Stress Impairs Murine Intestinal Barrier Function: Improvement by Glucagon-Like Peptide-2

Abstract

Stress-induced intestinal barrier dysfunction may be involved in chronic intestinal disorders. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic growth hormone that can rapidly improve intestinal epithelial barrier function. Here, we investigated whether mouse intestine is responsive to chronic psychological stress and whether pretreatment with GLP-2 can ameliorate stress-induced changes. Mice were subjected to water avoidance stress (WAS; 1 h/day for 10 days) with GLP-2 or saline administered 4 h before each WAS session. After the final stress period, the intestine was removed for assessment of physiological/morphological changes. Compared with controls (sham-stressed mice), stressed mice demonstrated enhanced ion secretion and permeability in the jejunum, ileum, and colon. In addition, increased numbers of bacteria were observed adhering to and/or penetrating the epithelium, associated with infiltration of mononuclear cells into the mucosa. GLP-2 treatment improved intestinal barrier function in stressed mice and ameliorated other aspects of impaired host defense. Our study extends previous findings in rats of stress-induced intestinal dysfunction and provides insights into potential novel therapeutics.

Full text

Stress Impairs Murine Intestinal Barrier Function: Improvement
by Glucagon-Like Peptide-2
Heather L. Cameron and Mary H. Perdue
Intestinal Disease Research Programme, McMaster University, Hamilton, Ontario, Canada
Received February 23, 2005; accepted March 24, 2005
ABSTRACT
Stress-induced intestinal barrier dysfunction may be involved in
chronic intestinal disorders. Glucagon-like peptide-2 (GLP-2) is
an intestinotrophic growth hormone that can rapidly improve
intestinal epithelial barrier function. Here, we investigated
whether mouse intestine is responsive to chronic psychological
stress and whether pretreatment with GLP-2 can ameliorate
stress-induced changes. Mice were subjected to water avoid-
ance stress (WAS; 1 h/day for 10 days) with GLP-2 or saline
administered 4 h before each WAS session. After the final
stress period, the intestine was removed for assessment of
physiological/morphological changes. Compared with controls
(sham-stressed mice), stressed mice demonstrated enhanced
ion secretion and permeability in the jejunum, ileum, and colon.
In addition, increased numbers of bacteria were observed ad-
hering to and/or penetrating the epithelium, associated with
infiltration of mononuclear cells into the mucosa. GLP-2 treat-
ment improved intestinal barrier function in stressed mice and
ameliorated other aspects of impaired host defense. Our study
extends previous findings in rats of stress-induced intestinal
dysfunction and provides insights into potential novel therapeu-
tics.
Psychological stress has been shown to influence the clin-
ical course of chronic intestinal disorders such as inflamma-
tory bowel disease (Levenstein et al., 2000; Collins, 2001;
Ringel and Drossman, 2001) and irritable bowel syndrome
(IBS) (Mayer, 2000). Clinical and experimental studies have
documented significant gut pathophysiology in response to
acute and chronic psychological stress. In particular, stress-
induced changes described in rats include increased secre-
tory state, altered colonic motility, increased epithelial per-
meability to small and large probes, damaged mitochondria
in epithelial cells, altered epithelial/bacterial interactions,
and increased inflammatory infiltrate (Saunders et al., 1997,
2002; Kiliaan et al., 1998; Santos et al., 2000; Mazzon et al.,
2002; Soderholm et al., 2002). Together, the evidence sug-
gests that intestinal dysfunction and inflammation can be
initiated by psychological stress in a naı¨ve host (Soderholm
et al., 2002). The mechanism of stress-induced onset or re-
lapse of intestinal dysfunction is largely unknown, but one
possible link is mucosal barrier dysfunction.
Mucosal barrier function is maintained largely by the ep-
ithelial lining of the gastrointestinal tract: gut epithelial cells
(enterocytes) joined at their apical poles by tight junctions to
form a physical barrier. This barrier is not inert but rather is
regulated by neuroendocrine and immunological factors.
