary to
establish the validity of this approach for studies of enterocyte
metabolism.
Flux of glucose through complex pathways or through specific steps of a
single pathway has been assessed by measuring the rate at which
3H2O
is produced from specific
[3H]glucose isotopes.
Detritiation of
[2-3H]glucose is known
to occur during the isomerization of the hexose phosphates,
detritiation of
[3-3H]glucose occurs
during isomerization of the triose phosphate intermediates and during
flux through the pentose phosphate pathway, and detritiation of
[5-3H]glucose occurs
during both the triosephosphate isomerization and the enolase steps
(see Refs. 5, 17, and 51 for review). Rates of glycolysis and glucose
utilization have been measured by quantifying
3H2O
release from
[2-3H]glucose (5, 16)
and [5-3H]glucose (33,
53). Under conditions in which flux through the pentose phosphate
pathway is insignificant, flux through phosphofructokinase-1, the
flux-generating step for glycolysis in many tissues, has been measured
using [3-3H]glucose
(23, 29, 37, 38). Although this approach has a strong theoretical
basis, and the techniques are straightforward, the results may be
confounded by incomplete detritiation and isotope discrimination (51).
Retention of 3H in metabolic
products other than
3H2O
has been reported previously (51), and the presence of nonglucose metabolites has been shown to influence the degree of detritiation (44).
To determine the reliability of using
3H-glucose techniques to quantify
flux of glucose through glycolysis in intestinal epithelial cells, it
was necessary to determine whether
3H would be retained in
metabolites other than
3H2O,
and it was also necessary to determine whether detritiation would be
influenced by the presence of nonglucose substrates. From these
experiments, we determined that
[3-3H]glucose, but not
[2-3H]glucose,
could be used to reliably estimate flux of glucose through the
glycolytic pathway under a range of experimental conditions. Glycolytic
flux data and 14C-metabolite data
then could be used to calculate the relative energy contribution of
glucose vs. glutamine for intestinal epithelial cells, and differences
could be assessed due to intestinal segment, animal age, and substrate
availability.
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MATERIALS AND METHODS |
Animals.
To determine the effect of aging, young (4 mo) and aged (24-25 mo)
male Fischer 344 rats were purchased from the National Institute on
Aging breeding colony maintained under barrier-reared conditions
(Harlan Industries, Indianapolis, IN). They were given NIH 31 stock
diet (Western Research Products, Hayward, CA), since they had been fed
this diet throughout their lifetimes. For other glycolysis and
14C-metabolism experiments,
Fischer 344 rats (Simonsen Laboratories, Gilroy, CA) weighing
250-300 g were used. Until the day of experimentation, these
animals were allowed free access to commercial diet (Rat Chow no. 5012, Ralston Purina, St. Louis, MO). The animals were anesthetized with
pentobarbital sodium (5 mg/100 g rat; Abbott Laboratories, North
Chicago, IL) and killed by thoracotomy. Animal handling procedures were
approved by the Animal Care and Use Committee, University of California
(Berkeley, CA).
Chemicals.
Radiochemicals obtained from Dupont NEN (Boston, MA) included
high-performance liquid chromatography (HPLC)-purified
[3-3H]glucose,
HPLC-purified
[2-3H]glucose,
3H2O,
[U-14C]aspartate,
[U-14C]citrulline,
[U-14C]glucose,
[U-14C]glutamine,
[U-14C]glutamate,
[U-14C]lactate,
U-14C-labeled
-methylglucose,
[U-14C]ornithine,
[U-14C]proline, and
[1,4-14C]succinate.
[5-3H]glucose and
[U-14C]alanine were
obtained from Amersham Life Sciences (Arlington Heights, IL). Ion
exchange resins and antibiotics and antimycotics including streptomycin
sulfate, penicillin G, kanamycin monosulfate, and amphotericin B
were purchased from Sigma Chemical (St. Louis, MO). Other
chemicals and reagents were obtained commercially and were of reagent
grade.
Glucose isotopes were purified using thin-layer chromatography. The
labeled isotope was applied to a 20 × 20 cm glass plate coated
with MN400 microcrystalline cellulose (250 µm, Avicel; Analtech,
Newark, NJ) and developed in a solvent system containing n-butanol, glacial acetic acid, and
H2O (24:4:10, vol/vol/vol), as
described previously (49). The presence of radiolabeled compounds was
detected using a radioactive plate scanner (Bioscan, Washington, DC).
The region of the plate containing glucose was scraped from the plate
and the glucose was eluted from the cellulose using Krebs-Henseleit
(KH) buffer and stored at
20°C. Radioactivity of the extract
was measured, and this information was used to achieve the desired
specific activity of the final substrate-containing solutions. In
controlled experiments (data not shown), the purification procedure
reduced the radioactivity in control flasks (without cells) and
reduced variability among replicates but did not significantly alter
values for glycolytic flux.
Preparation of isolated cells.
Animals were killed in the fed state on the morning of experimentation.
