Metabolism of short-chain fatty acids by rat colonic mucosa in
vivo
Mark D.
Fitch and
Sharon E.
Fleming
Department of Nutritional Sciences, University of California,
Berkeley, California 94720-3104
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ABSTRACT |
To determine the influence of substrate
concentration and substrate interactions on short-chain fatty acid
metabolism in vivo, a surgical procedure was established. Rats were
surgically operated to cannulate a 5-cm segment of proximal colon,
isolate the vasculature, and cannulate the right colic vein draining
this segment. Thus metabolism was restricted to the defined colonic
segment. The appearance of total
14C and
14CO2
in the mesenteric blood stabilized after 30 min of perfusion. Increasing luminal concentrations of butyrate from 2 to 40 mmol/l resulted in linear increases in total
14C, but
14CO2
production from
[14C]butyrate
increased as a function of concentration only up to 10 mmol/l and was
stable at higher butyrate concentrations. In addition to
CO2, 3-hydroxybutyrate and lactate
were major metabolites of acetate and butyrate in vivo. The presence of
a mixture of alternative substrates in the lumen had no influence on
the metabolism of butyrate to CO2
but significantly reduced the metabolism of acetate to
CO2. When compared with young (4 mo old) animals, transport of butyrate was significantly lower for aged
(48 mo old) animals, as evidenced by the rate of appearance in blood of
total 14C
(P = 0.04) and
14C in butyrate
(P = 0.03), but metabolism was
similar, since differences were not significant for
14C in the major metabolites
3-hydroxybutyrate (P = 0.06) and
CO2 (P = 0.17). These results show that
important aspects of short-chain fatty acid transport and metabolism
are not predicted from data using isolated colonocytes but require
study using an in vivo model.
acetate; butyrate; intestine; oxidation; ketone bodies
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INTRODUCTION |
SHORT-CHAIN FATTY ACIDS (SCFA), including acetate,
propionate, and butyrate, are produced in the cecum and colon of
nonruminant animals and humans via the fermentation of unabsorbed
carbohydrates and dietary fiber. The type of fermentation substrate and
the rate of fermentation can influence the luminal concentrations of
SCFA and the relative proportion of the individual acids (reviewed in
Ref. 14). The rate at which SCFA are transported across the mucosa of
the large bowel has been shown to be concentration dependent (15, 38),
suggesting that increased production rates will result in greater
availability of these compounds to the intestinal cells and other tissues.
Butyrate is known to influence the proliferation and differentiation of
multiple cell types, including colonocytes and intestinally derived
cell types (reviewed in Ref. 45). Also, butyrate withdrawal from guinea
pig colonic mucosa has been shown to cause time-dependent hypoplasia
and rapid triggering of massive apoptosis (27), providing evidence that
butyrate has the capacity to modulate survival and death of
colonocytes. Recently, experimental colitis was shown to enhance
carcinogenesis using a rat model, and butyrate was reported to reduce
the incidence and size of tumors in this study (10), suggesting that
butyrate may be useful in long-term therapy to reduce colon cancer risk
in ulcerative colitis.
From in vitro studies, it appears also that butyrate is an important
fuel for colonic epithelial cells. Butyrate has been shown to be
oxidized more readily to CO2 than
other potential substrates such as acetate, propionate, glucose,
glutamine, long-chain fatty acids, and ketone bodies (15, 17, 18).
Also, butyrate oxidation is not suppressed by the presence of other
energy-providing substrates (3, 6, 15), whereas the presence of
butyrate reportedly suppressed the oxidation of other SCFA including
acetate (6, 18). Recently, colonocytes from aged rats were reported to
oxidize fatty acids at an abnormally high rate, and in both the young
and aged animals butyrate was found to provide 50% of the energy for
isolated colonocytes (18). These and earlier observations have caused
butyrate to be recognized as the preferred energy-providing substrate
for colonic epithelial cells, leading to the speculation that butyrate
deprivation jeopardizes colonocyte and mucosal health. The work in aged
animals suggested abnormal fatty acid oxidation by colonocytes of these
senescent animals.
With the use of in vitro techniques, SCFA have been shown to be
metabolized by the colonic epithelial cells to
CO2 and other metabolites. The
large bowel of nonruminants appears to resemble the rumen epithelium in
its ketogenic capability (31). Specifically, instilling butyrate into
the rat cecum increased acetoacetate and 3-hydroxybutyrate
concentrations in the aorta blood (32), and butyrate increased net
production of ketone bodies by isolated human (34) and rat colonocytes
(2, 6, 35). In rat colonocytes, labeled butyrate was shown to be
incorporated into ketone bodies (13).
Whether the in vitro data accurately reflect the metabolism of SCFA by
the colonic epithelia in vivo is unknown, since in vivo data are not
available. Because the metabolic characteristics of cells in culture
are often found to differ quite considerably from their metabolism in
situ, in vivo studies of colonic SCFA metabolism were needed. To do
these studies, it was necessary to establish appropriate methodologies.
