Articles in PresS. Am J Physiol Gastrointest Liver Physiol (January 15, 2015). doi:10.1152/ajpgi.00269.2014
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Effects of obesity on severity of colitis and cytokine expression in mouse mesenteric fat.
Potential role of adiponectin receptor 1
Aristea Sideri1,7, Dimitris Stavrakis1, Collin Bowe1, David Q. Shih2, Phillip Fleshner2, Violeta
Arsenescu3, Razvan Arsenescu4, Jerrold R. Turner5,6, Charalabos Pothoulakis1, Iordanes
Karagiannides1.
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Inflammatory Bowel Disease Center, and Neuroendocrine Assay Core, Division of Digestive
Diseases, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095; 2Inflammatory
Bowel and Immunobiology Research Institute, Cedars Sinai Medical Center, Los Angeles, CA;
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Inflammatory Bowel Diseases Center, Division of Gastroenterology, Hepatology & Nutrition,
Wexner Medical Center, OSU, Columbus, OH 43210; 4Department of Internal Medicine,
Division of Gastroenterology, Hepatology & Nutrition, Wexner Medical Center, OSU,
Columbus, OH 43210; 5Department of Pathology and 6Medicine, The University of Chicago,
Chicago, IL 60637; 7Postgraduate Program: “Molecular Medicine”, University of Crete, Medical
School, Greece.
Short title: Fat tissue and colitis during obesity
Corresponding Authors: Iordanes Karagiannides, Ph.D., Inflammatory Bowel Disease Center,
Division of Digestive Diseases, David Geffen School of Medicine, University of California at
Los Angeles, 675 Charles E Young Dr., South MRL building 1220, Los Angeles, CA 90095.
Tel: 310 825-8557; Fax: 310 825-3542; e-mail: [email protected]
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Copyright © 2015 by the American Physiological Society.
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ABSTRACT
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In Inflammatory Bowel Disease (IBD), obesity is associated with worsening of the course of
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disease. Here we examined the role obesity in the development of colitis and studied mesenteric
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fat-epithelial cell interactions in patients with IBD. We combined the diet-induce obesity (DIO)
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with the Trinitrobenzene Sulfonic Acid (TNBS) colitis mouse model to create groups with
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obesity, colitis, and their combination. Changes in the mesenteric fat and intestine were assessed
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by histology, myeloperoxidase (MPO) assay and cytokine mRNA expression by real-time PCR.
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Medium from human mesenteric fat and cultured preadipocytes was obtained from obese and
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IBD patients. Histological analysis showed inflammatory cell infiltrate and increased
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histological damage in the intestine and mesenteric fat of obese mice with colitis compared to all
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other groups. Obesity also increased the expression of proinflammatory cytokines including IL-
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1β, TNFα, MCP-1 and KC while it decreased the TNBS-induced increases in IL-2 and IFNγ in
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mesenteric adipose and intestinal tissues. Human mesenteric fat isolated from obese and IBD
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patients demonstrated differential release of adipokines and growth factors compared to controls.
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Fat conditioned media reduced adiponectin receptor 1 (AdipoR1) expression in human NCM460
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colonic epithelial cells. AdipoR1 intracolonic silencing in mice exacerbated TNBS-induced
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colitis. In conclusion, obesity worsens the outcome of experimental colitis and obesity and IBD-
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associated changes in adipose tissue promote differential mediator release in mesenteric fat that
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modulate colonocyte responses and may affect the course of colitis. Our results also suggest an
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important role for AdipoR1 for the fat-intestinal axis in the regulation of inflammation during
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colitis.
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Key words: Obesity, Adipose tissue, Adipokines, Colitis
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INTRODUCTION
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Obesity is an epidemic affecting one out of three Americans (8, 38) and a major risk factor for
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chronic diseases such as diabetes, cardiovascular diseases, and cancer (17, 30). Moreover,
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obesity-associated metabolic syndrome affects approximately one fourth of the US population,
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with resulting co morbidities burdening the healthcare system (8, 38). Obesity involves a “low
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grade inflammatory state”, mostly attributed to altered function of hypertrophic adipocytes.
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Adipose tissue is an active endocrine organ (1) and a source of cytokines such as TNF (tumor
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necrosis factor) α, interleukins, and the adipokines adiponectin, leptin, and ghrelin (7, 9, 25, 44,
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47). These mediators play pro-inflammatory, anti-inflammatory or appetite-controlling roles
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depending on the conditions during their release (34, 42). Circulating levels of adipokines are
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also deregulated in obese patients (10), and this response may contribute to the pathophysiology
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of obesity-related diseases.
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Frequency of IBD, which includes ulcerative colitis (UC) and Crohn’s Disease (CD), is elevated
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in the developed world and associated with increasing morbidity (36). Anatomically, the affected
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intestine is in immediate proximity to the intra-abdominal mesenteric and omental fat depots
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which contain lymph nodes and are well vascularized. Although poorly understood, the presence
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of adipose tissue wrapping around intestinal lesions in CD patients (creeping fat) has been well
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documented during surgery (15), while fat wrapping in UC has not been reported. Moreover,
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patients with higher body mass index (BMI) at diagnosis demonstrate increased need for
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hospitalization during the course of the disease and shorter time span between diagnosis and
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surgical intervention (6, 20). Recent studies demonstrated similarities in the expression patterns
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between adipocytes isolated from whole mesenteric fat depots obtained from obese and CD
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patients, with inflammation- and lipid metabolism-associated pathways showing the highest
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degree of convergence between the two groups (51). A recent report failed to establish a
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causative relationship between obesity and IBD (12). This investigation (12), however, was
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focused on BMI as a risk factor for developing IBD without assessing directly the effect of
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obesity on IBD outcome. Despite all the indications favoring a link between obesity and IBD
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outcome, evidence for this association is still limited.