Crohn’s disease is associated with increased intestinal per-
meability (Olaison et al., 1990; Meddings et al., 1994; Soder-
holm et al., 1999), which precedes relapse (Wyatt et al.,
1993). In addition, long-term sustained stress increases the
number of relapses in patients with ulcerative colitis (Lev-
enstein et al., 2000). Defective barrier function has been
described for at least one subgroup of individuals with IBS,
and mild inflammation has also been described in IBS
(Spiller et al., 2000). That increased epithelial permeability
precedes intestinal inflammation supports the concept of bar-
rier dysfunction as an early or initiating event. Increased
permeability could expose the mucosal immune system to an
increased load of luminal antigens from foods and/or bacte-
ria, increasing immune stimulation that may then lead to
intestinal inflammation. Improvement of intestinal barrier
function may be a novel strategy to prevent or ameliorate
stress-induced intestinal dysfunction.
Glucagon-like peptide-2 (GLP-2) is an intestinotrophic
growth hormone that promotes many aspects of intestinal
function, including enhancement of mucosal growth and pro-
motion of nutrient absorption (for review, see Drucker, 2002).
Importantly, a unique property of this growth hormone is its
This research was supported by a grant to M.H.P. from the Crohn’s and
Colitis Foundation of Canada and by Astra Ha¨ssle AB. H.L.C. was the recip-
ient of an Ontario Graduate Scholarship. H.L.C. is a Ph.D. student in the
Medical Science Program at McMaster University. This work was presented at
the 2003 American Gastroenterology Association Digestive Disease Week,
Orlando, FL, May 17–22 (abstract #100619). Gastroenterology 124 (Suppl
1):A300.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.105.085373.
ABBREVIATIONS: IBS, irritable bowel syndrome; GLP-2, glucagon-like peptide-2; WAS, water avoidance stress; HRP, horseradish peroxidase.
0022-3565/05/3141-214–220$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 314, No. 1
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ability to rapidly enhance mucosal barrier function, reducing
intestinal permeability of the epithelial barrier via both
transcellular and paracellular routes (Benjamin et al., 2000).
Given that altered mucosal barrier function is an early event
in stress-induced intestinal pathophysiology, and the known
importance of mucosal barrier function in maintaining intes-
tinal integrity, we sought to examine whether enhancement
of epithelial barrier function with GLP-2 could prevent or
ameliorate stress-induced mucosal pathophysiology. For this
purpose, we developed a mouse model of chronic psychologi-
cal stress based on our established model of water avoidance
stress (WAS) in rats (Santos et al., 2000, 2001; Soderholm et
al., 2002). We found that exposure of mice to chronic stress
dramatically altered barrier function, impaired host defense,
and initiated inflammation in the jejunum, ileum, and colon.
Furthermore, we documented that GLP-2 treatment in this
model was able to ameliorate the stress-induced intestinal
abnormalities. This information may be useful in designing
novel therapeutic strategies for treating various gastrointes-
tinal disorders.
Materials and Methods
Animals
Male BALB/c mice were housed in pairs and maintained on a
12:12-h dark-light cycle and provided with food and water ad libi-
tum. Mice were allocated at random to one of four groups: control,
control ⫹GLP-2, stress, and stress ⫹GLP-2. On the day of the
experiments, mice were sacrificed by cervical dislocation immedi-
ately after the final stress or sham-stress procedure. All procedures
were approved by the Animal Care Committee at McMaster Univer-
sity.
Stress Protocol
Mice were handled daily by the same investigator for 7 days before
the study and then submitted to WAS or sham stress (control). Mice
were given a subcutaneous injection of protease-resistant GLP-2
[h[Gly
2
]-GLP-2, 5
␮
g (in 0.5 ml); Astra Ha¨ssle AB, Mo¨lndal, Sweden]
(or saline) 4 h before WAS or sham stress. The dose of GLP-2 was
based on our previous experiments showing enhanced barrier func-
tion 4 h after a single dose of GLP-2 (Benjamin et al., 2000; Cameron
et al., 2003). The procedure involved placing the mouse on a platform
(3 ⫻6 cm) in the center of a container (56 ⫻50 cm) containing 3 cm
of room temperature water. Mice avoided the aversive stimulus
(water) by remaining on the platform for 1 h. Control mice (sham
stress) were placed on the same platform in a waterless container for
1 h where mice were free to move off the platform and explore the
container. Mice were subjected daily to 1 h WAS or sham stress for
10 consecutive days. Body weight, as an index of growth was mea-
sured daily (expressed in grams).