These techniques were used to 1) determine major metabolites
of luminal butyrate in vivo, 2)
determine how luminal butyrate concentration influences butyrate
transport and metabolite formation by the proximal colon, 3) evaluate whether butyrate
metabolism is influenced by the presence of other oxidizable
substrates, 4) compare metabolite
formation from butyrate vs. acetate in a mixture of substrates, and
5) compare butyrate oxidation in the
proximal colon of young adult vs. aged animals in vivo.
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MATERIALS AND METHODS |
Chemicals.
All chemicals were obtained from commercial suppliers and were reagent
grade. Radioisotopes
([1,2-14C]acetate and
[1-14C]butyrate) were
obtained from American Radiolabeled Chemicals (St. Louis, MO) and
DuPont NEN Research Products (Boston, MA). Unlabeled substrates,
acetylcysteine, and antibiotics were obtained from Sigma Chemical (St.
Louis, MO). Pentobarbital sodium (50 mg/ml) was obtained from Abbott
Laboratories (North Chicago, IL). Sodium heparin, injectable, was
obtained from Elkins-Sinn (Cherry Hill, NJ). All silicone tubing was
medical grade (Baxter-Scientific Products, McGaw Park, IL). Siliconized
glass wool was obtained from Alltech Associates (Deerfield, IL).
Animals.
For experiment 1, male Sprague-Dawley
rats weighing 350-450 g (6-9 mo of age) were obtained from
Simonsen Laboratories (Gilroy, CA). They were allowed free access to
commercial rat diet (chow no. 5012, Ralston Purina, St. Louis, MO). For
experiment 2, young (4 mo) and aged
(24-25 mo) male Fischer 344 (F344) rats were purchased from the
National Institute on Aging breeding colony maintained under
barrier-reared conditions (Harlan Industries, Indianapolis, IN). They
were allowed free access to National Institutes of Health 31 stock diet
(Western Research Products, Hayward, CA) because they had been fed this
diet throughout their lifetimes. All procedures involving animals were
reviewed and approved by the Animal Care and Use Committee at the
University of California (Berkeley, CA).
Substrates and solutions.
All substrate solutions were prepared in Krebs-Henseleit buffer (26)
but without Ca2+ and with
antibiotics (2.5 µl/ml amphotericin B, 100 µg/ml kanamycin monosulfate, 250 U/ml penicillin G, and 250 µg/ml streptomycin sulfate) and acetylcysteine (10 mmol/l). Unlabeled sodium butyrate was
dissolved in this solution to produce final concentrations of 2, 5, 10, or 40 mmol/l butyrate. Ethanolic solutions of
[1-14C]butyrate were
evaporated to near dryness and then added to the butyrate solutions to
produce a final specific activity of 0.1 µCi/µmol for the 2, 5, and
10 mmol/l substrate concentrations and of 0.06 µCi/µmol for the 40 mmol/l substrate concentration. Two substrates were also tested in a
mixture of 10 mmol/l each of acetate, propionate, butyrate, glucose,
and glutamine. The first contained
[1-14C]butyrate at a
specific activity of 0.1 µCi/µmol, and the second contained
[1,2-14C]acetate at a
specific activity of 0.3 µCi/µmol.
Dulbecco's PBS (12) was used during the surgical preparation to keep
abdominal tissues moistened. PBS containing 10 mmol/l acetylcysteine
was used as a flush solution for the cannulated colon lumen before the
infusion of labeled substrate solution.
Surgical procedures and experimental protocols.
The surgical techniques were based on the protocols developed for
studies in the rat small intestine (46-49). Details of the experimental procedures have been fully described (50). Modifications needed to adapt these procedures to the proximal colon are described below. A schematic of the overall surgical setup is provided (Fig. 1).

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Fig. 1.
Schematic representation of the surgical setup for catheterizing the
right colic mesenteric vein and cannulating the proximal colon of the
laboratory rat. BP, blood pressure.
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On the evening before the experiment, four or five animals were
anesthetized by intraperitoneal injection of pentobarbital sodium (5 mg/100 g body wt), and blood was collected using cardiac puncture.
During surgery, donor blood was reinfused into the operated experimental animal using a peristaltic pump equipped with an in-line
blood filter and bubble trap. The silicone tubing terminated in a 25 gauge × 11-mm needle that was inserted into the left saphenous vein.
On the day of the experiment, one rat was anesthetized by
intraperitoneal injection of pentobarbital sodium and prepared for surgery as described (50). To prepare the colon for cannulation, the
lower and middle portions of the colon were detached from the back body
wall by selectively severing the mesentery at the point of attachment.
Fatty tissue was moved to expose the underlying surface of the proximal
colon and associated vasculature. The middle colic artery and vein were
tied off and then cut to allow repositioning of the middle region of
the colon within the body cavity and facilitate installation of the
lumen cannula. With this one exception, care was taken not to interfere
with colonic innervation. The dorsal aorta was exposed and cleared of
fat and connective tissue from the left renal artery to the iliac
bifurcation, at which point a blunt trocar was introduced into the
abdomen and tunneled toward the base of the tail. An incision through the skin and muscle at this location allowed a saline-filled cannula to
be introduced and then positioned alongside the aorta, as the aorta
would be cannulated at a later time. This tubing was connected to an
electronic monitor (VT-15, Winston Electronics, Millbrae, CA) for
measuring blood pressure. The right colic vein was then exposed, taking
care to avoid trauma. Three 6-0 silk sutures were looped under the
vessel between the superior mesenteric vein and the branch point of the
vessel on the colon proper. These sutures were left loosely draped
while the lumen cannulas were installed, giving the right colic vein
time to relax and return to normal blood flow. The stainless steel tips
of the cannulas were inserted into the lumen of the proximal colon at
the position of the middle colic vein and at the cecocolonic junction.
The segment was then flushed with PBS containing 10 mmol/l
acetylcysteine to remove digesta and excess mucus.
In preparation for the installation of three vascular cannulas, the
animal was injected with 200 units of sodium heparin into the right
saphenous vein. The cannula supplying donor blood was installed into
the left saphenous vein, and the previously positioned aorta cannula
was installed into the aorta between the left renal artery and the
iliac bifurcation as previously described (50). The right colic vein
was then cannulated, and the 6-0 silk sutures were tightened to hold
the cannula tightly in place. The cannula consisted of an 11-mm portion
of a 23-gauge stainless steel needle with a 90° bend attached to 2 cm of silicone tubing (1.2 mm OD × 0.62 mm ID) and then to 30 cm
of polyethylene tubing (0.96 mm OD × 0.58 mm ID; Intramedic
PE-50, Becton Dickenson, Parsippany, NJ). Once blood flow was
established in the right colic vein cannula, the intestinal segments
and abdominal fat were placed in their final positions within the body
cavity and the abdominal opening was covered with plastic wrap.
Substrate solution labeled with
14C was delivered to the colon
segment through a 37°C warming loop at 2.0 ml/min for 1 min to displace the PBS flush solution. The flow rate was then reduced to 1.0 ml/min, and blood collections were started. Blood samples were
collected on ice at 10-min intervals under 300 µl mineral oil in 12 × 75-mm tubes. Luminal perfusion continued for 60 min.
Whole blood was analyzed immediately after collection for total
14C and for
14CO2.
Whole blood total 14C was
determined by dissolving 50-µl aliquots in alkaline tissue solubilizer (TS-2, RPI, Mt. Prospect, IL). They were decolorized with
300 µl of 15% benzoyl peroxide in toluene and counted with 5 ml
Hionic-Fluor scintillation cocktail (Packard Instruments, Meriden, CT).
Whole blood
14CO2
was determined in 200-µl aliquots using 25-ml Erlenmeyer flasks fitted with stoppers and disposable center wells. Proteins were precipitated with 0.8 ml methanol, and the flask contents were acidified with 1.0 ml of 1 M
NaH2PO4.
Carbon dioxide was trapped in 0.45 ml of 10 N NaOH in the center well
during a 2-h room temperature incubation and counted with 1.0 ml of
water and 15 ml of Hionic-Fluor cocktail.
Metabolites for HPLC analysis were extracted from frozen whole blood
aliquots using two volumes of 8%
HClO4 followed by neutralization with 5.0 N and 1.0 N KOH. The supernatant was lyophilized, and the
dried residue was extracted three times with 93% ethanol. The ethanol
supernatant was evaporated to dryness and derivatized using
bromoacetophenone (BAP, 50 mg/ml) and 18-crown-6 ether (25 mg/ml) in
acetonitrile (4). The BAP derivatives were dried and redissolved in
30% acetonitrile, and acid compounds were analyzed by reverse-phase
HPLC using a 25 × 0.46-cm, 5-µm particle size octadecylsilane
column (Ultrasphere, Beckman, Fullerton, CA). The elution rate (1.0 ml/min) and gradient (5 min at 30% acetonitrile, then a linear
increase in acetonitrile concentration of 1.53% per min, ending at
68% acetonitrile for 30 min) conditions were sufficient to resolve
lactic, acetic, propionic, butyric, acetoacetic, and 3-hydroxybutyric
acids. The HPLC column effluent fractions were collected and counted in
15 ml of 3a70b scintillation cocktail (RPI). All scintillation counting
was done in a 1600TR liquid scintillation counter (Packard). Metabolism
was quantified as the rate at which substrate carbon appeared in a
specific metabolite in mesenteric blood, and data are reported as
nanomoles of substrate carbon atoms per gram wet weight per minute,
where wet weight refers to the weight of the cannulated intestinal segment.
Expired breath from the animal was continually collected through a nose
cone and bubbled under slight vacuum through a liquid trap (800 ml of
0.5 N NaOH). At the conclusion of the experiment, 14CO2
was quantitated by counting 1-ml aliquots of the trap solution using
Hionic-Fluor scintillation cocktail.
