Am J Physiol Gastrointest Liver Physiol 297: G869–G877, 2009.
First published September 10, 2009; doi:10.1152/ajpgi.00164.2009.
Differential adipokine response in genetically predisposed lean and obese rats
during inflammation: a role in modulating experimental colitis?
Niall P. Hyland,1,2* Adam P. Chambers,1* Catherine M. Keenan,1 Quentin J. Pittman,1
and Keith A. Sharkey1
1
Snyder Institute of Infection, Immunity and Inflammation, Hotchkiss Brain Institute and Department of Physiology
and Pharmacology, University of Calgary, Calgary, Alberta, Canada; 2Alimentary Pharmabiotic Centre and Department
of Pharmacology and Therapeutics, University College Cork, Cork, Ireland
Submitted 30 April 2009; accepted in final form 3 September 2009
diet-induced obese; body weight; leptin; plasminogen activator inhibitor-1; adiponectin; ghrelin
INFLAMMATORY BOWEL DISEASE (IBD) occurs in genetically predisposed individuals as a result of an incompletely understood
gene-environment interaction, also involving the intestinal microflora (3). Similarly, obesity, or more specifically diet-induced obesity (DIO), develops as a consequence of environmental factors, diet for example, but also genetic factors
because not all animals (27) or humans (7) who display a
preference for high-fat diets actually develop obesity. Studies
in DIO-prone rats implicate decreased central leptin sensitivity
(26), hypothalamic pituitary dysfunction (32), maternal obesity
(19), and postweaning exercise (24a) as among some of the
factors that contribute to the DIO phenotype.
* N. Hyland and A. Chambers contributed equally to this work.
Address for reprint requests and other correspondence: K. Sharkey, Dept. of
Physiology and Pharmacology, Univ. of Calgary, 3330 Hospital Dr. NW,
Calgary, Alberta, T2N 4N1, Canada (e-mail: [email protected]).
http://www.ajpgi.org
Although DIO animals display increased gene-expression
profiles for several immune response genes, including those
involved in antigen presentation, response to biotic stimuli, and
phagocytosis (29), there is also a growing recognition that
endocrine factors released from adipose tissue are involved in
the pathophysiology of many chronic inflammatory conditions
(17, 20, 42), including IBD (23). The accumulation of central
adipose tissue and changes in the expression of the proteins
synthesized and released by adipocytes (adipokines) are
thought to underlie the abnormalities that characterize aspects of the metabolic syndrome including leptin resistance,
chronic inflammation, and impaired fibrinolysis (17, 21).
Collectively, the growing list of adipokines has redefined
adipose tissue as a complex endocrine organ that regulates
immunity and inflammation (17, 20, 42), in addition to
energy balance (43).
As well as altering the course of colitis, adipokines can also
contribute to homeostatic imbalances during IBD (11). Plasminogen activator inhibitor-1 (PAI-1) is produced by a number
of tissues including adipocytes (1, 38) and is increased in obese
humans (22) and rodents (39). PAI-1, which promotes a hypercoagulant state, is significantly increased in plasma taken
from patients suffering from IBD (13) and may be associated
with coagulation abnormalities that accompany intestinal inflammation (11).
Studies in leptin-deficient, ob/ob mice (4, 40) further support
a role for adipokines as direct modulators of colonic inflammation. In these studies, it was shown that leptin infusion
worsened colitis and that leptin-deficient mice developed less
severe experimental colitis than wild-type controls. Leptin
circulates in direct proportion to fat stores and is almost
exclusively released into the circulation from adipocytes,
and its receptor is expressed both in the central nervous
system and periphery (16). The protective effect of the
ob/ob genotype was abolished by leptin infusion, suggesting
that leptin, not obesity per se, increased damage caused by
experimental colitis. Other adipokines such as adiponectin
may also influence the development of colitis. The role
played by this adipokine during colitis in gene-deficient
animals is controversial (15, 33).
A similar, somewhat ambiguous situation exists for the
feeding stimulant ghrelin. Administration of this adipokine
12 h after trinitrobenzene sulfonic acid (TNBS) treatment
significantly decreased TNBS-induced damage and proinflammatory cytokine production (18), indicative of an anti-inflammatory effect. However, in human colonic cell lines, ghrelin
increases TNF-␣-induced proinflammatory interleukin-8 production and activates NF-␬B (47).
0193-1857/09 $8.00 Copyright © 2009 the American Physiological Society
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Hyland NP, Chambers AP, Keenan CM, Pittman QJ, Sharkey
KA. Differential adipokine response in genetically predisposed lean
and obese rats during inflammation: a role in modulating experimental
colitis? Am J Physiol Gastrointest Liver Physiol 297: G869 –G877, 2009.
First published September 10, 2009; doi:10.1152/ajpgi.00164.2009.—
The relationship between a predisposition to obesity and the development of colitis is not well understood. Our aim was to characterize
the adipokine response and the extent of colitis in diet-induced obese
(DIO) rats. DIO and control, diet-resistant (DR) animals were administered either saline or trinitrobenzene sulfonic acid (TNBS) to induce
colitis. Macroscopic damage scores and myeloperoxidase (MPO)
activity were measured to determine the extent of inflammation.
Trunk blood was collected for the analysis of plasminogen activator
inhibitor-1 (PAI-1) as well as leptin, ghrelin, and adiponectin. Colonic
epithelial physiology was assessed using Ussing chambers. DIO rats
had a modestly increased circulating PAI-1 before TNBS treatment;
however, during colitis, DR animals had more than a fourfold increase
in circulating PAI-1 compared with DIO rats. Circulating leptin was
higher in DIO rats compared with DR animals, in the inflamed and
noninflamed states. These changes in TNBS-induced adipokine profile
were accompanied by decreased macroscopic tissue damage score in
DIO animals compared with DR tissues. Furthermore, TNBS-treated
DR animals lost significantly more weight than DIO rats during active
inflammation. Colonic epithelial physiology was comparable between
groups, as was MPO activity. The factors contributing to the decreased colonic damage are almost certainly multifold, driven by both
genetic and environmental factors, of which adipokines are likely to
play a part given the increasing body of evidence for their role in
modulating intestinal inflammation.
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OBESITY, ADIPOKINES, AND EXPERIMENTAL COLITIS
These studies demonstrate that circulating adipokines have
the potential to influence the development of IBD, but to date
it is unclear whether DIO in animals predisposed to this
condition alters the development of intestinal inflammation. A
number of factors may contribute positively or negatively to
the development of inflammation, including the enteric flora,
which is altered in obesity (3, 28, 45), and the extent of
mesenteric fat deposition. Understanding how the mechanisms
governing intestinal inflammation are altered in obesity is of
importance given its increasing prevalence throughout the
world (24). Therefore, the aim of the present study was to
determine whether experimental colitis is altered in DIO rats.
MATERIALS AND METHODS
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RESULTS
Development of DIO and DR. After exposure to the MHF
diet for a period of 9 wk, DIO rats weighed significantly more
than DR animals before the induction of colitis (Fig. 1A).
Although previous studies indicate increased circulating insulin and insulin resistance, in DIO-prone animals (25), fasted
serum insulin levels were comparable between DR and DIO
Table 1. Criteria used for assessment of microscopic tissue
damage
Parameter
Observation (Score)
Destruction of Normal
Architecture
Presence and Degree of
Cellular Infiltration
Extent of Muscle Thickening
Presence or Absence of
Crypt Abscesses
Presence or Absence of
Goblet Cell Depletion
normal (1), mild/moderate (2), extensive (3)
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normal (1), mild/moderate (2), transmural
infiltration (3)
normal (1), mild/moderate (2), extensive (3)
absent (0), present (1)
absent (0), present (1)
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Animals and diet. All methods used in this study were approved by
the University of Calgary Animal Care Committee and were carried
out in accordance with the guidelines of the Canadian Council on
Animal Care. Animals were housed in a temperature-controlled room
maintained on a normal 12-h:12-h light/dark cycle and were allowed
access to food and water ad libitum.