The entire small intestine was removed, and the appropriate segment was
excised (the proximal segment consisted of a 30-cm segment beginning 10 cm distal to the pylorus; the distal segment consisted of a 30-cm
segment proximal to the ileocecal junction). Cells were isolated from
the mucosa using chemical (EDTA) and gentle mechanical procedures (11,
13, 14, 25). The everted segments were filled with
Ca2+-free KH buffer containing
0.025% bovine serum albumin (BSA). The proximal segment required a
10-min EDTA incubation to completely remove the mucosal cells, whereas
the distal segment required a 20-min incubation. The cells were then
"peeled" from the underlying matrix by directing a pressurized
stream of ice-cold Ca2+-containing
KH buffer with 0.25% BSA and 5 mmol/l dithiothreitol at the mucosa.
Care was taken to maintain the cells at ~4°C throughout subsequent preparation steps. Antibiotics were included in the incubation solution (2.5 µg/ml amphotericin B, 100 µg/ml kanamycin monosulfate, 250 U/ml penicillin G, and 250 µg/ml streptomycin sulfate) and in the substrate solutions (0.25 µg/ml amphotericin B,
100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate). The
compositions of these mixtures were based on those used previously for
cells isolated from the intestine (25) and for preparing mammalian cell
cultures (14).
Lactate dehydrogenase (LDH; E.C.1.1.1.27) release (Sigma Diagnostics
kit DG1340-UV) was used to evaluate membrane integrity (13). Our
preparations contained primarily intact villi and rafts or clumps of
cells. During the 30-min incubation, leakage of LDH into the media
averaged 5-6% of the total cell content. Uptake of
-methylglucose (22) and
14CO2
production were used to assess metabolic integrity (20). The
-methyl-glucose uptake ratio averaged 23, indicating that
-methyl
glucose was concentrated inside the cell by 23-fold and showing that
the cell membranes were intact and the cells capable of pumping
substrate into the cells against a concentration gradient. Data are
reported on a dry weight basis (100°C, 2 h).
Incorporation of 3H from glucose into
3H2O and other
metabolites.
Cell suspensions (2-4 mg cells, dry wt for ion exchange chromatography
analyses; 5-7 mg cells for thinlayer chromatography analyses) were
incubated for 30 min at 37°C in KH buffer containing 0.25% BSA,
substrates, and trace quantities of
[2-3H]glucose,
[3-3H]glucose, or
[5-3H]glucose.
The specific activity of tracers ranged from 0.1 to 0.2 × 106 dpm/µmol glucose for ion
exchange chromatography and from 1-2 × 107 dpm/µmol glucose for
thin-layer chromatography. Each incubation included one of the
following substrates: glucose (0.01-5 mmol/l); glucose (5 mmol/l)
and glutamine (5 mmol/l); or glucose (5 mmol/l) and alanine
(5 mmol/l).
In experiments to determine metabolite production using ion exchange
chromatography, cell incubation reactions were stopped by the addition
of 5% ZnSO4 (47) followed by the
addition of an equimolar amount of 0.3 N
Ba(OH)2, according to the method of Bontemp et al. (4), and the mixture was vigorously agitated. In
these experiments, an aliquot of the supernatant (attained by
centrifugation at 500 g, 2-3
min), was passed through a column (0.7 ml) of Dowex 1 (borate form) and
3H2O
was rinsed from the resin with
H2O. This technique was used previously to separate
3H2O
quantitatively from 3H-glucose
(15). Radioactivity in aliquots of the eluate was determined using
liquid scintillation counting (Packard 1600TR, Packard Instruments,
Meriden, CT).
Dowex 1×8-400 was converted to the borate form in 100-g batches
by successive washes with 1 N NaOH (2 times),
H2O (4 times), 0.9 M boric acid (2 times), 0.05 M
Na2B4O7
(2 times), and H2O (4 times). Columns containing 0.7 ml of resin were rinsed
with H2O before sample loading.
Incorporation of glucose and glutamine carbon into metabolites.
Epithelial cells from the proximal small intestine of fed Fischer 344 rats were incubated with substrates and trace quantities of
[U-14C]glucose or
[U-14C]glutamine. Each
incubation included 4-6 mg cells (dry wt), tracer (2 × 106 dpm to quantify
CO2 production or 2 × 107 dpm to quantify other
metabolites), and one of the following substrates (in mmol/l): 5 glucose, 5 glutamine, 5 glucose and 5 glutamine, 5 glucose and 0.3 alanine, or 5 glucose and 5 alanine. After a 30-min incubation,
metabolism was stopped by the addition of
HClO4 (10%).
CO2 production was determined
using previously described procedures (11, 21).
To determine the incorporation of substrate into metabolites other than
CO2, the acidified incubates were
centrifuged at 4,300 g for 2 min.