Sections of liver, lung, and abdominal fat weighing 1-2 g were
excised and minced by hand. Aliquots (200-250 mg) were digested in
a tissue solubilizer (TS-2) at 50°C for 16 h, decolorized with 250 µl of 15% benzoyl peroxide in toluene, and counted using
Hionic-Fluor scintillation cocktail.
Experimental design and statistical analyses.
Data from two experiments are reported. In experiment
1, 21 Sprague-Dawley rats were randomly assigned to
seven treatments (substrates) so that a total of 3 animals were exposed
to each treatment. In experiment 2,
six young F344 rats and six aged F344 rats were randomly assigned to
two treatments (substrates) so that three young and three aged animals
were exposed to each treatment. (The substrates evaluated in
experiments 1 and
2 are listed in Tables 2 and 3,
respectively.)
Because of unequal variance among treatments in
experiment 1, differences among group
means for experiment 1 were determined on log-transformed data using one-way ANOVA and the
Tukey-HSD procedure as follow-up. Differences among group
means for experiment 2 were determined
on nontransformed data using ANOVA with two main effects (age and
substrate) and their interaction as sources of variation. The one-way
and ANOVA procedures in SPSS (42) were used to perform these
statistical analyses. Differences were considered to be statistically
significant at P
0.05.
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RESULTS |
Assessing whether the perfusion technique limits metabolism to the
cannulated segment.
To assess the validity of this technique, it was necessary to determine
whether the surgical procedure effectively prevented transport of the
luminal perfusate into tissues other than the cannulated intestinal
segment. To do this, breath gases were quantitatively collected
throughout experiments, and tissue specimens were taken after a 1-h
perfusion with 14C-labeled
substrate. Breath gases taken from animals throughout the 1-h perfusion
contained a small amount of radioactivity that comprised ~0.05% of
radioactivity in perfusates or 3% of the radioactivity transported
into the mesenteric blood (Table 1). Minute
quantities of radioactivity were also detected in the liver and in
blood taken from the aorta, and radioactivity in these tissues
comprised <1% of the radioactivity transported into the mesenteric
blood. Radioactivity in the visceral fat pad of experimental animals was equivalent to radioactivity in control animals that had never been
exposed to radioactivity (data not shown). The sum of radioactivity in
breath, fat, blood, and liver was calculated to be <5% of the radioactivity transported into the mesenteric blood during the 60-min
perfusion period.
Steady-state metabolic conditions.
Sufficient substrate was perfused through the cannulated colonic
segment to prevent substantial changes in the availability of substrate
to the epithelia during experimentation. This is evidenced by observing
that <2% of substrate in the perfusate was transported into
mesenteric blood during the 60-min perfusion period (Table 1). Also,
direct measurements of the perfusate at 0 vs. 60 min indicated that
substrate concentration changed on average by 2.6 ± 1.5% for data
from experiment 1 (n = 21).
Total 14C and
14CO2
are presented as a function of perfusion time for selected treatments
in which the lumen was perfused with 10 mmol/l butyrate. Total
14C and
14CO2
increased during the first 30 min of perfusion and then stabilized for
at least 20-30 min (Fig. 2). In
subsequent experiments, metabolite analyses were conducted on blood
samples taken from the 40- to 60-min time points.

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Fig. 2.
Influence of perfusion time on cumulative appearance in mesenteric
blood of total carbon and CO2 from
butyrate. Data were taken from experiment
1. Proximal colon was perfused continuously with trace
quantities of
[1-14C]butyrate in
either 10 mmol/l butyrate (n = 3) or a
5-substrate mixture (10 mmol/l each of acetate, propionate, butyrate,
glucose, and glutamine). Mesenteric blood was collected at 10-min
intervals during the 60-min perfusion period. An aliquot of each blood
collection was analyzed for total
14C and for
14CO2,
and specific activity of the substrate was used to calculate the rate
of appearance in blood of total butyrate carbon and of
CO2 from butyrate. Because
differences between the two substrates were not significantly
different, data were pooled by time. Values are means ± SE;
n = 6 for each time point. Values
( , ) with different superscripts are significantly different at
P < 0.05.
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Influence of butyrate concentration on
14C in mesenteric blood.
Four of the seven treatments evaluated in experiment
1 were used to determine the influence of luminal
butyrate concentration on the rate of appearance in mesenteric blood of
total 14C and its constituent
metabolites. In these treatments, 95-99% of total
14C in mesenteric blood was
accounted for as acetate, propionate, butyrate,
CO2, 3-hydroxybutyrate, or
lactate. Differences among the seven treatments in
experiment 1 were statistically
significant for total 14C and for
14C in constituents, including
acetate, butyrate, CO2,
3-hydroxybutyrate, and lactate (Table 2).
Follow-up analysis of differences among the mean values for the four
treatments (butyrate at 2, 5, 10, and 40 mmol/l) relevant to this
objective indicated that total 14C
in mesenteric blood increased linearly
(r = 0.998) with luminal butyrate
concentration (Fig.