DIO and DR animals. Male selectively bred diet-resistant (DR) and
DIO rats [strain Crl: CD(SD) DIO or DR; Charles River Laboratories,
Montréal, QC, Canada] were 6 wk of age when they were exposed to
a medium-high-fat diet (MHF, 31.8% fat, 16.8% protein, and 51.4%
carbohydrate per Kcal; D12266B, Research Diets, New Brunswick,
NJ) ad libitum for a period of 9 wk.
Plasminogen activator inhibitor knockout mice. Four- to six-weekold wild-type male C57BL/6 mice and PAI-1⫺/⫺ mice (B6.129S2Serpine1/J) on a C57BL/6 background were obtained from Jackson
Laboratories (Bar Harbor, ME) and were housed in a temperaturecontrolled room (22°C). Mice were maintained on a normal 12-h:12-h
light/dark cycle and were allowed free access to standard lab chow
and water before treatment with TNBS as outlined below.
Induction of colitis. Randomly assigned DR and DIO rats and
wild-type and PAI⫺/⫺ mice were briefly anesthetized with isoflurane
(induced at 4% and maintained at 2%) and subjected to administration
of either TNBS [rats: 0.5 ml of 50 mg/ml in 50% (vol/vol) ethanol,
mice: 0.1 ml of 40 mg/ml in 40% (vol/vol) ethanol; Caledon Laboratories, Edmonton, AB, Canada] or an equivalent volume of sterile
saline (0.9%) into the lumen of the colon through a polyethylene
catheter inserted rectally 7 cm (for rats) or 3 cm (for mice) proximal
to the anus. Following the induction of colitis, animals were housed
individually, and daily food intake and body weight were monitored.
Animals were allowed to recover for a period of 7 days.
Tissue collection. At the time of tissue collection (9:30 –10:30
AM), DR and DIO rats were anesthetized with sodium pentobarbital
(80 –100 mg/kg ip) and decapitated, and trunk blood was collected in
tubes containing EDTA. After immediate centrifugation, serum was
removed and then snap frozen and stored at ⫺80°C for subsequent
assay of leptin, insulin, ghrelin, adiponectin, and PAI-1. Mice were
euthanized by cervical dislocation, and the entire colon of both rats
and mice was removed and assessed for macroscopic damage (31);
colonic samples (rat, 82–294 mg; mice, 48 –230 mg) were taken from
damaged sites and snap frozen for later assessment of myeloperoxidase (MPO) activity, an enzyme found in cells of myeloid
origin, and used as a marker of neutrophil infiltration as previously
described (31).
Assessment of adipokines, insulin, and ghrelin. All serum samples
were sent to Linco Diagnostics (now Millipore; St. Charles, MO) for
either radioimmunoassay (RIA) or LINCOplex analysis. Nonacidified
samples were analyzed for total ghrelin and adiponectin by RIA. The
interassay variability for the ghrelin assay was 4.1–10.0% coefficient of variation (CV), intra-assay variability 14.7–17.8% CV,
and the lower limit of detection was 93 pg/ml. For the adiponectin
RIA, the interassay variability was 3.7– 4.4% CV, intra-assay
variability 6.6 – 8.2% CV, and the lower limit of detection was 1
ng/ml. Serum leptin, insulin, and PAI-1 levels were measured
using a rat adipokine LINCOplex assay (the characteristics of
which are available via www.millipore.com).