Supernatants were neutralized with KOH, lyophilized, ethanol extracted,
and chromatographed on 20 × 20 cm cellulose thin-layer
chromatography plates using two-dimensional chromatography [solvent
1 composed of phenol,
H2O, and NaCN (3:1:0.003,
wt/vol/wt; Ref. 48; dried overnight);
solvent
2 composed of
n-butanol, glacial acetic acid, and
H2O (24:8:6,
vol/vol/vol)]. The Rf values for 22 amino acids, lactate, and succinate were previously
determined. Samples were spiked with a mixture containing
seven unlabeled amino acids (Ala, Gln, Glu, Asp, Pro, Cit, and Orn) to
facilitate visualization by spraying the developed plates with 0.05%
fluorescamine (48). Radioactivity was visualized using a radioactive
plate scanner. Regions of the plate corresponding to known metabolites were scraped and placed in scintillation vials, and compounds were
eluted with 400 µl of 0.1 M NaOH, 1 ml
H2O, and aqueous compatible scintillation cocktail (3a70b; RPI, Mt. Prospect, IL). Aspartate and
glutamate values were confirmed by processing samples through Dowex 50 and Dowex 1 (formate) ion exchange columns. Effluent containing
aspartate and glutamate was lyophilized, redissolved, and analyzed by
one-dimensional thin-layer chromatography
(solvent 1), then visualized, scraped, and
counted as described previously.
The specific activity of substrate [dpm/µmol carbon of
substrate
dpm/(µmol substrate × number of carbons/molecule
of substrate)] and the amount of radioactivity in each metabolite
were used to calculate the incorporation of substrate carbon into
metabolites.
Glucose utilization.
Net glucose disappearance was determined by measuring glucose
concentrations in the media before and after the 30-min incubation. Glucose concentrations were determined enzymatically using assay kits
obtained from Sigma Chemical.
Statistical analyses.
Two-way analysis of variance was used to analyze data describing the
effects of substrate and age (or segment) on glycolytic flux. When a
factor was found to be statistically significant (P < 0.05), follow-up tests were
executed using Tukey's Studentized range test at a procedure-wise
error rate of 0.05. All analyses were performed using the SPSSX
statistical package (40).
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RESULTS |
Reliability of detritiation techniques for quantifying glycolytic flux.
Differences in the rate of detritiation due to the position of the
3H on the glucose molecule were
investigated. Initially, cells were incubated with 5 mmol/l glucose,
and trace quantities of either
[2-3H]glucose,
[3-3H]glucose, or
[5-3H]glucose and
3H2O
were measured in the eluate after ion exchange chromatography. Production of
3H2O
from [2-3H]glucose
(8.2 ± 0.92 µmol · g
1 · min
1;
means ± SE, n = 3) was
significantly lower than from
[3-3H]- and
[5-3H]glucose isotopes
(12.7 ± 2.0 and 12.7 ± 2.9 µmol · g
1 · min
1,
respectively). In a subsequent experiment, the rate of detritiation of
these three isotopes was measured as a function of glucose concentration, and differences among the isotopes were observed across
a wide range of glucose concentrations (Fig.
1).

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Fig. 1.
Influence of glucose concentration on rate of appearance of
3H. Epithelial cells, isolated
from proximal small intestine of rats, were incubated 30 min in
presence of glucose at concentrations ranging from 0.1 to 5.0 mmol/l.
Cells were incubated in presence of 1 of the following radioisotopes:
[2-3H]glucose,
[3-3H]glucose, or
[5-3H]glucose.
Supernatants were applied to Dowex 1 borate columns, and radioactivity
was quantified in eluates.
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Glucose utilization by the isolated intestinal cells was calculated by
measuring the concentration of glucose in the media both before and
after incubation. Glucose utilization averaged 13.5 µmol · g
1 · min
1
when cells were incubated in glucose alone. This agrees well with
glycolytic flux data when measured using
[3-3H]- or
[5-3H]glucose.
However, these data suggested that glycolytic flux was being
underestimated when the
[2-3H]glucose isotope
was used.
The efficiency with which the Dowex 1-borate ion exchange resin
separated glucose from its metabolites was evaluated by quantifying the
recovery of radiolabeled standards from the columns. Glucose was found
to be entirely retained by the resin, and
H2O was found to be entirely
excluded (Table 1). Lactate and alanine
standards were not clearly discriminated, however, and these two major
metabolites of glucose were found in the eluate at ~5% and 50%,
respectively, using conditions identical to those for eluting
3H2O.
This raised the question as to whether, in cell incubations, there was
incomplete detritiation of some isotopes of glucose resulting in
retention of label as lactate or alanine and inaccurate measures of
glycolytic flux.
To ascertain this, radioactivity was determined in lactate and alanine.
In parallel experiments using
[U-14C]glucose as
tracer, we found that 76-78% of the metabolized glucose carbon
could be recovered in these products (Fig.