3A).
Increasing luminal butyrate concentration also significantly increased
the rate of appearance in mesenteric blood of
14C in butyrate (Fig.
3B) and in metabolites including
acetate (Fig. 3C) and
3-hydroxybutyrate (Fig. 3E).
Differences among these four treatments were not statistically
significant for CO2 (Fig.
3D) or lactate (Fig.
3F).
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Table 2.
Experiment 1: significance of differences regarding influence of
luminal substrate on production of metabolites
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Fig. 3.
Influence of luminal butyrate concentration on the rate of appearance
(nmol carbon atoms · g wet wt
intestine 1 · min 1)
in mesenteric blood of total carbon atoms from butyrate
(A) and on the rate of appearance of
butyrate carbon in metabolites
(B-F).
Data were taken from experiment 1.
Proximal colon was continuously perfused with
[1-14C]butyrate in
butyrate at 2, 5, 10, or 40 mmol/l. Aliquots of mesenteric blood
collected during the 40- to 60-min perfusion periods were analyzed for
total 14C and for
14C in metabolites. Values are
means ± SE; n = 3 for each
concentration. These data are part of experiment
1 as presented in Table 2. Values ( ) with different
superscripts are significantly different at
P < 0.05.
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Luminal butyrate concentration influenced the relative proportions of
14C in the main metabolites. When
the lumen was perfused with 2 mmol/l butyrate,
14CO2
accounted for more than 50% of all
14C in metabolites, but this
proportion decreased with increasing butyrate concentration until, at
10 and 40 mmol/l, CO2 represented only 37 and 35%, respectively, of all metabolites (Fig.
4). Increasing butyrate concentration also
decreased the proportion of butyrate metabolized to lactate. In
contrast, 3-hydroxybutyrate represented only 28% of metabolites at 2 mmol/l butyrate but 51 and 50%, respectively, at 10 and 40 mmol/l
butyrate.

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Fig. 4.
Influence of butyrate concentration on the relative proportion of
substrate carbon in metabolites (%). Data were taken from
experiment 1. Proximal colon was
continuously perfused with
[1-14C]butyrate in
butyrate at 2, 5, 10, or 40 mmol/l. Aliquots of mesenteric blood
collected during the 40- to 60-min perfusion periods were analyzed for
total 14C and for
14C in metabolites, and these
values were used to calculate percentage of total substrate carbon in
each metabolite (substrate carbon identified as butyrate was excluded
from this calculation). Values are percent total substrate carbon in
nonsubstrate metabolites; n = 3 for
each concentration.
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Colonic metabolism of acetate vs. butyrate.
As part of experiment 1 (Table 2),
colonic metabolism of acetate and butyrate was compared. To do this,
data from four treatments were considered:
[14C]acetate in
acetate, [14C]butyrate
in butyrate,
[14C]acetate in a
mixture of five substrates (acetate, propionate, butyrate, glucose and
glutamine), and
[14C]butyrate in the
mixture of five substrates.
The rate of appearance in mesenteric blood of total
14C and of
14CO2
was not significantly lower for acetate than for butyrate when only
acetate or butyrate was present in the lumen (Fig.
5A). When a mixture of substrates was present, however, the rate of appearance of total butyrate carbon was 3.5-fold higher than the rate
of appearance of total acetate carbon, and the rate of appearance in
CO2 was 8.25-fold higher for
butyrate than for acetate carbon. The rate of appearance of
[3-14C]hydroxybutyrate
was significantly higher for butyrate than for acetate when the lumen
was perfused with a single substrate or with the mixture (Fig.
5E), and values for butyrate were
more than 12-fold higher than the values for acetate. Similarly,
[14C]lactate
appearance rate was nearly fourfold higher for butyrate than for
acetate when the lumen was perfused with the mixture of substrates
(Fig. 5F).

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Fig. 5.
Metabolite appearance from acetate vs. butyrate and influence of
substrate composition on metabolite appearance rate (nmol carbon
atoms · g wet wt
intestine 1 · min 1)
from both substrates. Data were taken from experiment
1. Proximal colon was perfused with trace quantities of
[14C]butyrate in 10 mmol/l butyrate (butyr) or in a mixture of substrates (10 mmol/l each
of acetate, propionate, butyrate, glucose, and glutamine) and trace
quantities of
[14C]acetate in 10 mmol/l acetate (Ac) or in a mixture of substrates. Aliquots of
mesenteric blood collected during the 40- to 60-min perfusion periods
were analyzed for total 14C
(A) and for
14C in metabolites
(B-F).
Values (bars) are means ± SE; n = 3 for each treatment. Values (bars) with different superscripts are
significantly different at P < 0.05.
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Of the 14C present in mesenteric
blood, metabolites accounted for a greater proportion of total
14C for butyrate than for acetate
(~45% for the two butyrate treatments vs. 20 and 34% for the two
acetate treatments). This difference was particularly evident when the
lumen was perfused with the mixture of substrates: only 54% of total
butyrate carbon was found in butyrate, whereas 80% of total
acetate carbon was found in acetate. The relative proportion of carbon
in the main metabolites also differed for acetate and butyrate (Fig.