Epithelial Physiology
Ussing Chamber Studies. The intestine was excised and cut
into segments of proximal jejunum (distal to the ligament of Treitz),
ileum, and colon. Each segment was immersed in 37°C oxygenated
Kreb’s buffer and opened along the mesenteric border into flat
sheets. Two adjacent pieces of jejunum and a single piece of ileum
and colon from each mouse were mounted in Ussing chambers (WPI,
Sarasota, FL). The chamber opening exposed 0.6 cm
2
of tissue sur-
face area to 8 ml of circulating oxygenated Kreb’s buffer at 37°C. The
buffer contained 115 mM NaCl, 1.25 mM CaCl
2
, 1.2 mM MgCl
2
, 2.0
mM KH
2
PO
4
, and 25 mM NaHCO
3
, pH 7.35. In addition, the serosal
buffer contained 10 mM glucose as an energy source that was osmot-
ically balanced by 10 mM mannitol in the mucosal buffer. The cham-
bers contained agar-salt bridges to monitor the potential difference
across the tissue and to inject the required short-circuit current (I
sc
)
to maintain a zero potential difference as registered via an auto-
mated voltage clamp (WPI). I
sc
(microamperes per square centime-
ter) was recorded by a computer connected to the voltage-clamp
system. Tissue conductance (G), a measure of passive permeability
to ions, was calculated according to Ohm’s law and expressed as
millisiemens per square centimeter.
HRP Flux. Permeability to macromolecules was assessed by mea-
suring the mucosal-to-serosal flux of protein horseradish peroxidase
(HRP) (44 kDa). HRP (type II; Sigma-Aldrich, St. Louis, MO) was
added to the luminal buffer 15 min after the tissues were mounted at
a final concentration of 4.5 ⫻10
⫺5
M and allowed to equilibrate for
30 min. Serosal samples (0.5 ml) were obtained at 30-min intervals
for 2 h and were replaced with Kreb’s buffer to maintain a constant
volume in the chambers. HRP activity was determined using a mod-
ified Worthington method as described previously (Kiliaan et al.,
1998) using a kinetic assay in a 96-well plate reader. Mucosal-to-
serosal fluxes of HRP were calculated according to standard formu-
las and expressed as picomoles per hour per square centimeter.
Electron Microscopy. Tissue segments from jejunum, ileum,
and colon to be used for transmission electron microscopy were
immediately fixed in 2.5% glutaraldehyde in 0.1mol/l sodium-caco-
dylate buffer, pH 7.4, for2hatroom temperature, rinsed for 18 h
(4°C) with 0.05 Tris buffer, pH 7.6, washed three times, 5 min each
time, and subsequently processed for routine transmission electron
microscopy. For quantification, bacterial-epithelial cell interactions
(defined as bacteria in contact with or inside epithelial cells, associ-
ated with disappearance of microvilli, and/or condensation of the
cytoskeleton) were evaluated in 30 fields per mouse in a blinded
manner by one investigator (H. L. Cameron).
Histology. Full-thickness segments of jejunum and colon adja-
cent to the tissue segments mounted in Ussing chambers were coded
and processed for morphological microscopic analysis. Tissues were
fixed in paraformaldehyde, embedded in paraffin, and subsequently
stained with H&E. In the light microscopy studies, tissues from four
mice per group were scrutinized, three tissues per mouse, and for
each tissue, 12 contiguous nonoverlapping areas above the muscu-
laris mucosa were evaluated in a blinded manner by investigator
H. L. Cameron. As a marker of inflammation, mononuclear cells
were identified. Cells were counted at 400⫻magnification and ex-
pressed as number of cells per square millimeter.
Statistical Analysis. Results are expressed as mean ⫾S.E.M.
For all statistical comparisons, when several observations were ob-
tained from the same mouse, the mean value was calculated before
group means were obtained; i.e., nvalues represent the number of
mice in each group. One-way analysis of variance was used with
Newman-Keuls as a subsequent multiple comparison test. pvalues
⬍0.05 were considered significant.