2). When
[2-3H]glucose was used
as tracer and cell supernatants were analyzed by quantitative
two-dimensional thin-layer chromatography,
3H was detected in both alanine
and lactate (Table 2). The retention of
tritium in alanine ranged from 0.2 to 0.5 µmol · g
1 · min
1
under the three substrate conditions evaluated. The retention of
tritium in lactate differed by substrate, however, and ranged from 0.95 µmol · g
1 · min
1
in the presence of 5 mmol/l each of glucose and alanine, to 4.8 µmol · g
1 · min
1
in the presence of glucose alone. When
[3-3H]glucose was used
as tracer, retention of 3H in
either alanine or lactate was negligible under the range of conditions
evaluated.

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Fig. 2.
Incorporation of glucose carbon into metabolites. Epithelial cells,
isolated from proximal small intestine of rats, were incubated 30 min
in presence of 5 mmol/l glucose, 5 mmol/l each of glucose and
glutamine, 5 mmol/l glucose and 0.3 mmol/l alanine, and 5 mmol/l each
of glucose and alanine. Trace quantities of
[U-14C]glucose were
present also. Specific activity of glucose in incubation medium was
used to calculate incorporation of glucose carbon into metabolites as
indicated ("others" refers to fraction of radioactive glucose
that was detected on regions of the plate not ascribed to substrate or
to 1 of the identified metabolites). Results are reported as µmol
glucose carbon atoms incorporated · g dry wt isolated
cells 1 · min 1.
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Table 2.
Appearance of 3H in alanine and lactate after
incubation of isolated jejunal epithelial cells with
[2-3H]- or [3-3H]glucose in presence of
glucose with or without other substrates
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The rates at which the total of all
3H-labeled metabolites of glucose
were produced was calculated using the following data: amount of
3H eluted from the Dowex 1 column,
the efficiency with which the major metabolites of glucose were
excluded from or retained by the Dowex resin, and the amounts of
3H-labeled alanine and lactate
produced. Using
[2-3H]glucose as
tracer, we found that the rate of
3H-elution from the Dowex column
was significantly higher in the presence of glutamine or alanine vs.
glucose alone (Table 3). When the retention
of 3H-labeled alanine and lactate
by the Dowex column was considered, however, the major metabolites of
glucose (sum of
3H2O,
[3H]lactate, and
[3H]alanine) were
produced at similar rates for
[2-3H]glucose under
the conditions evaluated. Because
[3H]lactate and
[3H]alanine were minor
metabolites of
[3-3H]glucose, values
for 3H eluted from the Dowex
column and for total glucose metabolized were similar when
[3-3H]glucose was used
as tracer (Table 3). Also, calculated values for total
3H-metabolite production were
similar when either
[2-3H]glucose or
[3-3H]glucose were
used as tracers, and these values were unaffected by the presence of
the nonglucose substrates glutamine and alanine. Based on these
results, future glycolytic flux experiments were conducted using
[3-3H]glucose, since
the tritium did not appear to be carried into major products such as
lactate and alanine, as was observed when [2-3H]glucose was
used.
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Table 3.
Total 3H-labeled metabolite production from glucose after
incubation of isolated jejunal epithelial cells with
[2-3H]- or [3-3H]glucose in presence of
glucose with or without other substrates
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Influence of animal age and intestinal segment on glycolytic flux.
Glycolytic flux in cells of the proximal small intestine was not
significantly different for young (4 mo) vs. aged (24 mo) animals, when
determined with glucose at 5 mmol/l (Table
4). Also, adding glutamine to the media at
5 mmol/l had no significant effect on flux of glucose through the
glycolysis pathway for animals at either of these two ages.
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Table 4.
Glycolytic flux rate using [3-3H]glucose for mucosal
cells of the proximal and distal small intestine from young and aged
rats
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When cells of the distal small intestine were incubated in glucose at 5 mmol/l, glycolytic flux was only 15% of the rate in cells of the
proximal small intestine (Table 4). A similar degree of difference
between segments was observed when both glucose and glutamine were
present.
Metabolites of glucose and glutamine using
14C-labeled substrates.
The formation of products from the metabolism of glucose and glutamine
was measured to consider both energy-producing and energy-consuming
processes when calculating the energetic contribution to intestinal
epithelial cells of these two major substrates. Major metabolites of
glucose included lactate, alanine, and
CO2 (Fig. 2). The relative
proportions and absolute amounts of these metabolites were influenced
by the composition of the media. In the presence of glucose alone,
lactate and CO2 predominated.
Including glutamine in the media reduced the formation of lactate and
CO2 from glucose and increased
alanine formation. Including alanine in the media had similar effects
to including glutamine, but the magnitude of the effects was influenced
by alanine concentration; increasing alanine concentration decreased
glucose conversion to lactate and
CO2 and increased conversion of
glucose to alanine. The presence of alanine did not influence
production of citrulline and CO2
from glucose. For all four substrate treatments, quantifiable but
insignificant amounts of glucose carbon (a total of <1 µmol carbon
atoms · g
1 · min
1)
were detected in compounds including ornithine, proline, glutamate, aspartate, and succinate.