6). When these SCFA represented the sole
luminal substrate, CO2 was the
most abundant metabolite of acetate, whereas both
CO2 and 3-hydroxybutyrate were
main metabolites of butyrate. Also, more than 20% of metabolized acetate was recovered in lactate vs. 8% for butyrate.

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Fig. 6.
Influence of perfusate composition on the relative proportion of
acetate and butyrate carbon in metabolites (%). Data were taken from
experiment 1. Proximal colon was
perfused with trace quantities of
[14C]butyrate in 10 mmol/l butyrate or in a mixture of substrates (10 mmol/l each of
acetate, propionate, butyrate, glucose, and glutamine) and trace
quantities of
[14C]acetate in 10 mmol/l acetate or in a mixture of substrates. Aliquots of mesenteric
blood collected during the 40- to 60-min perfusion periods were
analyzed for total 14C and for
14C in metabolites, and these
values were used to calculate percentage of total substrate carbon in
each metabolite (substrate carbon identified as either acetate or
butyrate was excluded from these calculations). Values are percent
total substrate carbon in nonsubstrate metabolites;
n = 3 for each concentration.
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Influence of alternative fuels on metabolism of acetate and
butyrate.
To determine the influence of alternative fuels on the metabolism of
acetate and butyrate, data for the single-substrate treatments were
compared with data for the corresponding treatments in the mixture of
substrates
([14C]acetate in
acetate vs.
[14C]acetate in a
mixture of five substrates;
[14C]butyrate in
butyrate vs.
[14C]butyrate in a
mixture of five substrates).
The rate of appearance in mesenteric blood of butyrate carbon was
not significantly different when butyrate was the sole luminal substrate from when the mixture of five substrates was present in the
lumen for total 14C,
14C in butyrate, or for
14C in any of the metabolites
including acetate, CO2,
3-hydroxybutyrate, or lactate (Fig. 5). For acetate carbon, however,
the rate of 14CO2
appearance was significantly lower when the mixture of substrates was
present than when only acetate was provided in the lumen (Fig. 5D). Differences were not
statistically significant for other metabolites.
The presence of the mixture of substrates had little, if any, effect on
the relative distribution of butyrate carbon among the main metabolites
(Fig. 6) but generally reduced the proportion of acetate carbon in
CO2 and increased the proportion
transported as 3- hydroxybutyrate.
The metabolic advantage of having more than one SCFA available in the
lumen was assessed by summing data for the two treatments in which the
lumen was perfused with the mixture of substrates ([14C]acetate or
[14C]butyrate in
mixture of five substrates). A comparison of these data with those for
treatments in which the lumen was perfused with either acetate or
butyrate as sole substrates indicates that the rate of appearance of
total substrate carbon from acetate plus butyrate was higher when the
mixture of substrates was present than when either was the sole
substrate (Fig. 7). The appearance rate of
acetate and butyrate carbon in the main metabolites
(CO2, 3-hydroxybutyrate, and
lactate), however, was similar to their rates of appearance when
butyrate was the sole substrate, suggesting that acetate made only a
minor contribution to the metabolic pool when butyrate was available.
Considerably more acetate appeared in blood, however, when the lumen
was perfused with the mixture of substrates, and this increment was
largely responsible for the higher rate of appearance of total
14C.

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Fig. 7.
Rate of appearance in mesenteric blood of carbon from acetate or
butyrate or of the sum of acetate plus butyrate carbon when both
substrates are provided. Data were taken from
experiment 1. For bar labeled
[14C]acetate in
acetate, proximal colon was perfused with trace quantities of
[14C]acetate in 10 mmol/l acetate. For bar labeled
[14C]butyrate in
butyrate, proximal colon was perfused with trace quantities of
[14C]butyrate in 10 mmol/l butyrate. For bar labeled
[14C]acetate plus
[14C]butyrate in
mixture, data from 2 treatments were added together. These 2 treatments
included [14C]acetate
in the 5-substrate mixture and
[14C]butyrate in the
5-substrate mixture as described more fully in Table 2. Values are
means; n = 3 for each treatment.
|
|
Influence of animal age on transport and metabolism of butyrate.
Animal age significantly influenced the rate of appearance in
mesenteric blood of butyrate carbon in several metabolites (Table 3). Because interactions between animal age
(young vs. aged) and perfusion substrate (butyrate alone vs. butyrate
in a mixture of five substrates) were not statistically significant,
differences due to animal age could be determined using data pooled for
the two perfusates. The pooled means for young animals were
significantly higher than those for aged animals for total
14C,
14C in butyrate, and
14C in lactate (Fig.
8). Animal age, however, did not
significantly influence the metabolism of butyrate to
CO2 or 3-hydroxybutyrate.
View this table:
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|
Table 3.
Experiment 2: significance of differences regarding influence of animal
age and luminal substrate on production of metabolites from butyrate
|
|

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|
Fig. 8.