Ussing chamber experiments. To assess colonic epithelial physiology, rat distal colons were immediately placed in fresh Krebs solution
of the following composition (in mM) 117 NaCl, 4.8 KCl, 2.5 CaCl2,
1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 11 D-glucose. Mucosal
preparations (with intact submucosal plexus) were obtained by opening the colon along the mesenteric border and removing the circular
and longitudinal smooth muscle layers plus myenteric plexus by blunt
dissection. Preparations were subsequently mounted in Ussing chambers (exposed mucosal area of 0.6 cm2) containing 10 ml oxygenated
(95% O2-5% CO2) Krebs solution that was maintained at 37°C.
Tissues were voltage clamped at 0 mV using an automatic voltage
clamp (EVC 4000; World Precision Instruments, Sarasota, FL), and
the short-circuit current (Isc) required to maintain the 0-mV potential
was monitored using DataTrax software (World Precision Instruments) and resistance calculated using Ohms law.
Histology. Tissues from the inflamed regions were fixed by overnight immersion in Zamboni’s fixative, washed in PBS (3 ⫻ 10 min),
cryoprotected in PBS-sucrose (20%), and embedded for cryostat
sectioning in optimal cutting temperature compound. Cross sections
(12–14 ␮m) were cut and stained with hematoxylin and eosin to
reveal structural features. Scoring of sections was based on a semiquantitative scoring system taking into account the following five
features: mucosal architecture, muscle thickness, presence and degree
of cellular infiltration, crypt abscesses, and goblet cell mucus depletion as outlined in Table 1.
Statistical analysis. Changes in body weight and food consumption
for each day were compared using a two-way ANOVA with repeated
measures with phenotype and treatment as between factors (independent variables) and time from treatment (day, dependent variable) as
the repeated measure. Where a significant effect of treatment was
found, one-way ANOVAs with Bonferroni’s test were conducted
comparing the groups on each day. Macroscopic tissue damage score
was analyzed by nonparametric Mann-Whitney test, and total length
of damage, length of severe damage, and serum, leptin, insulin,
adiponectin, ghrelin, and PAI-1 concentrations were determined using
an unpaired two-tailed Student’s t-test. If adipokine levels fell
below the detection limit of an assay, zero was recorded for
statistical analysis. In each case, data were considered statistically
significant when P ⱕ 0.05. Data are presented as means ⫾ SE.
OBESITY, ADIPOKINES, AND EXPERIMENTAL COLITIS
animals in our study (Fig. 1B). Consistent with previous studies
(37), DIO-prone rats displayed significantly increased circulating leptin concentrations compared with DR animals (Fig. 2A).
No significant differences in circulating PAI-1, ghrelin, or
adiponectin (summarized in Fig. 2, B–D) were observed between noninflamed DIO and DR animals; however, DIO animals tended to have increased circulating PAI-1 (twofold), and
adiponectin (1.4-fold) and decreased circulating ghrelin (1.5fold; P ⫽ 0.06).
Serum adipokine and ghrelin measurements in TNBStreated DIO and DR rats. Leptin levels remained significantly
increased in DIO rats after TNBS treatment (P ⬍ 0.05, Fig. 2A)
relative to DR TNBS-treated rats; however, in DIO animals,
leptin was reduced by ⬃2.5-fold by TNBS treatment. TNBS
treatment caused a greater than 20-fold increase in PAI-1 levels
in DR animals and a twofold increase in DIO rats. Between DR
and DIO inflamed rats, PAI-1 levels were significantly increased in DR animals (P ⬍ 0.001, Fig. 2B). Inflammation had
a tendency to increase circulating total ghrelin in both DR and
DIO animals; however, the relative increase in ghrelin after
TNBS treatment was similar between DR and DIO animals
(DR saline vs. DR TNBS, 39.2% increase in ghrelin; DIO
saline vs. DIO TNBS, 41.8% increase in ghrelin). With respect
to this adipokine, the difference between DR and DIO animals
only reached significance in TNBS-treated DIO rats relative to
DR TNBS-treated animals (Fig. 2C). Serum adiponectin, on
the other hand, was significantly increased after TNBS treatment in DIO rats (Fig. 2D).
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TNBS-induced damage was significantly reduced in DIO
rats. Macroscopic tissue damage scores were significantly
increased in TNBS-treated DIO and DR rats compared with
saline-treated controls (data not shown). Only colonic thickness contributed to the score observed in saline-treated animals
(DIO, 0.4 ⫾ 0.03 mm; DR, 0.4 ⫾ 0.04 mm). Comparisons
between TNBS-treated groups revealed a significantly lower
macroscopic damage score in inflamed DIO tissues compared
with DR colon (Fig. 3A, P ⬍ 0.05). Breakdown of the macroscopic damage scores indicated that the total involved length
(Fig. 3B, P ⬍ 0.05) and length of severe TNBS-induced
damage (Fig. 3C, P ⫽ 0.01) were significantly less in DIO
compared with DR rats. MPO activity was significantly increased by TNBS treatment in both DR and DIO colon (data
not shown); however, no significant difference was observed
between TNBS-treated DIO and DR animals [DIO, 51.0 ⫾ 9.6
U/g, (n ⫽ 5); DR, 62.3 ⫾ 13.0 U/g, (n ⫽ 6)]. Microscopic
tissue damage scores were also comparable between TNBStreated DIO and DR tissues [DIO, 8.5 ⫾ 0.8, (n ⫽ 6); DR,
10.5 ⫾ 0.3, (n ⫽ 6); Fig. 4].
DIO rats lose significantly less weight than DR rats after
TNBS treatment. DIO rats lost significantly less weight than
TNBS-treated DR animals 4 (P ⬍ 0.001) and 5 (P ⬍ 0.001)
days posttreatment (Fig. 5A) and, compared with DR rats, ate
significantly more food on days 2–7 after TNBS treatment
(Fig. 5B, P ⬍ 0.05– 0.001).
TNBS colitis in PAI-1⫺/⫺ mice. Because DR rats displayed
a striking elevation in circulating PAI-1 levels postinflammation relative to DIO animals, we hypothesized that PAI-1 may
play a protective role during inflammation in obese rodents by
potentially limiting the extent of inflammation. In the absence
of a commercially available PAI-1 antagonist, we investigated
the course of TNBS colitis 7 days posttreatment in wild-type
and PAI-1⫺/⫺ mice. Seven days after TNBS treatment, inflammatory indices were reduced in PAI-1⫺/⫺ animals (Fig.
6, A–D); however, no significant differences in any of these
parameters were observed.
Colonic secretory function in saline- and TNBS-treated DIO
and DR tissues. Brun et al. (8) described alterations in the
electrical resistance and permeability parameters of obese
ob/ob and db/db mouse small intestine compared with their
lean counterparts. Because alterations in colonic permeability
and secretory function can exacerbate TNBS-induced colitis,
we examined basal electrical properties in DIO and DR salineand TNBS-treated colon.
In both DR and DIO tissues, TNBS treatment significantly
decreased basal Isc (P ⬍ 0.05, Table 2); however, we did not
observe any significant effect of either phenotype or treatment
on the transepithelial barrier resistance, a measure of colonic
permeability (Table 2).
DISCUSSION
Environmental factors, such as lifestyle and diet, have been
implicated in the development of colitis (2, 5). These environmental factors very likely interact with the gut microflora in
genetically predisposed individuals to shape the expression of
inflammation (2). Although obesity is not frequently associated
with colitis, we have demonstrated its impact on the inflammatory adipokine response during colonic inflammation.
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Fig. 1. Bodyweight of diet-resistant (DR) and diet-induced obese (DIO)
animals at the time of saline or trinitrobenzene sulfonic acid (TNBS) treatment
following ⬃9-wk exposure to a medium-high-fat diet (A, n ⫽ 12, P ⬍ 0.001)
and serum insulin levels recorded seven days after saline or TNBS treatment
(B, n ⫽ 6). ***P ⬍ 0.001.
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Fig. 2. Serum leptin (A), plasminogen activator inhibitor-1 (PAI-1, B), ghrelin (C), and adiponectin (D) in DR and DIO saline-treated (left) and TNBS-treated
(right) rats. Serum leptin was significantly increased in saline-treated DIO rats compared with saline-treated DR controls (n ⫽ 6, P ⬍ 0.001). Saline-treated DIO
animals tended to have increased circulating PAI-1 (twofold) and adiponectin (1.4-fold) and decreased circulating ghrelin (1.5-fold; P ⫽ 0.06). After TNBS
treatment, serum leptin (note y-axis is half that of saline-treated animals) and adiponectin were significantly greater in DIO animals (n ⫽ 6, P ⬍ 0.05) compared
with TNBS-treated DR animals. PAI-1 (B) significantly increased in DR animals after TNBS treatment (n ⫽ 6, P ⬍ 0.001; note the y-axis is tenfold that in
saline-treated animals); ghrelin was significantly decreased in DIO rats (n ⫽ 6, P ⬍ 0.05). *P ⬍ 0.05; ***P ⬍ 0.001.
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The observation that DIO rats lost less weight and ate more
food than DR rats provided the first indication that DIO rats
were less susceptible to the effects of TNBS. This increased
intake of a MHF diet may itself protect against the effects of
TNBS colitis because oral nutrient intake (Modulen IBD;
Nestle, Milan, Italy; 42 Kcal% fat), when used as a first-line
treatment for Crohn’s disease (CD), results in decreased endoscopic and histological damage (6). Exposure to a high-fat diet
has been proposed to disrupt the balance among intraepithelial
lymphocytes, increasing susceptibility to colitis in rodents
relative to animals fed a normal fat content diet (30). Although
we cannot rule a differential effect of diet on the inflammatory
response observed in DR- and DIO-prone animals, we do not
believe this was a confounding factor in our study because both
groups consumed the same diet. Furthermore, DIO animals
consumed more food by weight than DR animals yet displayed
decreased colonic damage indicative of a protective response
in DIO animals.
Changes in the expression of circulating adipokines have led
to the characterization of obesity as a state of chronic inflam-
Fig. 4. Representative photomicrographs of hematoxylin and eosin staining in saline- and TNBS-treated colon
7 days posttreatment. Criteria used for microscopic
tissue damage scoring are outlined in MATERIALS AND
METHODS and Table 1. Note the complete destruction of
the mucosa, the absence of colonic crypt structure, and
the presence of extensive inflammatory infiltrate in
TNBS-treated DR and DIO colonic tissues. Scale bar ⫽
200 ␮m.
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Fig. 3. Macroscopic tissue damage score (A), total involved length (B), and length of severe damage (C) were all significantly decreased in DIO (open bars)
compared with DR (solid bars) TNBS-treated rats 7 days posttreatment. Only colonic thickness contributed to the macroscopic damage score in saline treated
rats; n ⫽ 6 in each group. *P ⬍ 0.05, **P ⬍ 0.01.
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OBESITY, ADIPOKINES, AND EXPERIMENTAL COLITIS
mation (17). In our study, noninflamed DIO rats had significantly higher circulating leptin levels than age-matched DR
control rats. PAI-1 also tended to be increased in DIO animals,
whereas differences in circulating adiponectin and ghrelin were
nonsignificant. In TNBS-treated animals, this adipokine profile
was altered (Fig. 2A), and these changes were accompanied by
decreased TNBS-induced damage in DIO animals without an
associated change in either MPO or microscopic damage.
Whether differences in MPO activity or microscopic damage
occurred earlier in the inflammatory response, before euthanasia, is however unknown.
The fact that PAI-1 is significantly increased in plasma taken
from patients suffering from IBD (13), is associated with
coagulation abnormalities that accompany intestinal inflammation (11), and displayed the greatest changes postinflammation
in our study suggests that this procoagulant factor and adipokine may influence the severity or course of colitis. As anticipated, PAI-1 tended to be higher in noninflamed DIO rats
compared with DR controls. However, the opposite was observed in DR TNBS-treated animals. Deficits in the clotting
system are now recognized as having a potential role in
increasing the inflammatory process during IBD (11). Prothrombotic plasma PAI-1 is significantly elevated in patients
with IBD (10, 13), and vascular lesions are prominent features
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Table 2. Basal electrical properties of DIO and DR colon
DR
Saline
DIO
TNBS
Saline
TNBS
Basal
Isc (␮A/cm2) 25.1⫾8.6 (5) 7.6⫾2.1 (6)* 21.8⫾3.3 (5) 12.3⫾5.9 (5)*
Resistance
(⍀/cm2)
38.1⫾4.9 (5) 57.9⫾4.2 (6) 50.2⫾4.1 (5) 81.4⫾24.6 (6)
Values are means ⫾ SE. Values in parentheses are n values. DR, diet
resistant; DIO, diet-induced obesity. *Saline vs. trinitrobenzene sulfonic acid
(TNBS), *P ⬍ 0.05.
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Fig. 5. TNBS-treated DIO animals (E) lost significantly less weight than DR
(F) animals 4 and 5 days after TNBS treatment, and this was accompanied by
a significant decrease in food intake in DR TNBS-treated rats on all days after
TNBS treatment; n ⫽ 6 in each group. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.
of CD, characterized by fibrin deposits; in some cases, fibrin
thrombi were found to occlude the vascular lumen of blood
vessels in the ileum (14). Decreased capillary blood flow was
also reported during the acute phase of TNBS colitis in rats.
Because we observed an ⬃26-fold increase in PAI-1 during
active TNBS colitis in DR rats, it is tempting to speculate that
this may lead to increased coagulation in DR inflamed tissues
and potentially localized hypoxia, giving rise to the increased
extent of tissue damage or necrosis.
Changes in the circulating PAI-1 profile in DR rats may be
due to the decreased food intake observed in these animals. DR
animals ate significantly less food over several days (days 1–7)
after TNBS treatment, and food deprivation in mice, while
increasing PAI-1 message in epididymal and intestinal fat,
does not alter circulating PAI-1 in obese mice but does so in
lean animals (34). Nonetheless, our data obtained in PAI-1⫺/⫺
mice, while demonstrating a moderate protective effect for this
adipokine during TNBS colitis, did not reach significance,
perhaps supporting the notion that several adipokines, as observed in our models, are likely to have synergistic or antagonistic effects in terms of modulating the inflammatory response to TNBS.
Significantly elevated circulating levels of adiponectin have
been identified in patients with CD, the source of which is
likely to be hypertrophied mesenteric adipose tissue or the
creeping fat associated with CD (36, 46). Yamamoto et al. (46)
also identified an inverse relationship between adiponectin and
proinflammatory IL6 production, suggesting that adiponectin
may have anti-inflammatory properties. We did observe a
significant increase in circulating adiponectin in DIO inflamed
rats 7 days after TNBS treatment when the colon was actively
inflamed; however, given the conflicting results obtained in
genetically modified animals with respect to the effects of this
adipokine on the severity of colitis (15, 33), it is difficult to
conclude what role adiponectin plays in modulating the inflammatory response in DR or DIO animals. Seven days after
TNBS treatment a significant increase in serum ghrelin in both
DIO and DR rats was observed, and this was significantly
greater in DR compared with DIO TNBS-treated animals. This
data is consistent with decreased food intake and weight loss
observed during inflammation. To date the role ghrelin plays in
contributing to intestinal inflammation is not well characterized
(18, 47). However, recently De Smet et al. (12) demonstrated
a proinflammatory role for ghrelin during colitis in mutant
mice lacking the peptide as well as in mice exogenously treated
with ghrelin. Consistent with this finding, we observed a
significantly decreased ghrelin response in DIO animals postcolitis that was accompanied by decreased macroscopic dam-
OBESITY, ADIPOKINES, AND EXPERIMENTAL COLITIS
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age in keeping with the findings of De Smet et al.; however, the
percentage change in ghrelin in DR and DIO animals after
TNBS treatment was similar between the groups. Therefore,
the precise role for ghrelin in contributing to decreased colonic
damage in DIO animals, while plausible, is likely to be more
complex in the DIO model in which several adipokines are
altered in response to inflammation.
At this point, we can only speculate as to what aspect of
the DIO phenotype confers protection during inflammation,
but, because leptin is significantly altered in DIO animals
both before and during inflammation and can alter colonic
neutrophil activity during colitis through activation of the
hypothalamic pituitary adrenal axis (9), as well as modulating inflammation directly though receptors on most immune
cells (42), it is tempting to question the role of this adipokine in modulating the course of TNBS colitis in DIO rats.
Although the proinflammatory effect of leptin (41) makes
the observed decreased colonic damage in DIO animals
seem counterintuitive, leptin resistance has also been observed in the immune system of DIO animals; leptin receptors on T lymphocytes taken from DIO rodents are resistant
to leptin activation, suggesting that T lymphocytes develop
leptin resistance during DIO (35), potentially leading to a
decreased immune response to TNBS. It is therefore likely,
although remains to be investigated, that the DIO rats used
in our study may have developed T lymphocyte resistance to
leptin and are less responsive to the proinflammatory effect
induced by circulating leptin that occurs after TNBS treatment (4). Previous studies have indicated that, as early as
AJP-Gastrointest Liver Physiol • VOL
8 h after TNBS treatment, circulating leptin significantly
increased above that of saline-treated control animals and
may contribute to the initial decrease in food intake and
body weight in TNBS-treated rats (4). However, by 6 days
after TNBS treatment, leptin levels were comparable in
saline- and TNBS-treated rats (4).
We found no preexisting defect in colonic barrier or basal
secretory function that may predispose tissues to altered
TNBS-induced damage. Our data in the DIO model of obesity
differs from that in genetically obese, ob/ob and db/db mice,
which displayed decreased epithelial resistance and increased
permeability (8). This may reflect differences between obese
models in that leptin deficiency or decreased signaling, in
ob/ob and db/db mice, respectively, from birth may have
long-term consequences on the development of tight junctions,
whereas DIO rats have normal circulating levels of leptin
during development and generally do not display significant
increases in leptin until exposure to a MHF diet.
This is the first study to investigate the adipokine response
following inflammation in a physiological model of obesity
compared with that in lean animals. We have described significant changes in postinflammatory circulating adipokine profile
that was accompanied by decreased colonic damage after
TNBS treatment in obese animals, without an associated
change in colonic physiology. The factors contributing to the
decreased colonic damage are almost certainly multifold,
driven by both genetic and environmental factors, of which
adipokines are likely to play a part given the increasing body
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Fig. 6. Indices of tissue damage observed in PAI-1 knockout mice (n ⫽ 4 – 6) 7 days after TNBS treatment. No significant differences in macroscopic tissue
damage score (A), length of severe damage (B), myeloperoxidase activity (C), and microscopic tissue damage score (D) were observed.
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OBESITY, ADIPOKINES, AND EXPERIMENTAL COLITIS
of evidence for their role in modulating intestinal inflammation.
ACKNOWLEDGMENTS
K. Sharkey and Q. Pittman are Alberta Heritage Foundation for Medical
Research (AHFMR) Medical Scientists. K. Sharkey is the Crohn’s and Colitis
Foundation of Canada Chair in inflammatory bowel disease research at the
University of Calgary, and Q. Pittman is a University Professor. N. Hyland was
a recipient of Canadian Association of Gastroenterology/AstraZeneca/CIHR
Postdoctoral Fellowship, and A. Chambers was a recipient of an AHFMR
graduate studentship.
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