Major metabolites of glutamine included glutamate and
CO2, which were produced in
similar quantities regardless of whether or not glucose was added to
the media (Fig. 3). Lactate and alanine were produced to lesser extents, and the addition of glucose appeared to increase incorporation of glutamine carbon into lactate but decrease
incorporation into alanine. Small amounts of glutamine carbon were also
incorporated into aspartate, citrulline, proline, ornithine, and
succinate. Overall, the presence of exogenous glucose did not influence
the incorporation of glutamine carbon into these metabolites.

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Fig. 3.
Incorporation of glutamine carbon into metabolites. Epithelial cells,
isolated from proximal small intestine of rats, were incubated 30 min
in presence of 5 mmol/l glutamine or 5 mmol/l each of glucose and
glutamine. Trace quantities of
[U-14C]glutamine were
present also. Specific activity of glutamine in incubation medium was
used to calculate incorporation of glutamine carbon into metabolites as
indicated ("others" refers to fraction of radioactive glutamine
that was detected on regions of the plate not ascribed to substrate or
to 1 of the identified metabolites). Results are reported as µmol
glutamine carbon atoms incorporated · g dry wt of
isolated
cells 1 · min 1.
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Energetic contribution of glucose vs. glutamine.
The relative energy contributed by the metabolism of glucose vs.
glutamine was calculated using glycolytic flux and substrate oxidation
data (Table 4, Figs. 2 and 3). Several assumptions were used in these
calculations. In intestinal cells, as in other cell types, the
conversion of glucose to pyruvate would produce 8 mol ATP/mol glucose,
assuming that reducing equivalents were transferred into the
mitochondria via the malate/aspartate shuttle. The conversion of 1 mol
pyruvate to 3 mol CO2 was assumed
to produce 15 mol ATP/mol pyruvate. The production of lactate from
pyruvate was assumed to require 3 mol ATP/mol lactate since 1 mol NADH would be utilized. Also, 1 mol glutamine was assumed to be oxidized to
2 mol CO2, with an energy yield of
9 ATP/2 CO2. This low rate of
oxidation was based on values for "A + T" reported previously for
intestinal cells incubated under similar conditions (13). Values for A + T are calculated from either the acetate or succinate CO2 ratios (e.g.,
14CO2
from
[1-14C]acetate/14CO2
from [2-14C]acetate) and predict
the probability that a molecule entering the tricarboxylic acid cycle
will be oxidized to CO2 (18).
Values for A + T averaged 0.4 for cells of the proximal small intestine when incubated in 5 mmol/l glutamine or in 5 mmol/l of both glutamine and glucose (13). This finding was recently verified in our laboratory
by calculating the ratio of
14CO2
from
[U-14C]glutamine/[1-14C]glutamine
(data not shown). Similar observations were reported previously by
Watford (45). We assumed that the remaining three carbons of the
glutamine molecule would be detected in other metabolites, allowing
independent consideration of their energetic value. The energy cost of
producing lactate was considered. However, due to the low flux rate of
glutamine into other metabolites that was observed (Fig. 3), the energy
cost of producing compounds such as citrulline was ignored in these
calculations. The formulae used to perform these calculations are
provided in the Table 5 legend.
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Table 5.
Contribution of glucose and glutamine to the energy supply of small
intestine mucosal cells from young rats
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In cells of the proximal small intestine, total ATP production from
exogenous substrates was higher when both substrates were present than
with either glucose alone (30% higher) or glutamine alone (161%
higher, using values in Table 5). With both substrates present, glucose
provided 62% of the net ATP so that the relative energy contribution
from glucose:glutamine was 62:38 for cells of the proximal intestine
(Table 6). Because lactate production from
glucose and glutamine was not determined for cells of the distal small
intestine, only a maximum ATP production from these two substrates
could be estimated. Using this approach, it appears that net ATP
production from glucose and/or glutamine would be at least two
times higher for cells of the proximal small intestine than for cells
of the distal segment (Table 5).
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Table 6.
Contribution of glucose to the energy supply for mucosal cells of the
proximal small intestine of young rats
|
|
The glycolytic flux data reported here (Table 4) and the glucose and
glutamine oxidation data reported previously (13) were used to
calculate energy production from glucose by jejunal epithelial cells of
aged vs. young animals under conditions in which both substrates were
present at 5 mmol/l. As a percentage of ATP from glucose and glutamine,
glucose contributed 55% vs. 58% of net ATP from young vs. aged
animals, respectively, assuming that lactate production was not
influenced by the aging process. Total energy produced from exogenous
substrates by cells of aged animals was calculated to average 90% of
the value for young animals, and this difference was due to lower
oxidative decarboxylation of both glucose and glutamine.
 |
DISCUSSION |
[3-3H]glucose but not
[2-3H]glucose can be used to
quantify glycolytic flux in intestinal cells.