Influence of animal age on rate of appearance in mesenteric blood of
metabolites of butyrate (nmol carbon atoms · g wet wt
intestine 1 · min 1).
Data were taken from experiment 2.
Proximal colons of young (4 mo) and aged (24 mo) Fischer 344 rats were
perfused continuously with trace quantities of
[1-14C]butyrate in
either 10 mmol/l butyrate (n = 3) or a
5-substrate mixture (10 mmol/l each of acetate, propionate, butyrate,
glucose, and glutamine). Aliquots of mesenteric blood collected during
the 40- to 50-min perfusion period were analyzed for total
14C and for
14C in metabolites. Because
differences between the 2 substrates were not significantly different,
data were pooled by animal age; values are means ± SE, and
n = 6 for young and
n = 5 for aged. Significance of
differences (P values) are provided
for total butyrate carbon and key metabolites.
|
|
 |
DISCUSSION |
To our knowledge, this is the first model that simultaneously restricts
metabolism to a selected colonic segment and permits the metabolism to
be studied in vivo. With the use of a perfusion model based on the
jejunal model developed by Windmueller and Spaeth (47), metabolism in
the proximal colon could be studied under steady-state conditions. Our
results suggest that labeled metabolites did not escape from the lumen
of the perfused segment. Thus it seems unlikely that metabolites
originated from tissues other than from the perfused segment.
Influence of butyrate concentration on metabolite production.
Absorption of butyrate carbon into the mesenteric blood increased
linearly with luminal butyrate concentration. Similar observations have been made previously using the human rectum (38) and rat cecum
(15). Our observations do not preclude carrier-mediated transport, as
observed in the small intestine (39, 40) and colon (21, 29, 30, 33,
41), but demonstrate the important and direct effect of luminal
butyrate concentration on the net rate of transport across the intact
proximal colon of the rat in vivo.
The appearance in mesenteric blood of
CO2 from butyrate was not
significantly influenced by luminal butyrate concentration, presumably
due to the high variability among the small number of replicates. Thus
the current data suggest that luminal butyrate concentrations may limit
butyrate oxidation, since the mean values for the 2 mmol/l treatment
was only 50% of the value for the 10 mmol/l treatment. Butyrate
concentrations in cecal fluid and colonic contents of rats, pigs, and
monkeys have been reported to be as low 3-7 mmol/l when diets
contained little or no fermentable dietary fiber (7, 11, 16, 24, 43)
and as high as 40 mmol/l when the diets provided ample fermentable
fiber (5, 43, 44). In studies that have used isolated colonocytes, the
Michaelis-Menten constant for butyrate oxidation to
CO2 has been reported to range from 0.06 to 0.3 mmol/l (6, 8, 25). From in vitro data, one would
predict that butyrate oxidation by colonocytes would always be maximal,
and this contrasts sharply with observations in vivo. There are several
possible explanations for these differences. First, a higher
concentration of butyrate may be needed in vivo than in vitro to
achieve a certain intercellular concentration of butyrate. This could
be due to the presence of mucus in vivo or to differences in the
absorptive surface per cell, since cells are exposed to butyrate at the
luminal surface only in the in vivo model but on all surfaces in the in
vitro model. Second, the butyrate concentration gradient between the
lumen and intracellular space is likely to be greater in vivo than in
isolated cells. In isolated cells, the transport of absorbed butyrate
out of the cell would be inhibited by butyrate in the medium, thus
maintaining a near equilibrium with the substrate in vitro. In vivo, a
large percentage of absorbed butyrate (up to 80%) is continually
secreted into the blood stream. Third, the in vivo procedure is unable to eliminate metabolic inhibitors from unintentionally entering the
cells via the basolateral membrane. Although further studies would be needed to determine the explanation for these differences, it
appears that the luminal concentration of butyrate needed for maximal
butyrate oxidation cannot be accurately assessed using isolated colonocytes.
In our studies, there was substantial incorporation of butyrate into
3-hydroxybutyrate but acetoacetate was undetectable. In studies with
isolated colonocytes, the presence of butyrate was reported to cause a
greater net production of acetoacetate than 3-hydroxybutyrate (2, 13,
35, 36) and low 3-hydroxybutyrate-to-acetoacetate ratios (8, 9).
Results of studies using epithelial slices (19, 20) have been more
consistent with the in vivo data presented here, however. Differences
among these studies are likely due to differences in the redox status
of the cells rather than due to differences in relative flux through
specific metabolic pathways. The current study demonstrates that ketone
bodies (predominantly 3-hydroxybutyrate) are major metabolites of
butyrate in vivo, since more than 50% of the metabolized butyrate was
incorporated into 3-hydroxybutyrate when the lumen was perfused with 40 mmol/l butyrate. This is the first report, to our knowledge, of ketone body production from butyrate by the rat colon in vivo, and these results suggest similarities in the metabolic fate of butyrate between
rats and ruminant animals (22, 31).