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Using intracolonic administration of 2,4,6-Trinitrobenzenesulfonic acid (TNBS), we previously
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demonstrated histologic changes in the mesenteric and epididymal fat depots that resemble
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changes described in CD, and showed increased expression of pro-inflammatory mediators in
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these fat depots (21). TNFα, leptin and adiponectin have also been implicated in the induction of
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morphological changes in “creeping fat” adipocytes (11, 15, 18). Moreover, circulating levels of
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adiponectin, ghrelin, and resistin are increased, whereas those of leptin are decreased in IBD
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patients (24), suggesting that adipose tissue-derived mediators may affect IBD pathophysiology.
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Another study showed that high fat diet (HFD) exacerbates the outcome of colitis and is
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associated with increased fat mass (31) . Mice on HFD also exhibited increased number of
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natural killer T cells, which produced higher amounts of TNFα and IFNγ, and a decreased
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number of regulatory T cells (31). HFD- adoptive transfer of regulatory T cells rescued colitis
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and lowered cytokine levels observed in the HFD group (31). These observations underline the
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potential involvement of adipose tissue-derived responses in IBD pathophysiology.
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In the current study, we examined the effects of HFD-induced obesity on colitis outcomes by
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employing the well characterized model of chronic TNBS-induced colitis in either lean or obese
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mice. We found striking differences in colitis-associated animal morbidity and mortality after
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TNBS administration between lean and obese mouse groups. More severe inflammatory changes
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and dramatically higher cytokine levels were also observed in the colon and the mesenteric
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adipose tissues in obese, compared to lean mice. Adiponectin (Adipoq) expression in the fat and
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adiponectin receptor (AdipoR) expression in the intestine were also different in obese vs lean
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mice with colitis. Moreover, media obtained from mesenteric fat and cultured preadipocytes
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isolated from control, obese and IBD patients exhibited significant differences in inflammatory
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mediator release and elicited condition-dependent changes on AdipoR1 expression in NCM460
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human colonic epithelial cells, while silencing of AdopoR1 in mice showed increased
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inflammatory changes following experimental colitis.
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METHODS
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Human subjects: Mesenteric fat tissues from IBD (13 UC, 12 CD) and non-IBD patients (12
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Control, 9 obese) were used. For the non-IBD individuals fat tissue was resected during gastric
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bypass (for the management of obesity), gynecological, adenocarcinoma surgery, other
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gastrointestinal complications, or vascular surgery. Human studies protocols have been approved
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by the UCLA Institutional Review Board for Human Research (IRB#11-001527-AM-00003).
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All participants gave informed consent before taking part. All subjects were fasted for at least 10
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hrs prior to surgery. Subjects with malignancies were not excluded, since they may constitute an
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important sub-population that could yield significant information for our intergroup comparisons.
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Tissues from Cedars-Sinai Medical Center were obtained after informed consent in accordance
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with procedures established by the Cedars-Sinai Institutional Review Board (IRBs 3358 and
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23705).
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Isolation of human preadipocytes: 2-5 grams of mesenteric fat tissue were obtained from each
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patient. The tissue was placed into sterile 50 ml polypropylene tubes containing 15 ml of
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collagenase solution (1 mg of collagenase/1 ml of PBS, 3 ml of solution/1 g of tissue) and
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minced to a fine consistency. The solution was then vortexed for 20 seconds and the tubes placed
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in a 37oC shaking water bath (100 rpm) for 40 min. The solution was vortexed and filtered
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through a double gauze-containing funnel. The homogenates were centrifuged (1000 rpm, 10
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min) and the top fatty layer collected and washed 3x with PBS. The pellet was then re-suspended
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in 10 ml of erythrocyte lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA), placed in
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a 37oC shaking water bath for 5 min at 100 rpm and centrifuged at 1000 rpm for 10 min. The
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pellet was re-suspended in 10 ml plating medium (DMEM, 0.1 mM penicillin, 0.06 mM
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streptomycin, 10% HI-fetal bovine serum [FBS], pH 7.4), vortexed, plated onto 100 mm dishes,
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and incubated at 37oC.
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Cell culture of human preadipocytes: After 20 hrs cells were washed 3x with 10 ml PBS and 1
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ml trypsin solution (Invitrogen, Carlsbad, CA) was added. Trypsin was inactivated with 5 ml
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plating medium and cells were centrifuged at 1000 rpm for 10 min. The supernatant was
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aspirated, cells were re-suspended in 10 ml plating medium, and plated at 5x104 cells /cm2 in
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plating medium. Cells were incubated at 37oC until they reached confluence and medium was
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changed with fresh medium every 48 hrs. This isolation procedure yields >99% pure
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preadipocyte populations (as determined by cloning of individual cells and counting of colonies
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derived from them that were able to accumulate lipid) (45). Cells were then sub-cultured 3 or 4
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times to ensure removal of macrophages (46). No ADAM8, F4/80, or macrophage inflammatory
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protein-1a mRNA, markers of macrophages, were detected by Affymetrix array analysis of
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human mesenteric or omental preadipocytes prepared using this protocol as we previously
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described (23).
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Exposure of NCM460 human colonocytes: Human mesenteric fat tissue media. After surgery
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100-200 mg of mesenteric fat tissue were removed with sterile scissors, placed in a 15ml sterile
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polypropylene tube with 2ml of FBS-free medium (MEM, 0.1mM penicillin, 0.06mM
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strepromysin, obtained as shown above), and kept in a 37oC shaking water bath for 24 hours at
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100 rpm. 200 μl of the conditioned media were then placed over confluent NCM460 colonocytes
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in 24-well plates for 24 hrs and RNA was isolated in Trizol reagent.
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Human preadipocyte media. Preadipocyte medium was removed from confluent preadipocyte
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cultures of control, UC and CD patients during the third passage after isolation and exposure of
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confluent plates to fresh medium (MEM, 10% FBS, 1% P/S) for 24 hrs. NCM460 were grown to
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confluence in 6-well plates and 1ml of preadipocyte media was added for 24 hrs. The wells were
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then washed 1X and RNA was isolated using Trizol reagent.
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Adiponectin. NCM460 cells were grown to confluence with culturing medium (M3D, 1% P/S
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and 10% FBS) in 24-well plates, and were exposed to 10μg/ml reconstituted Adiponectin
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(Sigma-Aldrich, Saint Louis, MO) or vehicle (PBS, 0.1% BSA)in treatment medium (M3D with
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1% P/S, FBS-free) for 24 hrs. The wells were then washed 1xPBS and RNA was isolated using
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Trizol reagent.
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Animal groups: Male C57BL/6 mice (18-20g), 8-12 weeks old (n=8-16 per group) were
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purchased from Jackson Labs (Bar Harbor, Maine). Mice were maintained on a normal light-
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dark cycle, and provided with food and water ad libitum. Two groups of C57BL/6 mice were
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kept on high fat diet (HFD) (Research Diets, NJ) for 6-8 wks, while two additional groups were
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fed low fat diet (LFD) for the same period (or until groups separated by 10 grams). After feeding,
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TNBS colitis was induced intracolonicaly to one of the HFD-fed and one of the LFD-fed groups
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while the other two groups received intracolonic ethanol (control) injections. Collectively we
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produced the following four groups: A. LFD-fed, non-TNBS (LFD-C) B. LFD-fed, TNBS (LFD-
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TNBS) C. HFDfed, non-TNBS (HFD-C) and D. HFD-fed, TNBS (HFD-TNBS).
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All animal protocols were approved by the Institutional Animal Care and Use Committee at the
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David Geffen School of Medicine at UCLA and studies were carried out in accordance with the
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National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH
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Publications No. 80 23, revised 1978).
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TNBS colitis: Age- and weight-matched mice were lightly anesthetized with isofluorene and a
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polyethylene cannula (Intramedic PE-20 tubing, Becton Dickinson, Parsippany, NJ) was inserted
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intracolonicaly (at a length of 4 cm). A solution of 40% ethanol (vehicle) or ethanol-containing
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TNBS (Sigma, St. Louis, MI) was instilled into the colon (3-4cm from the anus) using a syringe,
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while control animals were treated with vehicle alone. TNBS or vehicle injections were
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performed once a week for six weeks of 1, 1.5 and 2mg per 20 g (2 weeks/dose). Mice were then
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left untreated for two more weeks. At the end of the study, body weight was assessed, and mice
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were then euthanized with Isofluorane overdose. Pieces of mesenteric fat and intestine were
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either placed in formalin for immunohistochemistry or frozen for protein and RNA extraction.
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Colitis score was assessed as described in (28).
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Real-time PCR: RNA was isolated from mouse and human mesenteric whole fat tissue, and
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human mesenteric preadipocytes using the Trizol method. 1 μg of RNA was reverse-transcribed
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into cDNA as previously described (22) and incubated with dual fluorogenic probes (Applied
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Biosystems, Foster City, CA). GAPDH and 18s were used as endogenous controls and were also
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detected using dual labeled fluorogenic probe (5’-FAM/3’-MGB probe, Applied Biosystems,
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Foster City, CA). Target mRNA (all from Applied Biosystems) levels were quantified using a
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fluorogenic 5'-nuclease PCR assay as described in (30) using a 7500 Fast Real-Time PCR
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sequence detection system (Applied Biosystems, Foster City, CA).
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mRNA multiplex analysis: Total RNA was isolated as described above and inflammation-
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related gene expression was analyzed using the 42-plex FlexScript LDA inflammatory panel 3
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(Luminex, Austin, TX). 20 ng of total RNA were loaded in each well and following treatments
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described in the company manual were performed (FlexScript LDA). The plate was run using
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Bio-plex 3D suspension array system (Bio-Rad, Hercules, CA). In addition to total RNA
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concentration, data were normalized to endogenous controls (GAPDH, B2M, β-actin) included
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within the gene panels.
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Intracolonic AdipoR1 knock down via siRNA: 10-12 week old C57BL/6 mice were placed
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into 3 groups (sham-EtOH, scrambled, and siAdipoR1; n=5 mice per group). At day 0 mice in
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the sham-EtOH group received intracolonically 100μl of lipofectamine 2000 (1:50/total volume).
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Mice in the scrambled group received intracolonically 4 nmoles of scrambled nucleotides
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(SR30004, Origene Technologies, Rockville, MD) in lipofectamine 2000, and mice in the
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siAdipoR1 group received 1.33 nmoles of each of 3 anti-AdipoR1 siRNA duplexes (SR412651A,
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B, and C, Origene Technologies) in lipofectamine 2000. The same injections were repeated on
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Day 2 while on Day 3 mice were injected intracolonically with 5mg TNBS. Mice were sacrificed
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for analysis on Day 5. Figure 9A includes a schematic representation of the design of these
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studies.
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Immunohistochemistry: Paraffin-embedded colon sections from UC, CD and control patients
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(n=4 per group) were mounted on slides. AdipoR1 staining was detected using an anti-AdipoR1
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rabbit monoclonal antibody (1:100 dilution, ab126611, Abcam, Cambridge, MA) and the
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EnVision+ System- HRP Labeled Polymer Anti-Rabbit kit (DAKO, Carpinteria, CA). Staining
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was performed at the Translational Pathology Core, UCLA following a standard procedure
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described in Millipore’s manual for the primary antibody treatment .
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Multiplex cytokine and phospho-protein immunoassays: Human mesenteric fat tissue was
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isolated and plated as described above and media were collected at the end of the 24hr period.
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Cytokine concentrations in conditioned media were determined using the Bio-Plex ProTM Human
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Adipokine Magnetic Bead Panel 1 (Bio-Rad, Hercules, CA) and the final data were obtained and
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analyzed via the Bio-plex 3D Suspension array system (Bio-Rad). In addition to loading volume
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results were normalized for total protein as well as tissue weight.
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Statistical Analysis: Results were analyzed using the Prism professional statistics software
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program (Graphpad Software Inc., San Diego, CA). Analyses of variances (ANOVA, one-way)
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as well as Mann-Whitney (for comparisons between two groups) were used for intergroup
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comparisons. A p value of < 0.05 was considered statistically significant.
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RESULTS
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High fat diet (HFD)-induced obesity exacerbates the effects of TNBS colitis on mesenteric
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fat depot mass in C57BL/6 mice. We previously showed that mesenteric fat depots isolated
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from TNBS-treated mice express high levels of proinflammatory cytokines and increased
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inflammatory cell infiltrates (21). In this study we separated mice in four groups (n=8-16 per
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group) as described in the animal groups section above. We observed that mesenteric fat depot
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expansion was evident in both lean and obese mice with colitis (Figure 1). Obese mice with
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TNBS-induced colitis were the only group that had diarrhea and blood in the stool. Mice were
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sacrificed 48 hrs after induction of colitis and tissues were collected due to the high mortality of
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animals in the HFD-TNBS group. As expected, HFD-induced obesity alone was associated with
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increased mesenteric fat mass around the intestine compared to lean, LFD-fed mice (Figure 1C
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vs 1A). In addition, obesity exacerbated this response in the mesenteric fat depots with more
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mesenteric fat attachment in the HFD-TNBS group compared to LFD-TNBS – exposed mice
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(Figure 1D vs 1B). Indicative of the severity of colitis, mice in the obese group showed high
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mortality rates (50%) 48 hrs post-TNBS using a low TNBS dose commonly used to promote
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chronic disease (2mg/20gr). In comparison, lean mice that received the same low TNBS dose
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were unaffected in terms of viability, weight loss or diarrhea (data not shown). Thus, conditions
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associated with increased fat mass during obesity contribute to a dramatic worsening of
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experimental colitis.
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In separate experiments, we injected lower doses of TNBS (0.75, 1, and 1.25 mg/20gr) in groups
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of animals as described above in an attempt to reach the endpoint of 6 weeks before sacrifice.
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Although the HFD-TNBS group did survive for the duration of the study (6 weeks) at these low
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TNBS doses we observed very low levels of inflammatory responses even in the obese group
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with minimal differences between groups (LFD-TNBS vs HFD-TNBS, p=0.02 for KC, n=7).
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Moreover, no signs of colitis were observed at the gross morphological level in any of the groups
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(data not shown).
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HFD-induced obesity worsens TNBS-induced histologic changes in mouse colon. In the
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same experimental groups described above colon was removed after the completion of the study
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and histological sections were evaluated as described in Methods. There was significantly
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increased colonic inflammation in mice fed HFD and treated with TNBS (Figure 2D, E) as
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compared to mice in the other 3 groups (LFD-C, LFD-TNBS, HFD-C, Figure 2A-C, E).
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Together, these data indicate that obesity may induce alterations in inflammatory mediator
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release that worsens colitis. Furthermore, mice in the HFD-TNBS group demonstrated increased
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weight loss compared to the LFD-TNBS group (Figure 2F, p<0.05) and had increased mortality
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with 50% of mice not surviving after the first 24-48 hrs (Figure 2G). Tissues from mice that did
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not survive to the point of sacrifice were excluded from any analysis.
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HFD-induced obesity increases proteinase 3 mRNA in the colon of TNBS-exposed mice. To
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further evaluate increased obesity-associated increased immune cell infiltration in mice with
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colitis, we measured mRNA expression of neutrophil (proteinase 3) and macrophage (EMR1,
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human homologue of F4/80). Figure 3A (HFD-TNBS vs. LFD-TNBS) shows significant obesity-
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related increases in colonic proteinase 3 mRNA expression in TNBS-exposed mice, suggesting
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increased presence of leukocytes in the intestine during colitis in HFD-fed mice. Obesity alone
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did not lead to any increases in proteinase 3 expression (Figure 3A, HFD-C vs. LFD-C). In
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contrast, colonic EMR1 levels were decreased in HFD-TNBS group (Figure 3B, p<0.01, n=6-8).
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Thus diet-induced obesity (DIO) is associated with increased colonic neutrophil, but not
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macrophage infiltration.
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HFD-induced obesity is associated with increased cytokine expression in mouse intestine
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during TNBS colitis. We used real time PCR to measure mRNA expression of inflammatory
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cytokines that may affect the development of colitis in mouse colon of all four mouse groups
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described above. We observed significant obesity-associated increases in the expression of IL-1β,
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IL-6, and KC (Figure 4A-C, HFD-TNBS vs. LFD-TNBS, respectively, * p<0.05, **p<0.01, n=6-
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8), and IL-10 (Figure 4F, p<0.001, n=6-8/group) 48 hrs after TNBS treatment. In contrast, IFNγ
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and IL-2 expression were lower in obese mice exposed to TNBS (Figure 4D & E, HFD-TNBS,
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*p<0.05, **p<0.01, n=6-8/group), indicating an active mechanism that induces the observed
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changes rather than complete cytokine deregulation due to severe colitis. For some of these
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cytokines such as IL-1β, IL-6, IFNγ, IL-2 we observed increased RNA expression with obesity
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alone (Figure 4A, B, D & E, LFD-C vs. HFD-C, ##p<0.01, ###p<0.001, n=7-8). Such changes
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may be related to the exacerbated responses observed in the obese group during colitis (Figure
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4A-F, HFD-TNBS).
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HFD-induced obesity is associated with increased inflammatory cell infiltrate in mesenteric
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fat depots during TNBS colitis. CD-like increased infiltration of immune cells in adipose tissue
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can be replicated in the TNBS colitis model (21, 26). Mesenteric fat was removed from all mice
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groups at the end of the experiments and examined histologically. As in the intestine, H & E
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stained histological sections showed that TNBS-associated inflammatory changes were
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dramatically exacerbated in the mesenteric fat depots by HFD-induced obesity, evidenced by
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increased inflammatory cell infiltrates in fat depots from obese mice with colitis (Figure 5D),
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compared to mesenteric fat isolated from lean animals (Figure 5B). No apparent inflammatory
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infiltration at the gross morphological level was observed in obese alone vs lean controls in the
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absence of TNBS treatment.
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We also measured mRNA expression of inflammatory cell markers in the mesenteric fat depots
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in the different groups of mice. Using an MPO assay we show that the levels of myeloperoxidase
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(MPO), a marker of activated neutrophils, within fat depots were significantly higher during
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colitis only in the obese group (Figure 6A, LFD-TNBS vs HFD-TNBS, p<0.01, n=10-11). In
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agreement with our previous study (21) we observed increased proteinase mRNA levels in the
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mesenteric fat during colitis (Figure 6B, LFD-C vs. LFD-TNBS, p<0.05, n=10-11), while
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obesity exacerbated this response (Figure 6B, HFD-TNBS vs. LFD-TNBS, p<0.01, n=10-11).
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EMR1 mRNA is increased in obese mice without colitis vs lean controls, but not when compared
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to lean TNBS and obese control mice (Figure 6C). Thus, obesity increases neutrophil infiltration
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in the mesenteric fat during colitis.
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HFD-induced obesity alters cytokine responses in mouse mesenteric fat depots during
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TNBS colitis. Next we isolated RNA from mesenteric fat of all experimental groups and
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analyzed them for the expression of proinflammatory cytokines that may be involved in the
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generation of adipocyte-specific effects in the intestine during colitis. Our results from
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mesenteric fat depots demonstrate dramatic increases with obesity in the mRNA expression of
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several proinflammatory cytokines such as IL-1β, IL-6, MCP-1, TNFα, and KC (Figure 7A-E,
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HFD-TNBS, **p<0.01, n=5-8) 48 hrs after the induction of TNBS colitis compared to all other
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groups (n=6-8). As expected for fat depots during obesity (HFD-C) (25, 44), the expression of
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several of these cytokines were increased (Figure 7, TNFα, MCP-1, IL-1β, and KC, LFD-C vs.
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HFD-C #p<0.01, n=6-8). Again, the mRNA levels of INFγ (but not IL-2) were dramatically
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reduced in the mesenteric fat of obese mice (HFD-TNBS) following TNBS compared to all other
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groups (Fig 7F, *p<0.05, n=5-8). These results indicate that the effects of obesity in the
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mesenteric fat following colitis are specific and not due to complete dysregulation of the
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inflammatory response. Interestingly, the expression of the anti-inflammatory cytokine IL-10
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was dramatically increased in the adipose tissues of obese mice after TNBS treatment (Figure 7G,
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p<0.01, n=5-8).
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HFD-induced obesity alters the expression of adiponectin and its receptors during TNBS
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colitis in mice. Several adipokines have been implicated in IBD pathophysiology, including
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adiponectin. Adiponectin reduces inflammation by a) inhibiting macrophage function (34, 48), b)
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triggering proinflammatory cytokine secretion (34, 41) and c) up-regulating the protective
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cytokine IL-10. Its expression also decreases with obesity (2), while increased adiponectin
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during CD may facilitate mucosal healing. Here we demonstrate that adiponectin mRNA
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expression was increased in the mesenteric depots following intracolonic TNBS (Figure 8A,
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LFD-C vs. LFD-TNBS, #p<0.01, n=10-13), and decreased during obesity in the mesenteric fat
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depots of mice (Figure 8A, LFD-C vs. HFD-C, n=12-13). However, when we combined obesity
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with TNBS colitis (HFD-TNBS), the obesity-induced decrease in adiponectin expression was
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exacerbated (Figure 8A, p<0.001 compared to all groups). Moreover, the expression of its
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receptors AdipoR1 and AdipoR2 in the mesenteric fat was increased in response to TNBS
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(Figure 8B & C, LFD-C vs. LFD-TNBS, p<0.01 and p<0.05, respectively, n=12-13), but
396
remained unaffected by obesity (Figure 8B & C, HFD-C vs. LFD-C). In obese mice with colitis,
397
both adiponectin receptors AdipoR1 and AdipoR2 remained at significantly lower levels
398
compared to mice with TNBS alone (Fig 8B & C, LFD-TNBS vs. HFD-TNBS). In the intestine
399
both AdipoR1 and AdipoR2 mRNA levels decreased in obese mice with colitis (Figure 8D & E,
400
p<0.01 and p<0.001, respectively, HFD-TNBS compared to all other groups). In contrast to our
401
observations in mesenteric fat depots, TNBS colitis alone did not significantly alter colonic
402
mRNA expression of these receptors (Figure 8D & E, LFD-C vs. LFD-TNBS, strong trend
403
towards decrease), suggesting different roles for adiponectin in the colon and mesenteric fat
404
during colitis.
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Intracolonic AdipoR1 knock down worsens TNBS colitis in mice. To highlight the potential
407
role for AdipoR1 in intestinal inflammation we knocked down this receptor via intracolonic
408
administration of anti-AdipoR1 siRNAs prior to the induction of colitis (siAdipoR1, Figure 9A).
409
Expectedly, both the siAdipoR1 and scramble groups showed increased weight lost compared to
410
sham treatment due to the TNBS induction (Figure 9B, *p<0.05, n=5-6). However the
411
siAdipoR1 group lost more weight even when compared to the scrambled group at day 5 of the
412
study (Figure 9B, #p<0.05, n=6). H&E staining of intestinal sections from the 3 groups showed
413
worsening of colitis in the siAdipoR1 group compared to both the scrambled and sham (EtOH)
414
groups (Figure 9C). Colitis severity (Figure 9D, p<0.01 vs. sham, p<0.05 vs. scrambled),
415
mucosal damage (Figure 9E, p<0.01 vs. sham, p<0.05 vs. scrambled), and crypt formation
416
(Figure 9F, p<0.01 vs. sham, p<0.05 vs. scrambled) were also exacerbated.
417
418
Differential AdipoR1 expression of human colonic epithelial cells following exposure to
419
conditioned media from control, obese and IBD patient preadipocytes. To provide evidence
420
of potential adipose tissue-derived effects during obesity or IBD on adiponectin-associated
421
responses in colonocytes, we exposed NCM460 human colonic epithelial cells to conditioned
422
media derived from 8-19 mesenteric fat tissues of control, UC and CD patients and from cultured
423
human preadipocytes of 6-9 control, obese, UC and CD patients. We then examined the mRNA
424
levels of adiponectin receptors in these cells. We observed that, conditioned media derived from
425
mesenteric fat tissues of both UC and CD patients reduced the expression levels of AdipoR1 in
426
NCM460 colonocytes (Figure 10A, p<0.05, n=8-19). In addition conditioned media from UC
427
patient preadipocytes induced a significant decrease in AdipoR1 mRNA levels in NCM460
428
colonocytes (Figure 10B, p<0.01, UC vs C, n=6-9), but not AdipoR2 (not shown). Strong trends
429
towards decreased AdipoR1 levels were also observed in colonocytes exposed to conditioned
430
media from preadipocytes obtained from obese and CD patients (Figure 10B, n=6-9, p= 0.0667
431
and 0.0867 for obese and CD patients, respectively). Thus, changes in adipose tissue-derived
432
mediator secretion during obesity and IBD may alter the capacity of intestinal epithelial cells to
433
respond to adiponectin. Phospho-protein multiplex analysis revealed that phospho-insulin-like
434
growth factor 1 receptor (IGF-1R) levels of NCM460 cells decrease after exposure to
435
conditioned media from preadipocytes isolated from UC and CD patients compared to those
436
from control patients (Figure 10C, p<0.01 for UC and p<0.05 for CD, n=6-9). Conditioned
437
media from fat tissue isolated from IBD patients decreased mRNA expression of the
438
transcription factor PPARγ in NCM460 colonocytes (Figure 10D, p<0,01 for UC, trend for CD,
439
n=8-21). Finally, IGF-1 treatment increased mRNA expression of AdipoR1 in NCM460
440
colonocytes (Figure 10E, p<0.01, n=6) suggesting that AdipoR1 regulation by preadipocytes and
441
fat tissue conditioned media may be mediated via down regulation of IGF-1R signaling.
442
443
AdipoR1 levels increase in colonic biopsies of IBD vs. Control patients and adiponectin
444
reduces cytokine expression in human NCM460 colonocytes. Immunohistochemistry for the
445
detection of the levels of AdipoR1 revealed increased receptor – positive cells in the colonic
446
mucosa of UC, and CD patients compared to control subjects (Figure 11A). To investigate the
447
effects of adiponectin in the intestine we treated human NCM460 colonocytes and collected
448
RNA for the measurement of mRNA expression levels of several cytokines. Among the 42
449
cytokines measured we observed that adiponectin treatment led to decreased mRNA levels of IL-
450
2, IL-5, IL-8, IL-17, IL23 and TGFβ2 (Figure 11B-G, *p<0.05, **p<0.01, n=6), and increased
451
the mRNA levels of VEGFA (Figure 11H, p<0.01, n=6). Thus, obesity-associated regulation of
452
AdipoR1 in the intestinal epithelium may affect the ability of these cells to respond to
453
inflammatory regulation by adiponectin during colitis.
454
455
Human mesenteric fat depots demonstrate distinct mediator release when isolated from UC
456
and CD patients. Systemic inflammatory changes are considered a hallmark of obesity (5, 19,
457
44). Several of these responses were observed systemically and within fat depots during IBD
458
(51). We have obtained mesenteric fat depots of 8 control, 14 UC and 14 CD patients and
459
collected conditioned media for multiplex adipokine analysis after 24 hrs in culture. Analysis of
460
the 11 adipokines revealed IBD-associated changes in the release of mediators from human
461
mesenteric fat compared to controls (Figure 12A-F, *p<0.05, **p<0.01, #p<0.1, n=8-14). We
462
observed increased adiponectin, TNFα, and IL-1β (Figure 12A, D, & F) release in mesenteric fat
463
from both UC and CD patients. Moreover, compared to controls, release of leptin was increased
464
only in UC (Figure 12B), while IL-8 release was increased in CD patients (Figure 12E) Resistin
465
release was also decreased in mesenteric fat depots from CD patients compared to controls
466
(Figure 12F).
467
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477
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482
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486
487
DISCUSSION
488
Our data strongly support the notion that obesity affects the outcome of experimental colitis and
489
produces a dramatically altered inflammatory environment both in fat and the intestine. We
490
demonstrate significant increases in inflammatory cell infiltration in both the intestine and
491
mesenteric fat depots (Figures 3 and 6) in obese mice during colitis, likely due to effects of
492
increased adiposity in the production of inflammatory mediators in both tissues (Figures 4 & 7).
493
Mice were sacrificed only 2 days after TNBS administration and not 6 weeks as originally
494
planned. This adjustment was necessary because of the detrimental response of the obese mice to
495
TNBS even at low chronic doses which failed to cause any inflammatory changes in lean mice.
496
497
It is striking that, for the mediators reported here, the responses in the obese group during colitis
498
are almost identical for both the intestine and adipose tissue. However, not all mediators exhibit
499
the same patterns of expression with obesity alone (HFD-C), with the adipose tissue showing a
500
wider range of responses. Significant decreases in the expression of both IFNγ and IL-2 (Fig 4D
501
& E, respectively) demonstrate that the effect of obesity on cytokine expression during colitis is
502
a result of specific and controlled transcriptional modulation. Both IFNγ and IL-2 are involved in
503
T cell maturation processes, suggesting an important role for obesity in the modulation of T cell
504
responses during colitis. Interestingly, mRNA expression of these two mediators increases
505
significantly during obesity (IL-2, IFNγ, Figures 4D & E) in the colon, but not in adipose tissue.
506
In addition, several of the cytokines dramatically changed in the obese group with colitis (HFD-
507
TNBS) have been shown to participate in the development of experimental colitis and IBD
508
pathophysiology. Briefly, TNFα antagonism represents one of the main treatment modalities for
509
IBD (39). IL-1β has been shown to promote the accumulation and survival of pathogenic CD4(+)
510
T cells in the T cell transfer mouse model of colitis (13), while the levels of this cytokine are
511
elevated and are associated with increased disease activity in the colon of IBD patients (29, 32).
512
Similar associations were also demonstrated for the colonic levels of IL-8 in IBD patients (14),
513
while the IL-10 mouse knockout model provides one of the most common tools for the study of
514
colitis (43).
515
516
As expected, obesity induces differential responses in the expression of pro-inflammatory
517
mediators both in the adipose tissue and the intestine, suggesting interactions between these two
518
tissues during obesity that may alter the course of colitis. Indeed, novel experimental evidence
519
demonstrating that the intra-mesenteric adipose tissue mediator content could influence the
520
availability of macrophage subtypes in “creeping” fat during CD (27) also support this
521
hypothesis. Collectively, our study is the first to demonstrate direct (and adverse) effects of
522
obesity in the outcome of experimental colitis and elucidate that obesity-related mesenteric fat
523
responses, while not identical, resemble a proinflammatory phenotype during colitis.
524
525
Adiponectin has anti-inflammatory properties and its levels are adversely affected by obesity
526
(33). A potential protective role of adiponectin in IBD is suggested by its significant increase in
527
mesenteric fat depots (“creeping fat”) of CD patients (49). Our data in Figure 8A show an
528
obesity-induced reversal in increased adiponectin expression during colitis. Abolishment of a
529
potential protective role of adiponectin may be responsible for the dramatic exacerbation of
530
inflammatory responses observed in the obese group during colitis in our study. We also
531
observed differential expression of both adiponectin receptors in the intestine and adipose tissue
532
during obesity and colitis (Figure 8B-E, HFD-TNBS vs LFD-TNBS), with levels dropping
533
significantly below those of control animals in the intestine (HFD-TNBS vs LFD-C). The
534
potential importance of the regulation of AdipoR1 levels during colitis is also highlighted by our
535
data in Figure 9 demonstrating increased TNBS colitis-associated weight loss and colonic
536
damage after siRNA-induced AdipoR1 knockdown and by our human data in Figure 11A
537
showing increased colonic levels of AdipoR1 receptor protein in IBD patients.
538
539
Considering the highly conflicting reports on the effects of adiponectin ablation on the course of
540
experimental colitis stemming from studies employing null mice (16, 37, 40), our data, along
541
with reports on the anti-inflammatory and healing roles of this adipokine in the intestine, suggest
542
the need for additional studies employing more tissue specific or tissue limited approaches.
543
Arsenescu et al showed that overexpression of adiponectin increased serum and colonic level of
544
IL-10, while Th1 cytokines were downregulated (4). A plant homologue of adiponectin had an
545
identical effect on IL-10 production (4). Furthermore, increased adiponectin expression
546
correlated with resistance to development of colitis, upregulation of Treg response, and
547
downregulation of Th17 pathway mediators (3). Increased IL-10 expression in the colon and fat
548
in our study (Figures 4F and 7G) suggests a generalized anti-inflammatory damage control
549
mechanism potentially being activated in our colitis model with obesity.
550
551
Our results show altered levels of colonocyte adiponectin receptor in response to conditioned
552
medium from IBD patient preadipocytes and whole fat tissue (Figure 10) suggesting that changes
553
in mediator expression within fat depots with colitis could affect adiponectin colonocyte
554
signaling. Colitis-associated down regulation of AdipoR1 in the colon may involve IGF-1R
555
signaling-associated pathways as suggested by our data (Figure 10) showing that conditioned
556
media from preadipocytes and fat tissue from IBD patients reduce the levels of phosphor-IGF-1R
557
(Tyr1131), and PPARγ mRNA. Interestingly, PPARγ has been shown to affect the transcription
558
of both IGF-1R and AdipoR1 (35, 50). Furthermore, to establish a potential link between IGF-1R
559
and AdipoR1 we treated NCM460 cells with IGF-1 and observed increased AdipoR1 expression.
560
The potential importance of the reduction of colonocyte adiponectin receptor levels by adipose-
561
derived products included in the conditioned media is highlighted by the anti-inflammatory
562
effects of adiponectin treatment in the same cells (Figure 11). Such effects may be diminished
563
during colitis in obese patients depriving these individuals of potentially beneficial effects of
564
adiponectin (expression is increased in IBD patients, Figure 12A) and thus, exacerbating colitis
565
in these patients. It is thus conceivable that the intestine is exposed to intra-abdominal fat-
566
derived products due to the close proximity of these tissues or possibly via the circulation during
567
obesity. It is also likely that such exposure to differentially expressed mediators (Figure 12) takes
568
place during IBD or experimental colitis, especially in cases where the intestinal wall is
569
compromised. This may lead to exacerbation of colitis such as in our case where obese mice
570
show high mortality with a dose to which their lean counterparts remain unaffected. These
571
results suggest that fat-promoted alterations in adiponectin-AdipoR1 signaling may affect course
572
of colitis during obesity.
573
574
In summary, our results implicate obesity – associated changes in the mesenteric fat depots as an
575
important component of the severity of experimental colitis. Our data also provide the first link
576
between altered adipose tissue function during obesity or IBD and intestinal epithelial cell
577
responses and highlight that the adiponectin-adiponectin receptor axis may play a significant role
578
in the regulation of colitis in obese patients.
579
580
Acknowledgments: Dr Sarah Dry and the Translational Pathology Core Laboratory, Department
581
of Pathology, University of California at Los Angeles, for providing human mesenteric fat tissue
582
samples for our studies.
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Grant support: Research Fellowship Awards from the Crohn’s Colitis Foundation of America,
Inc (IK); Research Grant from the Broad Medical Foundation (IK); the Neuroendocrine Assay
Core and Project 2 supported by NIDDK P50 DK 64539 (IK & CP); NIH NIDDK grant RO-1
DK 47343 (CP).
Author conflict of interest disclosures: There is no conflict of interest to disclose
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760
761
762
Figure Legends
763
Figure 1. Macroscopic changes in mesenteric fat depots during experimental (TNBS) colitis
764
resemble those observed in patient’s with Crohn’s disease. (A) Normal mouse intestine without
765
obesity or colitis. (B) TNBS colitis induces infiltration of fat and “wrapping” of the intestine
766
around the affected area. (C) Increased mesenteric fat mass proximal to the intestine without
767
colitis. (D) Increased fat “wrapping” of the involved intestine during TNBS colitis in obese mice
768
with strong attachment to multiple sites and signs of extensive angiogenesis.
769
770
Figure 2. Obesity exacerbates experimental colitis in mice. We removed a 1 cm piece of colon
771
from mice that included the visibly inflamed area located approximately 3cm from the anus and
772
sectioned and stained with H & E stain. (A-D) