Departments of Internal Medicine and Physiology, Division of Digestive Diseases, The Ohio State University School of Medicine, Columbus, Ohio 43210
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ABSTRACT |
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Short-chain fatty acids (SCFA) have been demonstrated to
at least partially ameliorate chronic intestinal inflammation. However, whether and how intestinal SCFA absorption may be altered during chronic intestinal inflammation is unknown. A rabbit model of chronic
ileitis produced by coccidia was used to determine the effect of
chronic inflammation on ileal
SCFA/HCO3 exchange.
SCFA/HCO
3 exchange was present in the
brush-border membrane (BBM) of villus but not crypt cells from normal
rabbit ileum. An anion-exchange inhibitor, DIDS, significantly inhibited SCFA/HCO
3 exchange.
Extravesicular Cl
did not alter the uptake of SCFA,
suggesting that SCFA/HCO
3 exchange is
a transport process distinct from
Cl
/HCO
3 exchange.
In chronically inflamed ileum, SCFA/HCO
3 exchange was also present
only in BBM of villus cells. The exchanger was sensitive to DIDS and
was unaffected by extravesicular Cl
. However,
SCFA/HCO
3 exchange was significantly reduced in villus cell BBM vesicles (BBMV) from chronically inflamed ileum. Kinetic studies demonstrated that the maximal rate of uptake of
SCFA, but not the affinity for SCFA, was reduced in chronically inflamed rabbit ileum. These data demonstrate that a distinct SCFA/HCO
3 exchange is present on BBMV
of villus but not crypt cells in normal rabbit ileum.
SCFA/HCO
3 exchange is inhibited in
chronically inflamed rabbit ileum. The mechanism of inhibition is most
likely secondary to a reduction in transporter numbers rather than
altered affinity for SCFA.
inflammatory bowel disease; short-chain fatty acid transport; immune regulation of nutrient transport; anion exchanger
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INTRODUCTION |
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SHORT-CHAIN FATTY ACIDS (SCFA) are important energy
sources for the large intestinal epithelium. SCFA absorption has been described in guinea pig, human, mouse, rat, and rabbit colon (1, 4, 5,
8, 9, 11, 14, 15). These studies suggested two mechanisms of
incorporation of SCFA in the mammalian colon: 1) SCFA
absorption in its protonated form is passive and the proton is supplied
by the brush-border membrane (BBM) Na+/H+
exchanger (14, 15); and/or 2) SCFA is absorbed by
SCFA/HCO3 exchange on the BBM of
absorptive cells (1, 8, 9). SCFA/HCO
3 exchange has also been demonstrated in the human ileum (6, 13). It is
of note that this anion exchanger was not inhibitable by stilbene
derivatives in these studies (6, 8, 9).
Although there is some controversy about the exact mechanism of SCFA incorporation in the colon, the presence of an active transporter suggests that it may be susceptible to alterations in pathophysiological conditions such as inflammatory bowel disease (e.g., Crohn's disease). A reduction in the production of intestinal SCFA has been postulated to result in intestinal inflammation (7). Evidence to support this hypothesis largely comes from the observations that local instillation of SCFA in diversion or distal ulcerative colitis ameliorates the intestinal inflammation (2, 7, 12). However, the effect of chronic intestinal inflammation on SCFA absorption is unknown.
Previous studies have demonstrated that during chronic intestinal
inflammation unique alterations occur in coupled
Na+-Cl absorption and
Na+-dependent nutrient transport (17-20). Given this
background the aims of this study were to 1) demonstrate
SCFA/HCO
3 exchange in the rabbit
ileum; 2) determine the villus-crypt distribution of
SCFA/HCO
3 exchange in the rabbit
ileum; and 3) delineate the alterations in
SCFA/HCO
3 exchange during chronic
ileal inflammation.
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METHODS |
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Induction of chronic inflammation. Chronic ileal inflammation was produced in rabbits as previously reported (17). Pathogen-free rabbits were intragastrically inoculated with 10,000 Eimeria magna oocytes or sham inoculated with 0.9% NaCl (control animals). None of the sham inoculations and ~80% of inoculations with coccidia resulted in chronic ileal inflammation during days 13-15. Only enterocytes from those animals that had histologically confirmed chronic ileal inflammation were used for experiments.
Cell isolation.
Villus and crypt cells were isolated from normal and chronically
inflamed rabbit ileum by a calcium chelation technique as previously
described (16, 17). Previously established criteria were used to
validate good separation and viability of villus and crypt cells (16,
17). Cells used for BBM vesicle (BBMV) preparation were frozen
immediately in liquid nitrogen and stored at 70°C until required.
BBMV preparation. BBMV from rabbit ileal villus or crypt cells were prepared by CaCl2 precipitation and differential centrifugation as previously described (18-20). BBMV were resuspended in a medium appropriate to each experiment. BBMV purity was assured with marker enzyme enrichment (18-20).
BBMV uptake studies. Uptake studies were performed by a rapid filtration technique as previously described (18-20). In brief, BBMV were suspended in 100 mM N-methyl-D-glucamine (NMG) gluconate, 0.10 mM MgSO4, 50 mM HEPES-Tris (pH 7.5), and 50 mM KHCO3 or K gluconate. The reactions were started by adding 90 µl of reaction mix containing 100 mM NMG gluconate, 0.1 mM MgSO4, 10 µM valinomycin, 50 µM [14C]butyrate, 50 mM HEPES-Tris, pH 7.5 or pH 6.0, and 50 mM KHCO3 or K gluconate. At desired times uptake was arrested by mixing with ice-cold stop solution (50 mM HEPES-Tris buffer, pH 7.5, 0.10 mM MgSO4, 50 mM K gluconate, and 100 mM NMG gluconate). The mixture was filtered on 0.45-µm Millipore (HAWP) filters and washed twice with 3 ml of ice-cold stop solution. Filters with BBMV were dissolved in Optifluor, and radioactivity was determined in a Beckman LS-5 scintillation counter.
Data presentation. When data are averaged, means ± SE are shown except when error bars are inclusive within the symbol. All uptake studies were done in triplicate. The n for any set of experiments refers to vesicle or isolated cell preparations from different animals. Preparations in which cell viability was <85% were excluded from analysis. Student's t-test was used for statistical analysis.
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RESULTS |
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SCFA uptake in rabbit ileal BBMV may be secondary to nonionic diffusion or carrier-mediated transport. To determine whether SCFA uptake could occur via nonionic diffusion, uptake of [14C]butyrate at an alkaline pH (pH 8 where pHin = pHout) and an acidic pH (pH 6 where pHin = pHout) was performed. With a 100-fold increase in proton concentration, an increase in [14C]butyrate uptake was observed (92.5 ± 14.1 vs. 168.4 ± 12.4 pmol/mg protein at 1.5 s; n = 3, P < 0.05).
Next, to determine whether SCFA absorption could occur via a
carrier-mediated anion-exchange process such as
SCFA/HCO3 exchange, a series of
experiments were performed. [14C]butyrate
uptake was stimulated by a HCO
3
gradient in villus cell BBMV (Fig.
1A). Gradients of pH and
HCO
3 further stimulated
[14C]butyrate uptake in villus cell BBMV.
Although a smaller (25 mM) HCO
3
gradient did significantly promote [14C]butyrate uptake (340 ± 62 and 95 ± 14 pmol/mg protein at 3 s with and without gradient, respectively), it was
significantly less than that seen with a 50 mM
HCO
3 gradient (750 ± 75 compared
with 340 ± 62 pmol/mg protein at 3 s; n = 3, P < 0.01). The distribution of this anion exchanger along the villus-crypt
axis of the normal rabbit ileum was then determined. Neither the
HCO
3 gradient nor the pH and
HCO
3 gradients stimulated
[14C]butyrate uptake in normal ileal crypt cell
BBMV (Fig. 1B). Thus SCFA/HCO
3
exchange is present in the BBM of villus but not crypt cells in the
normal rabbit ileum.
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Unlike other anion exchangers (e.g.,
Cl/HCO
3), the
SCFA/HCO
3 exchange in human ileum and colon and rat colon has been suggested to be insensitive to
anion-exchange inhibitors such as DIDS or SITS. Therefore, the effect
of DIDS on rabbit ileal SCFA/HCO
3
exchange was studied. DIDS (1 mM) almost completely inhibited
HCO
3 gradient-stimulated
[14C]butyrate uptake in villus cell BBMV (Fig.
2A). DIDS also nearly completely
inhibited pH and HCO
3
gradient-stimulated [14C]butyrate uptake in
villus cell BBMV (Fig. 2A). Thus, unlike SCFA/HCO
3 exchangers in human ileum
and colon and rat colon, the rabbit ileal
SCFA/HCO
3 exchanger appears to be
sensitive to stilbene derivatives. Figure 2B demonstrates the
DIDS dose-response curve for the rabbit ileal
SCFA/HCO
3 exchanger.
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To ensure that the observed [14C]butyrate
uptake is not via the
Cl/HCO
3 exchanger,
which is also known to be present on the BBM of rabbit ileal villus
cells (16), the effect of extravesicular Cl
on
[14C]butyrate uptake was studied.
Extravesicular Cl
did not inhibit the uptake of
[14C]butyrate in BBMV (Fig.
3). These data indicated that
SCFA/HCO
3 exchange is distinct from
Cl
/HCO
3 exchange in
normal rabbit ileal villus cells.
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Next, to establish whether [14C]butyrate uptake
represented electrodiffusional coupling, voltage-clamp experiments were
performed. Voltage clamping with equal intra- and extravesicular
K+ with its ionophore, valinomycin, did not alter pH and
HCO3 gradient-stimulated
[14C]butyrate uptake in villus cell BBMV (Fig.
3). These data indicated that [14C]butyrate
uptake occurs via an electroneutral process in the villus cell BBMV.
Having demonstrated the presence of a DIDS-sensitive
SCFA/HCO3 exchange in normal ileal
villus but not crypt cells, we next looked at the effect of chronic
ileal inflammation on this transport process. Both
HCO
3 gradient-stimulated [14C]butyrate uptake and pH and
HCO
3 gradient-stimulated [14C]butyrate uptakes were present in BBMV
prepared from villus cells from the chronically inflamed ileum (Fig.
4A). However, similar to the normal
ileum, neither HCO
3 nor pH and HCO
3 gradient-stimulated
[14C]butyrate uptake was present in crypt cell
BBMV from the chronically inflamed ileum (Fig. 4B). These data
demonstrated that the villus-crypt distribution of
SCFA/HCO
3 exchange is not altered during chronic ileal inflammation.
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Next, to determine whether the functionality of the
SCFA/HCO3 exchanger may be altered in
the chronically inflamed ileum, the effect of the anion-exchange
inhibitor DIDS was determined. As shown in Fig.
5, similar to the normal rabbit ileum, DIDS
also significantly inhibited the
SCFA/HCO
3 exchange in the chronically
inflamed rabbit ileum.
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To ensure that the observed [14C]butyrate
uptake is not via
Cl/HCO
3 exchange,
which is known to be inhibited in the chronically inflamed rabbit ileum
(17), the effect of extravesicular Cl
on
[14C]butyrate uptake was studied. Similar to
the normal ileum, extravesicular Cl
did not inhibit
the uptake of [14C]butyrate in BBMV (Fig.
6). These data indicated that
SCFA/HCO
3 exchange is also distinct
from Cl
/HCO
3
exchange in the chronically inflamed rabbit ileum.
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To establish whether [14C]butyrate uptake
represented electrodiffusional coupling in the chronically inflamed
ileum, voltage-clamp experiments were performed. Voltage clamping did
not alter the pH and HCO3
gradient-stimulated [14C]butyrate uptake in
villus cell BBMV from the chronically inflamed ileum (Fig. 6). These
data indicated that butyrate uptake also occurs via an electroneutral
process in the chronically inflamed rabbit ileum.
Next, the effect of chronic ileal inflammation on
SCFA/HCO3 exchange in villus cells
from the normal and chronically inflamed ileum was compared.
HCO
3 gradient-stimulated
[14C]butyrate uptake was reduced in villus cell
BBMV from the chronically inflamed ileum (Fig.
7). Furthermore, pH and
HCO
3 gradient-stimulated
[14C]butyrate uptake was also reduced in villus
cell BBMV from the chronically inflamed ileum (Fig. 7). These data
indicated that SCFA/HCO
3 exchange is
inhibited in the chronically inflamed rabbit ileum.
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To decipher the mechanism of inhibition of
SCFA/HCO3 exchange in the chronically
inflamed ileum, kinetic studies were performed. Because pH and
HCO
3-dependent [14C]butyrate uptake in BBMV was linear for at
least 6 s in the normal as well as the chronically inflamed ileum (data
not shown), uptake for all concentrations were carried out at 3 s.
Figure 8 demonstrates the kinetics of
butyrate uptake in villus cell BBMV from the normal and chronically
inflamed ileum. The figure shows the uptake of butyrate as a function
of varying concentrations of extravesicular butyrate. As the
extravesicular concentration of butyrate was increased, the uptake of
butyrate was stimulated and subsequently became saturated in the normal
as well as the chronically inflamed ileum (Fig. 8A).
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With the use of Enzfitter, a Lineweaver-Burk plot of these data was
generated, and this is shown in Fig. 8B. Kinetic parameters derived from these data demonstrated that the affinity for
[14C]butyrate uptake was not different between
the normal and chronically inflamed ileum [Michaelis constant
(Km) for butyrate uptake in BBMV was 39.6 mM in
normal and 40.9 mM in inflamed ileum]. However, the maximal
velocity (Vmax) of
[14C]butyrate uptake was reduced severalfold in
the chronically inflamed ileum (Vmax for butyrate
uptake in BBMV was 16.0 and 3.75 nmol · mg
protein1 · 3 s
1
in normal and inflamed, respectively). These data indicated that SCFA/HCO
3 exchange was inhibited in
the chronically inflamed rabbit ileum secondary to a decrease in the
number of anion exchangers rather than altered affinity for butyrate.
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DISCUSSION |
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This study demonstrates that SCFA/HCO3
absorption occurs via SCFA/HCO
3
exchange in the rabbit ileum. This transporter is present on the BBM of
villus but not crypt cells. The rabbit ileal
SCFA/HCO
3 exchange is DIDS sensitive.
This anion exchanger is distinct from Cl
/HCO
3 exchange,
which is also known to be present on the BBM of villus and crypt cells
in the rabbit ileum.
In the chronically inflamed rabbit ileum the
SCFA/HCO3 exchanger is also distinct
from Cl
/HCO
3
exchange. It is only present on the BBM of villus cells and is also
DIDS sensitive. However, SCFA/HCO
3 exchange is inhibited during chronic ileitis and the mechanism of
inhibition is secondary to a reduction in the number of transporters and not secondary to an alteration in the affinity for SCFA.
Previous studies have demonstrated that
SCFA/HCO3 exchange is present in the
BBM in human ileum and rat and human colon. However, the villus-crypt
distribution of this anion exchanger was previously unknown. This study
demonstrates that SCFA/HCO
3 exchange
is localized to the BBM of villus but not crypt cells in the normal
rabbit ileum.
The BBM SCFA/HCO3 exchangers
previously demonstrated in the human and rat colon have been shown to
be insensitive to anion-exchange inhibitors of the stilbene-derivative
class (e.g., DIDS). However, the BLM
SCFA/HCO
3 exchanger in the rat colon
was shown to be sensitive to DIDS (10). The rabbit ileal BBM
SCFA/HCO
3 exchanger is sensitive to
DIDS and in this regard resembles the rat BLM
SCFA/HCO
3 exchanger more than the BBM
SCFA/HCO
3 exchanger in human or rat colon.