Results
Body Weight
Throughout the sham/stress protocol, weight was moni-
tored in all mice. Weight gain was not significantly different
between sham-stressed (1.1 ⫾0.1 g) and sham-stressed ⫹
GLP-2-treated mice (1.1 ⫾0.2 g). In stressed mice, weight
gain was significantly reduced over the 10-day period (0.5 ⫾
0.1 g; p⬍0.05). In GLP-2-treated stressed mice, the value for
weight gain over the 10-day period (0.7 ⫾0.2 g) was not
significantly different from either sham-stressed or stressed
mice.
Mucosal Physiology
Ion Transport. After the final stress/sham stress proce-
dure, baseline I
sc
, a measure of active ion transport was
recorded (Fig. 1A). WAS induced a significant increase in I
sc
GLP-2 Ameliorates Stress-Induced Intestinal Dysfunction 215
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in all regions of the gut. GLP-2 treatment of sham-stressed
mice had no effect on baseline I
sc
. In contrast, GLP-2 treat-
ment of stressed mice ameliorated the stress-induced in-
crease in I
sc
for all regions of the gut.
Permeability. G, a measure of passive permeability to
ions was measured for each tissue (Fig. 1B). Stress induced a
significant increase in conductance for all regions of the gut
compared with sham-stressed controls. GLP-2 treatment of
stressed mice ameliorated the stress-induced increase in con-
ductance in the jejunum but not in the ileum or colon. In
addition, GLP-2 treatment reduced conductance in the small
intestine in control mice.
Macromolecular Permeability
Chronic stress caused a dramatic permeability defect in all
regions of the gut (jejunum, 27.1 ⫾0.6; ileum, 23.0 ⫾1.2; and
colon, 17.1 ⫾0.9 pmol/h/cm
2
) compared with sham-stressed
controls (jejunum, 11.2 ⫾2.1; ileum, 10.5 ⫾1.0; and colon,
8.4 ⫾1.3 pmol/h/cm
2
;p⬍0.001) (Fig. 2). GLP-2 treatment
eliminated the stress-induced permeability defect (p⬍0.001)
and reduced flux values below those of sham-stressed mice
(p⬍0.01) (jejunum, 7.0 ⫾1.6; ileum, 6.4 ⫾1.9; and colon,
4.0 ⫾1.2 pmol/h/cm
2
). GLP-2 treatment of sham-stressed
mice also significantly reduced the amount of HRP trans-
ported across all segments of the gut (jejunum, 5.3 ⫾0.5;
ileum, 5.9 ⫾0.3; and colon, 2.6 ⫾0.5 pmol/h/cm
2
) compared
with sham-stressed controls (p⬍0.01).
Altered Host Defense to Bacteria
Segments of jejunum, ileum, and colon were processed for
transmission electron microscopy after the final stress/sham-
stress procedure. Figure 3 shows a representative electron
photomicrograph of the colonic mucosa from control (Fig. 3A)
and stressed mouse ileum (Fig. 3B). In control tissues, inter-
acting bacteria were rarely observed, whereas many bacteria
were observed in tissues from stressed mice. We observed a
lack of cellular organelles and condensation of cytoskeleton
at the site of epithelial-bacterial interaction. When the num-
Fig. 1. Effect of chronic stress ⫾
GLP-2 treatment on intestinal physi-
ology. Sham-stressed mice were given
saline (open columns) or GLP-2 (solid
columns) 4 h before1hofsham stress
for 10 consecutive days. WAS mice
were given saline (open columns) or
GLP-2 (solid columns) 4 h before1hof
WAS for 10 consecutive days. Imme-
diately after the final sham/stress ses-
sion, tissue from the jejunum, ileum,
and colon were excised and mounted
in the Ussing chamber, in which base-
line I
sc
(A) and G (B) were measured.
Data are presented as means ⫾S.E.
(n⫽6 mice/group, with two to four
tissues per mouse). ⴱ,p⬍0.05; ⴱⴱ,p⬍
0.01 versus sham; †, p⬍0.05; ‡, p⬍
0.01 versus WAS.
216 Cameron and Perdue
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ber of bacteria directly interacting with the intestinal mucosa
was counted and expressed as the number of penetrating
bacteria per field (Fig. 3C), bacteria were rarely observed in
sham-stressed controls (jejunum, 0.1 ⫾0.1; ileum, 0.2 ⫾0.1;
and colon 0.3 ⫾0.1). WAS significantly increased the number
of bacteria interacting with the intestinal mucosa (jejunum,
3.7 ⫾1.2; ileum, 4.5 ⫾0.4; and colon, 10.0 ⫾1.0; p⬍0.01
compared with sham-stressed controls). Stressed mice
treated with GLP-2 also showed an increased number of
interacting bacteria compared with sham-stressed controls
(jejunum, 0.3 ⫾0.2; ileum, 2.3 ⫾1.0; and colon 1.3 ⫾1.0; p⬍
0.001) but significantly fewer than stressed mice (p⬍0.01).
In addition, we observed changes in mitochondria in stressed
mouse intestine, including mitochondrial swelling and loss of
Fig. 2. Effect of chronic stress ⫾GLP-2
treatment on HRP flux. Sham-stressed
mice were given saline (open columns) or
GLP-2 (solid columns) 4 h before1hof
sham stress for 10 consecutive days. WAS
mice were given saline (open columns) or
GLP-2 (solid columns) 4 h before1hof
WAS for 10 consecutive days. Immedi-
ately after the final sham/stress session,
tissue from the jejunum, ileum, and colon
were excised and mounted in the Ussing
chamber, in which HRP flux was mea-
sured. Data are presented as means ⫾
S.E. (n⫽6 mice/group, with two to four
tissues averaged per mouse). ⴱⴱ,p⬍0.01
and ⴱⴱⴱ,p⬍0.001 versus sham; ‡, p⬍0.
01 versus WAS.
Fig. 3. Effect of chronic stress ⫾GLP-2 treatment on
bacterial penetration. Representative transmission
electron photomicrograph of ileal epithelium in a sham
stress mouse (A) and a WAS mouse (B). Note bacteria
(arrows) interacting with the enterocyte and rear-
ranged cytoskeleton adjacent to interaction. Also, note
the appearance of mitochondria (arrowheads). Scale
bar, 1
␮
m; magnification, 5000⫻. C, number of pene-
trating bacteria in the jejunum, ileum, and colon of
sham-stressed mice given saline (open columns), sham-
stressed mice given GLP-2 (solid columns), stressed
mice given saline (open columns), and stressed mice
treated with GLP-2 (solid columns). Data are presented
as mean number of bacteria observed penetrating the
mucosa/30 fields ⫾S.E. (n⫽6 mice per group). ⴱⴱ,p⬍
0.01 versus sham; ‡, p⬍0.01 versus WAS.
GLP-2 Ameliorates Stress-Induced Intestinal Dysfunction 217
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cristae architecture. These changes were not quantified but
were qualitatively similar to those reported previously in
rats subjected to chronic WAS (Soderholm et al., 2002).
Colonic Inflammatory Cells
Colonic segments adjacent to those used for Ussing
chamber studies were processed for histology after the
final stress/sham-stress procedure and the number of
mononuclear cells in the colonic mucosa was recorded and
expressed as cell number per square millimeter (Fig. 4).
WAS increased the number of mononuclear cells in the
mucosa of stressed mice approximately 2-fold (500 ⫾64)
compared with sham-stressed controls (p⬍0.001). GLP-
2-treated stressed mice showed reduced numbers of mono-
nuclear cells in the colonic mucosa (406 ⫾52) compared
with stressed mice (p⬍0.01). GLP-2 treatment in sham-
stressed mice had no effect on mononuclear cell number
(228 ⫾70) compared with sham-stressed controls (240 ⫾
47) (NS).
