Mechanism of inhibition of Na1-glucose cotransport
in the chronically inflamed rabbit ileum
U. SUNDARAM,1 S. WISEL,1 V. M. RAJENDREN,2 AND A. B. WEST3
of Gastroenterology, Departments of Medicine and Physiology, Ohio State University
School of Medicine, Columbus, Ohio 43210; 2Department of Medicine, Yale University School
of Medicine, New Haven, Connecticut 06320; and 3Department of Pathology, University of Texas,
Galveston, Texas 77555
1Division
glucose absorption; sodium absorption; electrolyte transport;
chronic intestinal inflammation; sodium-glucose cotransport;
sodium-potassium-adenosinetriphosphatase; inflammatory
bowel disease
Na1 and Cl2 absorption and malnutrition
have been well described in human inflammatory bowel
diseases (1, 3, 5, 22). However, the cellular mechanisms
of alterations of coupled NaCl and nutrient-dependent
Na1 absorption during chronic ileal inflammation are
not well understood. This is primarily because of the
lack of good animal models of chronic ileitis and the
inability to isolate viable villus and crypt cells from the
inflamed intestine suitable for the study of electrolyte
transport.
We recently reported a rabbit model of chronic ileal
inflammation from which viable and relatively pure
populations of villus and crypt cells can be isolated (27).
In this model of chronic ileitis specific alterations in
electrolyte transport pathways were demonstrated. For
example, it was shown that the mechanism of inhibition of coupled NaCl absorption was due to an inhibi1
1 exchange on the
tion of Cl2/HCO2
3 but not Na /H
brush-border membrane (BBM) of villus cells (27).
INHIBITION OF
Another important Na1-absorptive pathway in the
normal ileum is Na1-nutrient cotransport (e.g., Na1glucose cotransport). An alteration of this cotransport
process in the chronically inflamed ileum will not only
affect Na1 absorption but also the assimilation of
important nutrients.
Na1-glucose cotransport is known to be present on
the BBM of villus but not crypt cells in the normal
ileum. In the intact cell, Na1-K1-adenosinetriphosphatase (ATPase) provides the favorable Na1 gradient for
this cotransporter (7, 10, 15, 16, 25, 26, 28). Thus,
during chronic ileal inflammation, cellular alterations
in Na1-glucose cotransport may be at the level of the
cotransporter and/or Na1-K1-ATPase. Therefore, the
aims of this study were to test the hypothesis that
chronic inflammation alters Na1-glucose cotransport
and to determine the cellular mechanisms of this
alteration.
METHODS
Induction of chronic inflammation. Chronic ileal inflammation was produced in rabbits as previously reported (27).
Pathogen-free rabbits were intragastrically inoculated with
10,000 oocytes of the coccidian protozoan Eimeria magna or
sham inoculated with 0.9% NaCl (control animals). Oocytes
were isolated from feces of infected rabbits by the method of
Jackson (12). 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 utilized for experiments.
Measurement of epithelial dynamics. Histological sections
were analyzed to determine alterations in epithelial morphology during chronic ileal inflammation. The villus-to-crypt
ratio was defined as total villus height from the base of the
crypt divided by the total crypt height. Ten different villus/
crypt units were measured in three control or inflamed
intestines from different animals.
Cell isolation. Villus and crypt cells were isolated from the
normal and inflamed ileum by a Ca21-chelation technique as
previously described (26, 27). Established criteria were utilized to validate good separation of villus and crypt cells.
These criteria included 1) marker enzymes (e.g., thymidine
kinase), 2) transporter specificity, 3) differences in intracellular pH, 4) morphological differences, and 5) differing rates of
protein synthesis.
The following set of criteria was utilized to exclude cells
that showed evidence of poor viability: 1) trypan blue exclusion, 2) the demonstration of Na1/H1 and Cl2/HCO2
3 exchange activities, and 3) the ability of the cells to maintain a
baseline pH or imposed acid or alkaline gradient and return
to baseline pH after perturbations. The cells were maintained
in short-term culture for up to 6–8 h. For short-term culture,
cells were resuspended at a final concentration of 0.1 g cells in
0193-1857/97 $5.00 Copyright r 1997 the American Physiological Society
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Sundaram, U., S. Wisel, V. M. Rajendren, and A. B.
West. Mechanism of inhibition of Na1-glucose cotransport in
the chronically inflamed rabbit ileum. Am. J. Physiol. 273
(Gastrointest. Liver Physiol. 36): G913–G919, 1997.—In a
rabbit model of chronic ileal inflammation, we previously
demonstrated that coupled NaCl absorption was reduced
1
1 exbecause of an inhibition of Cl2/HCO2
3 but not Na /H
change on the brush-border membrane (BBM) of villus cells.
In this study we determined the alterations in Na1-stimulated glucose [Na1-O-methyl-D-glucose (Na1-OMG)] absorption during chronic ileitis. Na1-OMG uptake was reduced in
villus cells from the chronically inflamed ileum. Na1-K1adenosinetriphosphatase (ATPase), which provides the favorable Na1 gradient for this cotransporter in intact cells, was
found to be reduced also. However, in villus cell BBM vesicles
from the inflamed ileum Na1-OMG uptake was reduced as
well, suggesting an effect at the level of the cotransporter
itself. Kinetic studies demonstrated that Na1-OMG uptake in
the inflamed ileum was inhibited by a decrease in the
maximal rate of uptake for OMG without a change in the
affinity. Analysis of steady-state mRNA and immunoreactive
protein levels of this cotransporter demonstrates reduced
expression. Thus Na1-glucose cotransport was inhibited in
the chronically inflamed ileum, and the inhibition was secondary to a decrease in the number of cotransporters and not
solely secondary to an inhibition of Na1-K1-ATPase or altered
affinity for glucose.
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NA1-GLUCOSE COTRANSPORT INHIBITION IN CHRONIC ILEITIS
was used as Pi standard. Enzyme specific activity was expressed as nanomoles of Pi released per milligram protein per
minute.
Northern blot studies. Total RNA was extracted from rabbit
ileal villus cells by the guanidinium isothiocyanate-cesium
chloride method. After denaturation total RNA was electrophoresed on 1.8% agarose-formaldehyde gel, transferred to nylon
membrane (Schleicher and Schuell, Keene, NH), and incubated with prehybridization solution. Membranes were hybridized with 32P-labeled SGLT1 cDNA. The cDNA was
random labeled with [32P]CTP with Klenow polymerase.
b-Actin was used to ensure equal loading of total RNA onto
the electrophoresis gels. Hybridized membrane was exposed
to autoradiography film (NEN, Boston, MA). The SGLT1
cDNA was generously provided by Dr. E. M. Wright.
Western blot studies. BBMV (4 mg) were diluted in SDSreducing buffer, boiled, and electrophoresed on a 12% SDSpolyacrylamide gel electrophoresis. The gel was electroblotted onto polyvinylidene difluoride membrane (NEN) and
blocked for 2 h in 5% bovine serum albumin at room temperature. The membrane was incubated at room temperature
with 1:3,000 anti-rabbit SGLT1 anti-serum followed by goat
anti-rabbit immunoglobulin G coupled to horseradish peroxidase (1:10,000, Pierce, Rockford, IL). After each incubation
the membrane was washed extensively with phosphatebuffered saline-0.2% Tween 20. The signal was developed with
the chemiluminescence Western blot kit (NEN). The SGLT1
antibody was generously provided by Dr. E. M. Wright.
Data presentation. When data are averaged, means 6 SE
are shown except when error bars are inclusive within the
symbol. All uptakes were done in triplicate. The number (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.
RESULTS
The histological observation of villus blunting in the
inflamed ileum was confirmed by measurements of
crypt and villus height. Villus-to-crypt ratio was noted
to be diminished from 7.97 6 0.7 to 3.98 6 0.2 (P ,
0.0001) from the normal to the chronically inflamed
ileum. The morphological changes have been observed
in detail previously (27).
Crypt-villus cell separation is an important concern
in intestine in which the normal maturation process is
altered. Thus the following criteria were used to ensure
good cell separation (27): 1) marker enzymes (alkaline
phosphatase for villus cells and thymidine kinase for
crypt cells), 2) higher intracellular baseline pH in crypt
compared with villus cells, 3) presence of Na1/H1
exchange in the BBM of villus but not crypt cells, 4) a
better developed BBM on villus cells, 5) higher rate of
protein synthesis in crypt cells compared with villus
cells, and 6) the presence of Na1-nutrient cotransport
on the BBM of villus but not crypt cells.
Cell viability by trypan blue exclusion was observed
in 94 6 4% of villus cells from the normal rabbit ileum and
in 93 6 5% of villus cells from the inflamed ileum (n 5 11).
We have previously demonstrated that Na1-dependent glucose uptake is present in villus but not crypt
cells of the normal rabbit ileum (26). The initial study
was performed to confirm this observation. In villus
cells from the normal ileum 3-OMG uptake was signifi-
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40 ml of Leibowitz-15 medium (GIBCO) with 20 mM N-2hydroxyethylpiperazine-N8-2-ethanesulfonic acid (HEPES),
10% rabbit serum, 5,000 U/l penicillin, 5 mg/l streptomycin,
and 10 mg/l gentamicin and gassed with 100% O2, pH 7.4 at
37°C and kept in sterile flasks until needed. Cells used for
BBM vesicle (BBMV) preparation were frozen immediately in
liquid nitrogen and stored at 270°C until required.
BBMV preparation. BBMV from rabbit ileal villus cells
were prepared by CaCl2 precipitation and differential centrifugation (17). Frozen villus cells were thawed and suspended in
2 mM tris(hydroxymethyl)aminomethane (Tris)-HCl buffer
(pH 7.5) containing 50 mM mannitol. The suspension was
homogenized, and 10 mM CaCl2 was added. The homogenate
was centrifuged at 8,000 g for 15 min, and the supernatant
was centrifuged at 20,000 g for 30 min. Then the pellet was
resuspended in 10 mM Tris-HCl buffer (pH 7.5) containing
100 mM mannitol and homogenized. Vesicles were formed by
adding MgCl2 (10 mM). The homogenate was centrifuged at
2,000 g for 15 min to remove debris, and the BBMV were
precipitated by centrifugation at 27,000 g for 30 min. BBMV
were resuspended in a medium appropriate to each experiment. BBMV purity was assured with marker enzyme (e.g.,
alkaline phosphatase) enrichment.
Uptake studies in villus and crypt cells. Villus or crypt cells
(100 mg wet wt) were washed and resuspended in HEPES
buffer containing (in mM) 1.25 3-O-methyl-D-glucose (3OMG), 4.5 KCl, 1.2 KH2PO4, 1.0 MgSO4, 1.25 CaCl2, 20
HEPES, and either 130 mM NaCl or choline chloride and
were gassed with 100% O2 (pH 7.4 at 37°C). Ten microcuries
of [3H]OMG (Amersham) were added to 1-ml cell suspension
in the HEPES buffer, and 100-µl aliquots were removed at
desired time intervals. The uptake was arrested by mixing
with 3 ml ice-cold stop solution (choline-HEPES buffer). The
mixture was filtered on 0.65-µm Millipore (HAWP) filters.
After two washes with ice-cold stop solution, the filter was
dissolved in 4 ml Optifluor and the radioactivity was determined.
BBMV uptake studies. Uptake studies were performed by
the rapid filtration technique as previously described (17). In
brief, 10 µl of BBMV resuspended in (in mM) 100 choline
chloride, 0.10 MgSO4, 50 HEPES-Tris (pH 7.5), 50 mannitol,
and 50 KCl were incubated in 90 µl reaction medium that
contained 50 mM HEPES-Tris buffer (pH 7.5), 1 mM OMG, 20
µM [3]OMG, 0.10 mM MgSO4, 50 mM KCl, 50 mM mannitol, 100
mM of either NaCl or choline chloride, 10 µM valinomycin, and
100 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone.
At desired times, uptake was arrested by mixing with ice-cold
stop solution (in mM, 50 HEPES-Tris buffer, 0.10 MgSO4, 75
KCl, and 100 choline chloride, pH 7.5). The mixture was filtered on 0.45-µm Millipore (HAWP) filters and washed twice
with 3 ml ice-cold stop solution. Filters with BBMV were dissolved in Optifluor, and radioactivity was determined. Amiloride (0.1 mM)-sensitive 22Na1 uptake in BBMV was also
performed by rapid filtration as previously described (18).
Na1-K1-ATPase measurement. Na1-K1-ATPase was measured as Pi liberated by the method of Forbush (8) in cellular
homogenates from the same amount of cells from normal or
inflamed ileum as described below. The reaction was started
by adding 20 mM ATP-Tris to 10 mg membrane protein in 50
mM Tris-HCl (pH 7.2), 5 mM MgCl2, and as appropriate 20
mM KCl and 100 mM NaCl. The reaction was stopped by the
addition of ice-cold 2.8% ascorbic acid, 0.48% ammonium
molybdate, and 2.8% sodium dodecyl sulfate (SDS)-0.48 M
HCl solution and placed on ice for 10 min. The color was
developed by incubating at 37°C for 10 min after the addition
of 1.5 ml of 2% sodium citrate, 2% sodium arsenite, and 2%
acetic acid. Readings were obtained at 705 nM, and K2HPO4
NA1-GLUCOSE COTRANSPORT INHIBITION IN CHRONIC ILEITIS
Altered Na1 permeability would be expected to diminish the Na1 gradient across the membrane and may
explain the inhibition of 3-OMG uptake seen in BBMV
from the inflamed ileum. Therefore, 22Na1 uptake in
villus cell BBMV from the normal and inflamed ileum
was determined and was found not to be different (0.10
nmol · mg protein21 · 15 s21 in normal and 0.11 nmol · mg
protein21 · 15 s21 in inflamed, n 5 3, P not significant).
Unaltered amiloride- sensitive 22Na1 uptake (i.e.,
Na1/H1 exchange) also indicates that the villus cell
BBM from the inflamed ileum is not significantly
contaminated by crypt cell BBM, because it is known
that Na1/H1 exchange is only present on the BBM of
villus but not crypt cells in the rabbit ileum (16).
BBM purity was also ensured by a similar degree of
enrichment of the villus cell BBM marker enzyme,
alkaline phosphatase (11.0 6 1.0-fold enrichment in
normal villus cell BBM and 10.1 6 1.1-fold enrichment
in inflamed villus cell BBM, n 5 3).
To determine whether the inhibition of Na1-glucose
cotransport during chronic ileal inflammation was due
to an alteration in the affinity for glucose and/or in the
maximal rate of uptake (Vmax ) of glucose, kinetic studies were performed. Uptake for all the concentrations
was carried out at 6 s because in initial uptake studies
Na1-dependent glucose uptake in BBMV was linear for
at least 10 s (data not shown) in the normal and the
inflamed ileum. Figure 5 demonstrates the kinetics of
glucose uptake in villus cell BBMV from the normal
and inflamed ileum. Figure 5A shows the uptake of
Na1-dependent 3-OMG as a function of varying concentrations of extravesicular glucose. As the concentration
of extravesicular glucose was increased, the uptake of
Na1-dependent 3-OMG was stimulated and subsequently became saturated in the normal as well as in
the inflamed ileum. With use of Enzfitter, a LineweaverBurk plot of these data was generated and is shown in
Fig. 5B. Kinetic parameters derived from these data
demonstrate that the affinity [1/Michaelis constant
(1/Km )] for 3-OMG uptake was not different between
the normal and inflamed ileum (Km for 3-OMG uptake
in BBMV was 6.6 6 1.5 mM in normal and 7.0 6 2.0 in
inflamed ileum, n 5 3, P not significant). However, the
Vmax of 3-OMG was reduced severalfold in the inflamed
ileum (Vmax for 3-OMG uptake in BBMV was 5.0 6 0.5
nmol · mg protein21 · 6 s21 in normal and 1.2 6 0.4
nmol · mg protein21 · 6 s21 in inflamed ileum, n 5 3, P ,
0.05). These data suggested that Na1-glucose cotrans-
Fig. 1. Effect of extracellular Na1 on
3-O-methyl-D-glucose (3-OMG) uptake
as a function of time in intact villus and
crypt cells from chronically inflamed
rabbit ileum. A: villus cells, n 5 6, * P ,
0.005. Extracellular Na1 significantly
stimulated 3-OMG uptake at all time
points in villus cells from inflamed ileum. B: crypt cells, n 5 6. Similar to the
normal ileum, Na1-stimulated 3-OMG
uptake was also not present in crypt
cells from inflamed ileum. r, Na1 present; s, Na1 absent.
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cantly stimulated by extracellular Na1 (e.g., 8.56 6
0.90 nmol/mg protein at 15 min in the presence of Na1
and 1.03 6 0.13 nmol/mg protein in the absence of Na1,
n 5 6, P , 0.0001). However, Na1-stimulated 3-OMG
uptake was not observed in crypt cells from the normal
ileum (e.g., 1.10 6 0.36 nmol/mg protein at 15 min in
the presence of Na1 and 1.20 6 0.11 nmol/mg protein in
the absence of Na1, n 5 6, P not significant).
Na1-stimulated 3-OMG uptake in villus and crypt
cells from the inflamed ileum was next determined
(Fig. 1). In villus cells from the inflamed ileum 3-OMG
uptake was also significantly stimulated by extracellular Na1 (Fig. 1A). Similar to the normal ileum, Na1stimulated 3-OMG uptake was also not present in crypt
cells from the inflamed ileum (Fig. 1B). Thus these data
demonstrate that Na1-stimulated glucose uptake is
present in villus but not crypt cells from the normal and
inflamed ileum.
Figure 2 compares the Na1-dependent uptake of
3-OMG in villus cells from the normal and inflamed
ileum. Na1-dependent 3-OMG uptake was significantly
diminished in villus cells from the inflamed ileum.
These data indicate that Na1-glucose cotransport was
reduced in intact villus cells from the inflamed ileum.
Inhibition of Na1-glucose cotransport at the cellular
level may represent a direct effect on the cotransporter
located on the BBM and/or may be secondary to an
inhibition of Na1-K1-ATPase on the basolateral membrane (BLM), which provides the favorable Na1electrochemical gradient for this cotransport process.
Thus Na1-K1-ATPase activity was measured as previously described (14) in homogenates of villus cells from
normal and inflamed ileum. Na1-K1-ATPase activity
was reduced ,50% in villus cells from the inflamed
ileum compared with the normal ileum (Fig. 3). These
data suggest that the reduction in Na1-glucose cotransport in inflamed ileal villus cells may, at least in part,
be due to reduced electrochemical gradients of Na1
across the BBM resulting from an alteration in Na1
extrusion capacity by the cells.
To determine whether chronic inflammation has a
direct effect on the Na1-glucose cotransporter itself,
3-OMG uptake was determined in BBMV prepared
from villus cells from the normal and inflamed ileum.
Na1-dependent 3-OMG uptake was significantly reduced in villus cell BBMV from the inflamed ileum (Fig.
4). These data suggest that the cotransporter itself was
directly inhibited during chronic ileal inflammation.
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NA1-GLUCOSE COTRANSPORT INHIBITION IN CHRONIC ILEITIS
Fig. 4. Na1-dependent uptake of 3-OMG in villus cell brush-border
membrane vesicles (BBMV) as a function of time from normal (r) and
chronically inflamed (s) ileum. Na1-dependent glucose uptake was
significantly reduced in villus cell BBMV from inflamed ileum (n 5 3,
* P , 0.05).
port was inhibited in the chronically inflamed ileum
secondary to a decrease in the number of cotransporters rather than altered affinity for glucose. To confirm
these findings we next looked at steady-state levels of
mRNA for SGLT1 in villus cells.
Steady-state levels of mRNA transcripts for SGLT1
were markedly reduced in villus cells from the chronically inflamed ileum (Fig. 6). Because steady-state
mRNA levels may not directly correlate with functional
protein levels on the BBM, immunoreactive SGLT1
levels on the BBM were also determined. Western blot
analysis of BBMV showed that the anti-SGLT1 antibody recognized one major immunoreactive protein
band at the expected size of 70 kDa, which was reduced
in intensity in the chronically inflamed ileum (Fig. 7).
parameters indicate that while the affinity for glucose
is not affected, the Vmax of glucose is reduced. Diminished levels of steady-state mRNA for SGLT1 and
SGLT1 immunoreactive protein are observed in the
chronically inflamed ileum. These data indicate that
the mechanism of Na1-glucose cotransport inhibition is
due to a decrease in the number of Na1-glucose cotransporters in the chronically inflamed ileum.
Our laboratory had previously demonstrated the
presence of Na1-glucose cotransport in isolated villus
but not crypt cells from the normal rabbit ileum (16,
26). This distribution was shown to be preserved in the
chronically inflamed ileum as well (Fig. 1). However,
one previous study has indicated the presence of Na1glucose cotransport in both villus and crypt cells from
the normal rabbit ileum (19). Inadequate cell separation criteria resulting in the contamination of crypt
cells with villus cells may account for this finding.
Indeed, immunocytochemistry and in situ hybridization studies have demonstrated that the Na1-glucose
cotransporter (SGLT1) is present only in the mature
villus cells in the normal rabbit ileum (11, 28). Thus
most of the currently available evidence supports the
observation that Na1-glucose cotransport is limited to
villus cells in the normal ileum.
Although the location of Na1-glucose cotransport
along the crypt-villus axis in the ileum is fairly clear,
how it is altered during chronic ileal inflammation is
not known. Previous studies have looked at the effect of
acute inflammation on Na1-glucose cotransport in several animal models. Differing observations about the
effect of acute inflammation on Na1-glucose cotransport have been reported; Na1-glucose cotransport was
found to be unaltered in acute enteritis produced by
Giardia in gerbils (4), whereas inhibition of Na1glucose cotransport was observed in acute enteritis
produced by Cryptosporidium in pigs (2) and Yersinia
enterocolitica in rabbits (21). It was not possible to
address the mechanism of alteration in Na1-glucose
cotransport at the cellular level based on these intact
tissue studies because in acute enteritis there is near
complete loss of mature villus cells that contain the
DISCUSSION
This study demonstrates that Na1-glucose cotransport is inhibited in the chronically inflamed ileum. This
inhibition is not entirely a consequence of a reduction
in the capacity of the villus cells to extrude Na1. In the
chronically inflamed ileum there is an inhibition at the
level of the Na1-glucose cotransporter itself. Kinetic
Fig. 3. Effect of chronic inflammation on Na1-K1-ATPase in villus
cells. Na1-K1-ATPase activity was decreased ,50% in villus cells
from inflamed ileum (10.8 6 2.0 nmol · mg protein21 · min21 in normal
and 5.8 6 1.2 nmol · mg protein21 · min21 in inflamed, n 5 6, * P ,
0.05).
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Fig. 2. Effect of chronic inflammation on Na1-dependent uptake of
3-OMG in villus cells. Na1-dependent 3-OMG uptake as a function of
time is shown and is significantly inhibited in villus cells from
inflamed ileum. Six inflamed (s) and six control (r) animals were
studied. * P , 0.01.
NA1-GLUCOSE COTRANSPORT INHIBITION IN CHRONIC ILEITIS
G917
Na1-glucose cotransporter. In acute enteritis produced
by transmissible gastroenteritis virus in piglets (9, 13,
14) the inhibition of Na1-glucose cotransport has been
postulated to be secondary to the loss of high affinity
D-glucose carrier. However, multiple Na1-glucose cotransport systems have not been described in other
animals, including rabbits (19). Thus the mechanism of
alteration of Na1-glucose cotransport during acute
intestinal inflammation is not completely understood.
The alterations that occur in Na1-glucose cotransport during chronic ileal inflammation have not previously been investigated. Undoubtedly, this is a result of
a lack of good animal models of chronic ileal inflammation. Two other models of chronic small intestinal
inflammation, peptidoglycan polysaccharide-induced
enterocolitis in rats (23) and alloimmunization-induced
enterocolitis in guinea pigs (20), have not yet been
utilized for electrolyte transport studies. At present
there are no perfect animal models of chronic ileitis
Fig. 6. Northern blot analysis demonstrates that steady-state levels
of SGLT1 mRNA are reduced in villus cells from chronically inflamed
ileum. Representative of 4 experiments each with different animals.
comparable to the human disease. Although this rabbit
model of chronic intestinal inflammation should not be
considered as an example of the human disease, it does
possess many of the same features; the ileum is thickened with a cobblestone appearance, the villi are
blunted, the crypts are hypertrophied, and the immune
response is characterized by chronic rather than acute
inflammatory cells. Thus it may be a suitable animal
model to study the effect of chronic ileal inflammation
on electrolyte transport at the cellular level.
Inhibition of Na1-glucose cotransport in villus cells
from the chronically inflamed ileum may occur at the
level of the cotransporter and/or secondary to an alteration in Na1 extrusion from the cell facilitated by
Na1-K1-ATPase. This study indicates that during
chronic ileal inflammation the mechanism of inhibition
of Na1-glucose cotransport is at the level of the cotransporter and is not exclusively secondary to an alteration
in the Na1 extrusion capacity of the villus cell. Kinetic
studies and Northern and Western blot studies indicate
that the number of Na1-glucose cotransporter is reduced in villus cells from the chronically inflamed
ileum.
One possible explanation for the reduction in the
number of Na1-glucose cotransporters in the chronically inflamed ileum may be a significant contamination of the isolated villus cell population with crypt cells
or immunocytes. Fortunately, in the chronically inflamed ileum many of the characteristics of mature
villus cells are clearly preserved, which allowed us to
separate villus and crypt cells consistently (27). Furthermore, the cell isolation process did not result in any
Fig. 7. Western blot analysis demonstrates reduction in amount of
immunoreactive SGLT1 in BBMV from chronically inflamed ileum.
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Fig. 5. Kinetics of glucose uptake in villus cell BBMV from normal (r) and chronically inflamed (s) ileum. A:
representative of 3 experiments. Na1-dependent uptake of [3H]OMG is shown as a function of varying concentration
of extravesicular D-glucose. Isosmolarity was maintained by adjusting the concentration of mannitol. Uptake for all
concentrations was determined at 6 s. As concentration of extravesicular glucose was increased, uptake of glucose
was stimulated and subsequently became saturated in villus cell BBMV from both normal and inflamed ileum. B:
analysis of these data with Lineweaver-Burk plot yielded kinetic parameters. Affinity for 3-OMG uptake is not
affected during chronic ileal inflammation. However, maximal rate of uptake of 3-OMG is reduced severalfold in
inflamed ileum.
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NA1-GLUCOSE COTRANSPORT INHIBITION IN CHRONIC ILEITIS
We thank K. M. Sundaram and V. Annapurna for technical
assistance. We thank Dr. E. M. Wright for generously providing
SGLT1 cDNA and antibodies used in the Northern and Western blot
studies.
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant R29-DK-45062 and the American Gastroenterological Association Research Industry Scholar Award
to U. Sundaram.
Address for reprint requests: U. Sundaram, Div. of Gastroenterology, Ohio State Univ. School of Medicine, N-214 Doan Hall, 410 W.
Tenth Ave., Columbus, OH 43210.
Received 24 September 1996; accepted in final form 30 June 1997.
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significant contamination of epithelial cells with immunocytes (27). Thus it is unlikely that the inhibition of
Na1-glucose cotransport during chronic ileal inflammation demonstrated in this study is a result of the
contamination of villus with crypt cells or immunocytes.
Alterations in absorption and secretion by ileal villus
and crypt cells undoubtedly occur as a result of the
numerous immune-inflammatory mediators endogenously produced in the chronically inflamed intestine
(5, 6, 22, 24). In this model of chronic inflammation we
have previously demonstrated that coupled NaCl absorption, which occurs by the dual operation of Na1/H1
and Cl2/HCO2
3 exchange on the BBM of villus cells, is
inhibited as a result of impairment of Cl2/HCO2
3 but
not Na1/H1 exchange. Unlike the villus cells, in the
crypt cells Na1/H1 exchange, known to be present only
on the BLM of crypt cells, was stimulated in the
chronically inflamed ileum. The BLM Na1/H1 exchange stimulation alkalinizes the crypt cells, which
may subsequently stimulate the BBM Cl2/HCO2
3 exchange resulting in HCO2
3 secretion by these cells (27).
The previous findings (27) and this study demonstrate that specific transport pathways are altered in
villus and crypt cells during chronic ileal inflammation
to inhibit coupled NaCl and glucose-stimulated Na1
absorption and promote HCO2
3 secretion. Given the
numerous immune-inflammatory mediators known to
be released in the chronically inflamed intestine and
that at least some of them are capable of affecting
electrolyte transport pathways, it is hypothesized that
different immune-inflammatory mediators released in
the chronically inflamed ileum may have unique effects
on transport pathways in villus and crypt cells. Which
of these agents are responsible for the transport abnormalities observed in this model of chronic ileal inflammation has yet to be elucidated.
In conclusion, Na1-glucose cotransport is inhibited
during chronic ileal inflammation. The mechanism of
inhibition is not solely secondary to a diminution in the
Na1 extrusion capacity of the villus cell. In fact, our
studies suggest that the number of Na1-glucose cotransporters is reduced in villus cells in the chronically
inflamed ileum.
NA1-GLUCOSE COTRANSPORT INHIBITION IN CHRONIC ILEITIS
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p. 75–114.
G919
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26. Sundaram, U., R. G. Knickelbein, and J. W. Dobbins. pH
regulation in ileum: Na1-H1 and Cl2-HCO32 exchange in isolated crypt and villus cells. Am. J. Physiol. 260 (Gastrointest.
Liver Physiol. 23): G440–G449, 1991.
27. Sundaram, U., and A. B. West. Effect of chronic inflammation
on electrolyte transport in rabbit ileal villus and crypt cells.
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1997.
28. Wright, E. M., B. A. Hirayama, D. D. F. Loo, E. Turk, and
K. Hager. Intestinal sugar transport. In: Physiology of the
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