It is reasonably clear that SCFAs are important nutrients for
colonocytes and that they stimulate Na+ and fluid
absorption in the colon (1). It is also fairly well accepted that local
instillation of SCFA in diarrheal diseases characterized by chronic
inflammation of the colon alleviates, at least partially, the
malabsorption of fluid and electrolytes and improves the colonic
inflammation (2, 7, 12). On the basis of these observations it has been
hypothesized that SCFAs help to maintain the health and functional
integrity of the colonic epithelium. However, how SCFA absorption or
SCFA/HCO3 exchange may be altered
during chronic intestinal inflammation is unknown.
In a rabbit model of chronic ileal inflammation this laboratory has
demonstrated multiple unique alterations in electrolyte and nutrient
transport processes. NaCl absorption was inhibited by an inhibition of
Cl/HCO
3 but not
Na+/H+ exchange on the BBM of villus cells
during chronic ileitis (17). Na+-dependent nutrient
cotransport processes were also inhibited in the chronically inflamed
ileum by different mechanisms of inhibition. For example,
Na+-glucose cotransport was inhibited by a decrease in
cotransporter numbers without a change in the affinity for glucose
(18). In contrast, Na+-amino acid cotransport was inhibited
by a decrease in the affinity for amino acid without a change in the
cotransporter numbers (19). Unlike these two Na+-nutrient
cotransport processes, Na+-bile acid cotransport was
inhibited by both a decrease in cotransporter numbers and a decrease in
the affinity for the bile acid (20). In view of the unique nature of
the transport alterations observed in the chronically inflamed rabbit
ileum, it was hypothesized that different immune inflammatory mediators
may have unique effects on different transport processes during chronic
ileitis (19, 20).
The effect of chronic intestinal inflammation on SCFA transport has not
previously been studied. In one study, using intact tissue, acute
inflammation was noted to inhibit SCFA absorption in the rabbit colon
(3). However, this study did not demonstrate the presence of
SCFA/HCO3 exchange or delineate the
mechanism of inhibition of SCFA absorption. Furthermore, in acute
intestinal inflammation the severe loss of absorptive cells may be the
primary reason for the alterations in transport processes seen in these
conditions. In chronic intestinal inflammation the architectural
changes are not as severe as those seen on acute inflammation, and thus
the mucosa during chronic inflammation is a more suitable model to
determine the effect of immune-inflammatory mediators on transport
processes. Because local instillation of SCFA in
chronically inflamed colon improves the inflammation and malabsorption,
it may be hypothesized that the absorption of SCFA may not be
completely affected in these tissues. However, alterations in SCFA
absorption during chronic intestinal inflammation are at present
unknown. Similarly, the effect of chronic intestinal inflammation on
SCFA/HCO
3 exchange is also at present
unknown. This study demonstrates that
SCFA/HCO
3 exchange is inhibited during
chronic ileal inflammation. The mechanism of inhibition is secondary to
a reduction in transporter numbers rather than an altered affinity for SCFA.
In conclusion, this study presents several novel observations about
SCFA/HCO3 exchange: 1) it is
only present in the BBM of villus cells in the normal and chronically
inflamed rabbit ileum; 2) it is sensitive to anion-exchange
inhibitors such as DIDS in the rabbit ileum; 3) it is inhibited
during chronic ileitis; and 4) its mechanism of inhibition is
secondary to a reduction in transporter numbers in the chronically
inflamed ileum.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grant R29-DK-45062 and a Ohio State University Davis Medical Center grant to U. Sundaram.
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FOOTNOTES |
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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: U. Sundaram, Division of Digestive Diseases, Ohio State Univ. School of Medicine, N-214 Doan Hall, 410 W. 10th Ave., Columbus, OH 43210 (E-mail: sundaram-1{at}medctr.osu.edu).
Received 3 December 1998; accepted in final form 5 November 1999.
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