Discussion
To our knowledge, this is the first study examining the
effects of chronic psychological stress on intestinal function
in mice. The findings reported in this investigation indicate
that mouse intestine is indeed responsive to chronic psycho-
logical stress. Our previous studies in rats demonstrated that
psychological stress alters physiology and initiates intestinal
inflammation in the ileum (Santos et al., 2000; Soderholm et
al., 2002). In the present study, we extended these findings to
show intestinal dysfunction in mice in response to chronic
psychological stress. We identified the changes induced by
10-day chronic WAS in jejunum, ileum, and colon where
stress increased ion secretion and permeability to ions and
macromolecules, induced infiltration of mononuclear cells
into the mucosa, and increased mucosal-bacterial interac-
tions. In addition, we demonstrated that treatment of mice
with GLP-2 can prevent or ameliorate many of the observed
stress-induced intestinal changes in host defense.
We examined the ability of a daily GLP-2 treatment
during the stress protocol to affect intestinal function in
stressed and control conditions. In control (sham-stressed)
mice, 10 day GLP-2 treatment enhanced gut function to a
small but significant extent as measured by reduced per-
meability to both ions and macromolecules. In the intes-
tine of stressed mice, GLP-2 treatment restored the base-
line secretory state of the epithelium to control values and
significantly ameliorated stress-induced barrier dysfunc-
tion by reducing passive permeability to ions and dramat-
ically limiting penetration of macromolecules. GLP-2
treatment also ameliorated the stress-induced mononu-
clear cell infiltration and prevented abnormal bacterial
interactions with the epithelium.
In stressed mice, we recorded a decrease in weight gain
suggesting a failure to thrive during the 10-day stress period.
GLP-2 treatment did not affect weight gain in sham-stressed
mice, whereas in the stressed mice weight gain was not
significantly different from sham-stressed controls. The
stress-induced epithelial cell ion secretion in both small and
large intestine creates a driving force for water secretion that
may act to flush noxious materials out of the lumen, thus
contributing to host defense. However, when prolonged as in
chronic situations, ongoing secretion may be detrimental by
increasing water loss. Here, chronic stress caused an in-
crease in short-circuit current, whereas GLP-2 treatment in
stressed mice significantly reduced this increase in all re-
gions of the gut and further restored ion transport to control
levels in the jejunum.
Epithelial permeability as measured by conductance and
the penetration of an antigen-sized probe provide informa-
tion about the integrity of the epithelial barrier. The risk
of increased exposure to luminal antigens is most accu-
rately reflected by changes in HRP flux, whereas conduc-
tance indicates movement of ions. Intestinal permeability
to ions and macromolecules was significantly increased in
stressed mouse intestine, indicating that epithelial barrier
function is severely compromised by stress in mice. In rats
exposed to chronic psychological stress, a similar barrier
defect has been described previously (Santos et al., 2000,
2001; Soderholm et al., 2002). GLP-2 treatment of sham-
stressed mice enhanced barrier function by reducing pas-
sive permeability to ions and penetration of a macromolec-
ular probe. Moreover, in stressed mice, GLP-2 treatment
dramatically reduced macromolecular permeability in all
regions of the gut to levels significantly below both
stressed and sham-stressed values. We have previously
demonstrated that GLP-2 enhances barrier function by
reducing permeation of the intestine by both transcellular
and paracellular pathways (Benjamin et al., 2000). Thus,
GLP-2 treatment in stressed mice enhanced barrier func-
tion to completely prevent the stress-induced permeability
defect effectively reducing the amount of luminal antigen
that penetrates to the lamina propria, thereby limiting
immune stimulation (Cameron et al., 2003). It is known
that GLP-2 reduces fluid-phase endocytosis of macromol-
ecules and decreases paracellular permeability of smaller
probes (Benjamin et al., 2000), perhaps due to some effect
on cytoskeletal components or tight junction proteins. It is
also possible that the antiapoptotic/growth-promoting ef-
fects of GLP-2 (Drucker et al., 1996; Tsai et al., 1997)
contribute to the amelioration of stress-induced effects by
maintaining the integrity of the epithelial lining of the gut.
Fig. 4. Effect of chronic stress ⫾GLP-2 treatment on colonic inflamma-
tory infiltrate. Sham-stressed mice were given saline (open columns) or
GLP-2 (solid columns) 4 h before1hofsham stress for 10 consecutive
days. WAS mice were given saline (open columns) or GLP-2 (solid col-
umns) 4 h before1hofWASfor10consecutive days. Mononuclear cells
in the colonic mucosa were counted in coded light microscopy sections.
Tissues are from four mice per group, two tissues per mouse, and for each
tissue, 10 contiguous nonoverlapping areas above the muscularis mucosa
were evaluated. Data are presented as means ⫾S.E. ⴱⴱ,p⬍0.01 versus
sham; †, p⬍0.05 versus WAS.
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The ability of bacteria to access and adhere to the intes-
tinal epithelia is a key step in the pathogenesis of trans-
location and the initiation of infection (Katayama et al.,
1997). In this study, we noted a significant increase in
bacterial interaction with the intestinal mucosa in
stressed mice. We also observed mitochondrial changes
in tissues with increased bacterial interactions, including
mitochondrial swelling and distortion of cristae compara-
ble with our previous findings in rats (Soderholm et al.,
2002). Similar mitochondrial changes have been associated
with increased intestinal epithelial permeability after un-
coupling of oxidative phosphorylation (Somasundaram et
al., 1997). The stress-induced increase in bacterial adher-
ence and penetration is likely due, at least in part, to the
decreased protective capacity of the mucus layer. In mouse
colon, acute immobilization stress has been shown to in-
duce mucin secretion, leading to goblet cell depletion
(Castagliuolo et al., 1998). Therefore, increased bacterial
interaction with the epithelium may be the result of in-
creased epithelial exposure due to a thinned mucus layer.
Thus, we speculate that the modest ability of GLP-2 to
reduce this interaction in stressed mice may in turn be due
to some effect on mucus production or release. The ability
of GLP-2 to reduce bacterial penetration has previously
been documented in a report of reduced bacteremia in a
murine model of indomethacin-induced intestinal injury
(Boushey et al., 1999), likely due to enhanced mucosal
barrier function.
Several experimental studies examining the role of GLP-2
to treat diseases characterized by intestinal inflammation
(Boushey et al., 1999; Alavi et al., 2000; Kouris et al., 2001)
suggest a role for GLP-2 in ameliorating the severity of
inflammation, perhaps indirectly by enhancing the integrity
of the intestinal barrier. In this study, GLP-2 treatment of
stressed mice reduced the number of mononuclear cells re-
cruited to the mucosa, perhaps by limiting immune stimula-
tion via enhanced barrier function. Although there is some
controversy in the literature surrounding the location of the
GLP-2 receptor in the gut (Yusta et al., 2000; Bjerknes and
Cheng, 2001), it seems likely that one or several downstream
mediators mediate the diverse changes in intestinal function
that are attributed to GLP-2. Ongoing studies aim to under-
stand the underlying changes involved in GLP-2-induced
intestinal improvement and the mediators involved in these
changes.
In the setting of stress-induced intestinal dysfunction,
where a known permeability defect occurs, prophylactic
treatment to enhance barrier function, with GLP-2 can ame-
liorate the effects of stress on intestinal barrier and therefore
prevent barrier dysfunction-related sequelae. Compromised
intestinal barrier function may be an initiating or propagat-
ing factor in intestinal disease and as such may be a prom-
ising target for therapy. Improvement of barrier function
may then potentially prevent or delay onset of disease and
reduce the severity of disease.
In summary, our study demonstrates that chronic psycho-
logical stress induces intestinal dysfunction in mouse intes-
tine. Chronic stress induced changes in epithelial physiology,
barrier function, and bacterial interaction. In addition, we
showed that GLP-2 treatment in this model attenuates or
ameliorates these stress-induced changes. These findings
support the concept of altered intestinal permeability as an
initiating event in intestinal dysfunction and further support
the potential therapeutic usefulness of barrier-enhancing
treatment strategies to prevent or ameliorate intestinal dys-
function.
Acknowledgments
We acknowledge Dr. Ping Chang Yang for the preparation of
electron microscopy samples.
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Address correspondence to: Dr. Mary H. Perdue, Intestinal Disease Re-
search Programme, McMaster University HSC-3N5C, 1200 Main St. W., Ham-
ilton, ON, Canada L8N 3Z5. E-mail: perdue@mcmaster.ca
220 Cameron and Perdue
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