The production of
3H2O
from glucose, measured using traditionally accepted procedures,
differed among the three
[3H]glucose isotopes that were
used in these experiments. Using epithelial cells from the proximal
small intestine, we found that the rates of detritiation were
significantly higher for the
[3-3H]- and
[5-3H]glucose isotopes than for
the [2-3H]glucose isotopes (see
RESULTS and Fig. 1). Neither purifying these isotopes using conventional thin-layer chromatography techniques nor purchasing HPLC-purified isotopes altered this finding, and this
difference could not be explained using the technical information available from the suppliers regarding position of the
3H-label.
For several reasons, these results were surprising and not readily
explained. First, the rate of detritiation is usually expected to occur
at a greater rate (not lesser rate as we observed) for the
[2-3H]glucose isotope
than for the
[3-3H]glucose or
[5-3H]glucose
isotopes. The production of
3H2O
has been found to be higher from
[2-3H]glucose than
from [3-3H]glucose or
[5-3H]glucose in
studies of muscle (28), hepatocytes (10, 30), and oocytes (19).
Detritiation of the
[2-3H]glucose isotope
occurs during hexose phosphate isomerization, producing
fructose-6-phosphate that could have other fates, in addition to
proceeding through the glycolytic cycle and being converted to pyruvate
and other metabolites (as discussed earlier). By comparison,
detritiation of the
[3-3H]glucose isotope
occurs both during the pentose phosphate pathway and during flux
through glycolysis, whereas detritiation of the [5-3H]glucose isotope
occurs predominantly during flux through glycolysis (reviewed in Refs.
17 and 51). Secondly, the apparent stimulatory effects of glutamine and
alanine on
3H2O
production using the
[2-3H]glucose isotope
were unexpected, since these results imply that glutamine and alanine
stimulate flux of glucose through glycolysis in epithelial cells of the
proximal small intestine. In other studies with these cells, glutamine
did not increase net glucose utilization (7, 21), and glutamine also
did not influence relative flux of glucose through the pentose
phosphate pathway (21). The apparent stimulatory effect of alanine on
glycolysis was unexpected since alanine is known to inhibit glycolysis
by inhibiting pyruvate kinase in a variety of cells, including
intestinal epithelial cells (42).
The unexpected results with the
[2-3H]glucose isotope
are shown in these studies to be due to incomplete detritiation of the glucose molecule, resulting in partial retention of the tritium in
lactate and alanine (Table 2). Furthermore, the completeness of the
detritiation of the
[2-3H]glucose isotope
was influenced by the substrates present in the media (Table 2),
suggesting that the presence of glutamine and alanine exerted effects
on the transfer of 3H to
H2O during the hexose phosphate
isomerization reaction. As much as 31% of the
3H-label from glucose passing
through the glycolytic cycle could be retained on the Dowex 1-borate
resin, resulting in gross underestimation (Table 3). Consequently, the
[2-3H]glucose isotope,
when used in conjunction with traditional ion exchange chromatography
techniques, could not be used to quantify flux of glucose through
glycolysis in epithelial cells isolated from the proximal small
intestine. The extent to which these same phenomena occur in other
systems is not known, as data demonstrating the validity of this
procedure have not generally been presented, and warnings (17)
regarding the limitations of the detritiation techniques for assessing
metabolic flux have been generally ignored. Our experiences with this
system suggest the following: the
[2-3H]glucose isotope
is incompletely detritiated during glycolysis in some metabolic
systems, allowing the label to remain in lactate and alanine; the
completeness of detritiation is not consistent across a range of
metabolic conditions (e.g., variations in the presence and
concentrations of metabolites and substrates); and the traditional ion
exchange chromatography techniques do not allow
3H2O
to be quantitatively separated from all
3H-labeled metabolites of glucose
(Table 1). When these factors were accounted for, flux of glucose
through glycolysis (calculated as the sum of the three major
metabolites of
[2-3H]glucose:
3H2O,
[3H]alanine, and
[3H]lactate) was
unaffected by glutamine or alanine (Table 3), which is as we would
expect.
The problems observed with using
[2-3H]glucose to
measure glycolytic flux were avoided by using
[3-3H]glucose as tracer.
Consequently,
[3-3H]glucose can be
used in conjunction with Dowex 1 ion exchange chromatography to
quantify flux of glucose through glycolysis in intestinal epithelial
cells. The close agreement in glycolytic rates that were measured using
[3-3H]glucose and
[5-3H]glucose (Fig. 1)
supports the validity of the use of
[3-3H]glucose for
quantifying glycolysis in intestinal epithelial cells. This close
agreement is expected in tissues in which only a small fraction of
glucose utilization occurs via the pentose phosphate pathway, such as
in isolated intestinal epithelial cells (21, 26).
Glycolysis is an important energy-producing pathway for intestinal
epithelial cells.
When both glucose and glutamine were present at 5 mmol/l, the
metabolism of glucose by cells of the proximal small intestine was
calculated to provide 62% of the net ATP produced from these two
substrates (Table 6). Previously, glucose was found, in neonatal pig
enterocytes, to contribute 40% of the ATP produced from glucose and
glutamine when both were present at 2 mmol/l (34) and 75% of ATP when
glucose and glutamine were present at 5 and 2 mmol/l, respectively
(52).
Glycolysis makes an important contribution to the energy supply of
enterocytes, but this fact is not reflected in the
CO2 data alone. To illustrate, the
relative CO2 production from
glucose:glutamine has been reported to be ~20:80 and ~30:70 in the
proximal and distal small intestine, respectively (20, 21, 50), yet our calculations suggest that glucose may provide at least 60% of the net
ATP under similar conditions. Furthermore, the results of
13C NMR studies using intestinal
epithelial cells provided data that could be used to calculate the
proportion of glucose-derived lactate formed directly from the pyruvate
produced via glycolysis (7). These data showed that 70% of the lactate
was produced directly via glycolysis when mucosal cells of the proximal
small intestine were incubated either in glucose alone or in a media containing both glucose and glutamine. These earlier findings were used
to calculate the contribution of anaerobic glucose metabolism (defined
here as cytosolic metabolism of glucose to lactate via pyruvate) to the
overall energy production from glucose metabolism. Analysis of ATP
yield showed that anaerobic glucose metabolism was responsible for 78%
of the energy produced from glucose when glucose was the sole exogenous
substrate and 95% of the energy produced from glucose when both
glucose and glutamine were present (Table 6). These calculations
emphasize that the glycolytic pathway is much more important than the
tricarboxylic acid cycle in producing energy from glucose in these
cells. Cells in culture are commonly found to rely more heavily on
anaerobic metabolism than intact tissue. Substantial lactate and
alanine production from glucose has been reported for the jejunal
epithelium when studied in vivo (50), however, and the ratio of glucose
carbon in (lactate + alanine)/CO2
was similar for the in vivo study (ratio of 5.2; Ref. 50) and for our
in vitro study (ratio of 6.6; Fig. 2). This suggests that the high
anaerobic metabolism reported here (Table 6) may reflect enterocyte
metabolism in vivo.
The glycolytic flux data used in the calculations presented in Tables 5
and 6 were determined with glucose and glutamine at equimolar
concentrations of 5 mmol/l. The concentration was set at 5 mmol/l, the
postprandial plasma glucose concentration, and this concentration also
approximates the luminal glutamine concentration after consumption of a
high-protein meal (1). To simulate a condition in which luminal
nutrients are unavailable to jejunal epithelial cells, the energy yield
from glucose could be calculated using data that would mimic plasma
glucose and plasma glutamine concentrations at 5.0 and 0.5 mmol/l,
respectively (50). Under these conditions, flux of glucose through
glycolysis would be unaffected (interpolation of data in Table 4),
glucose oxidation would be increased (21), and glutamine oxidation
would be decreased (21). Thus, whereas glucose contributed 62% of the
ATP when glucose and glutamine were both present at 5 mmol/l, glucose
is expected to contribute an even greater proportion of the ATP under fasting conditions.
The energy yield from glucose immediately after meal consumption could
be estimated also. After food consumption, glucose concentrations in
the jejunal lumen have been reported to reach 40-60 mmol/l (9,
31), and luminal glutamine concentrations may be as high as 5 mmol/l
(1). We found that flux through glycolysis was relatively stable at
glucose concentrations exceeding 5 mmol/l (Fig. 1), and we reported
previously the influence of high glucose concentrations on glucose and
glutamine oxidation (20, 21). If the calculations are made using
estimated values for glycolytic flux, glucose oxidation, and glutamine
oxidation, glucose is still shown to contribute ~60% of the net ATP
for jejunal epithelial cells under these fed conditions, also. Thus it
appears that glucose may provide at least 60% of the net ATP from
these two major substrates under a range of usual physiological
conditions.
Substantially more energy was produced from exogenous substrates when
both glucose and glutamine were available to the cells than when only
glutamine was present (Table 5). Whether the presence of exogenous
fuels increases the total energy available (via metabolism of both
endogenous and exogenous fuels) is unknown, although previous work by
us (13), Ashy and Ardawi (2), Mallet et al. (27), and Watford et al.
(46) has shown that glucose and glutamine increase oxygen uptake by
small intestine epithelial cells. Providing exogenous substrates has
been shown to increase ATP turnover and spare endogenous fuels in
newborn pig enterocytes (34).
Alanine does not inhibit glycolysis in intestinal epithelial cells.
Alanine is known to inhibit pyruvate kinase in a variety of cells,
including intestinal epithelial cells (42). Because of the potential
for alanine concentrations in and around these cells to be elevated by
the presence of glutamine (Fig. 3), the potential for alanine to
influence energy production was considered. Alanine did not influence
flux of glucose through glycolysis (Table 3) even when alanine was
present at concentrations greatly exceeding the plasma concentration of
0.3 mmol/l (32, 50). Also, alanine did not influence the glucose
oxidation to CO2 (Fig. 2). Thus it
appears that alanine does not inhibit glycolysis in intestinal epithelial cells as it does in other organs. This difference may be of
strategic importance to intestinal cells since the intestine serves the
body by metabolizing glutamine, and alanine is a metabolite of both
glucose and glutamine (Figs. 2 and 3).
The malate/aspartate shuttle vs. glycerol phosphate shuttle.
The relative importance of the malate/aspartate shuttle vs. the
glycerol phosphate shuttle has been considered. For most of the
calculations (Table 5), the malate/aspartate shuttle was assumed to
predominate. The general conclusions are unaltered, however, when it is
assumed that reducing equivalents are transported from the cytosol into
the mitochondria via the glycerol phosphate shuttle. To reach this
conclusion, net ATP from glucose metabolism was recalculated assuming
that NADH, produced in the cytosol via glycolysis but not reconsumed in
the conversion of pyruvate to lactate, would be oxidized in the
mitochondria with a net ATP production of 2 mol ATP/mol NADH, rather
than 3 ATP as for the malate/aspartate shuttle. The relative
contribution of glucose:glutamine was 54:46 for the glycerol phosphate
shuttle vs. 62:38 for the malate/aspartate shuttle (Table 5). This
demonstrates that glucose generates considerable energy in intestinal
epithelial cells, regardless of which shuttle predominates. We expect
that the malate/aspartate shuttle will predominate, however, since the
jejunal mucosa has been reported to have similar activities for
aspartate aminotransferase (also known as glutamate-oxaloacetate
transaminase) and alanine aminotransferase (also known as
glutamate-pyruvate transaminase), and enzyme activities have been
reported in both the cytosolic and mitochondrial fractions (43). Our
data show, however, that negligible quantities of aspartate are
released as a metabolite of glucose or glutamine by the epithelial
cells, whereas alanine is a major metabolite of both substrates (Figs.
2 and 3). The most likely role for the aspartate aminotransferase
enzyme, therefore, is to facilitate the interconversions needed for the
malate/aspartate shuttle. It is possible that the glycerol phosphate
shuttle is present in these cells also, since glycerol phosphate
dehydrogenase activity, the rate-limiting enzyme for this shuttle, has
been reported in the mitochondrial fraction of the rat small intestine (24). Because of the high proportion of glucose-derived pyruvate that
is converted to lactate, however, the relative flux through the two
major shuttles appears to exert little influence on the calculated
relative energy contribution of glucose and glutamine to jejunal
epithelial cells.
Glycolytic flux is not different for aged vs. young rats.
Aging (4 vs. 24 mo) did not significantly influence the rate of
glycolytic flux in cells of the proximal small intestine (Table 4), and
the influence of glutamine was also not different for cells taken from
young adult vs. aged adult animals. We reported previously that, in
comparison to cells from young animals, jejunal epithelial cells from
aged animals consume oxygen at a lower rate (13) and produce
CO2 from glucose at a lower rate
(12, 13). Our calculations show that net ATP production from exogenous
glucose and glutamine by jejunal cells of aged animals was ~90% of
the value calculated for jejunal cells of young animals (data in
RESULTS). Nonetheless, the
proportion of energy calculated to be available to cells from glucose
and glutamine was similar for young vs. aged animals (data in
RESULTS). Taken together, these data
show that all metabolic processes in intestinal epithelial cells are not influenced similarly by the aging process since
CO2 production, but not
glycolysis, is influenced by aging.
In these studies,
[3-3H]glucose
detritiation was found to reliably quantify flux of glucose carbon
through glycolysis by isolated rat small intestine epithelial cells.
This technique was used to determine the influence of other pyruvate-
or acetyl CoA-producing substrates on the conversion of glucose to
pyruvate. Glycolytic flux was six- to sevenfold higher in cells of the
proximal small intestine than in cells of the distal small intestine,
yet, in both segments, glucose was calculated to provide >50% of the
net ATP derived from the two major exogenously provided substrates, glucose and glutamine. In the presence of both glucose and glutamine, anaerobic metabolism of glucose provided 95% of the energy derived from glucose.
 |
ACKNOWLEDGEMENTS |
We thank Dr. John D. Cremin, Jr. for helpful discussions and review
of this manuscript. We also thank J. Quan, R. Gill, and B. Young for
technical assistance, M. Hudes for statistical consultation, I. Hincenbergs for animal care, and Professor B. O. de Lumen for making
the radioactive plate scanner available to us. In addition, we thank
Dr. C. E. Kight for initiating the metabolite analyses using ion
exchange and thin-layer chromatography techniques.
 |
FOOTNOTES |
Address reprint requests to S. E. Fleming.
Received 4 February 1997; accepted in final form 26 June 1997.
 |
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AJP Gastroint Liver Physiol 273(4):G968-G978
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