Small amounts of butyrate were incorporated also into lactate, although
incorporation was not significantly different for the 2 vs. 40 mmol/l
butyrate treatments. Using isolated colonocytes, others have reported
that butyrate did not stimulate lactate production when presented alone
(2, 6, 35) or in the presence of other SCFA (28).
Influence of alternative fuels on butyrate metabolism and comparison
to acetate metabolism.
When the lumen was perfused with a mixture of five substrates,
significantly more carbon atoms from butyrate than acetate were
transported across the colonic mucosa, and significantly more butyrate
carbon was incorporated into CO2,
3-hydroxybutyrate, and lactate. If the 3.5-fold higher value for
butyrate carbon vs. acetate carbon in mesenteric blood is converted to
nanomoles of substrate transported, butyrate molecules were transported across the epithelium at a 1.7-fold higher rate than for acetate molecules. This observation is not consistent with previous data attained using the rat cecum, which showed that the three main SCFA
were transported out of the lumen at equivalent molar rates (15). The
transport kinetics may differ between the cecum and proximal colon,
since others have reported that SCFA transport differs along the
longitudinal axis of the colon (23, 37). Although the factor
responsible for these differences is not known, the current data
describe SCFA transport in the proximal colon for the first time, to
our knowledge, and the differences between acetate and butyrate are
noteworthy. The data show that acetate transport was not significantly
inhibited by the presence of other SCFA, whereas acetate metabolism to
CO2 was suppressed by the mixture
of substrates. This suppression effect may be due to the presence of
butyrate in the mixture, since others have shown, using isolated
colonocytes, that butyrate suppresses acetate oxidation (6, 18).
Influence of animal age on butyrate transport and metabolism.
The transport of total butyrate carbon and unmetabolized butyrate
across the proximal colon was significantly higher for young (4 mo)
than for aged (48 mo) animals. These differences were significant also
when transport was calculated in nanomoles per centimeter per minute
(data not shown), indicating that the aging effect was not likely due
to differences in the density (wt/length) of the epithelial cell layer
and verifying previous observations (18). Metabolism of butyrate to the
major metabolites, CO2 and 3-hydroxybutyrate, was not significantly influenced by age in these in
vivo studies, although there was a tendency for butyrate metabolism to
change in parallel with butyrate carbon transport. Previously, butyrate
oxidation to CO2 was reported to
be significantly lower for rat colonocytes isolated from young vs. aged
F344 rats (17, 18). Thus, for reasons that cannot be elucidated from the current studies, the effect of aging on colonocyte metabolism appears not to be accurately predicted using isolated colonocytes. It
is conceivable that changes in the cellular membrane may occur as a
consequence of the aging process, and these changes might reduce
butyrate transport across the cellular and subcellular membranes in
vivo, which, in turn, would reduce its metabolism. If colonocyte
membranes are influenced by the aging process, the chemical and
mechanical processes used to isolate colonocytes might have a different
effect on colonocytes of aged than on those of young animals. The
differences between in vitro and in vivo studies support this
suggestion and raise doubts that isolated cells can be reliably used to
evaluate the effects of aging on transport and metabolism.
In conclusion, a rat model was developed that permitted the metabolism
of SCFA by the mucosa of the proximal colon to be studied in vivo.
Transport of butyrate from the lumen into the mesenteric blood
increased linearly with increasing butyrate
concentrations, whereas butyrate metabolism followed
saturation kinetics. The major metabolites of both acetate and butyrate
were CO2, 3-hydroxybutyrate, and
lactate, and the proportions of these metabolites were dependent on
substrate concentration. Neither the transport nor metabolism of
butyrate was influenced by the presence of alternative substrates, including acetate or propionate. In contrast, the oxidation of acetate
to CO2 was suppressed by the
presence of alternative substrates. When present with other substrates,
transport and metabolism were significantly lower for acetate than for
butyrate. The transport of butyrate and, to a lesser extent, its
metabolism were lower in the proximal colon of aged than in that of
young animals. These results show that aspects of SCFA transport and
metabolism are not predicted from data using isolated colonocytes but
require study using an in vivo model.
 |
ACKNOWLEDGEMENTS |
We are indebted to Dr. John C. Cremin, Jr., for establishing the HPLC
technique in our laboratory and for helpful scientific discussion. We
also thank Dr. Mark Hudes for statistical consultation and A. Spaeth
for helpful discussion regarding the surgical procedures.
 |
FOOTNOTES |
This work was supported by National Institute on Aging Competitive
Grant RO1-AG-10765 and the Agriculture Experiment Station.
This work was presented in part as a poster at the annual meeting of
the Federation of American Societies for Experimental Biology, New
Orleans, LA, April 1997 (abstract in FASEB
J. 11: A380, 1997).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. E. Fleming,
Dept. of Nutritional Sciences, Univ. of California, Berkeley, CA
94720-3104 (E-mail: fleming{at}nature.berkeley.edu).
Received 27 October 1998; accepted in final form 23 March 1999.
 |
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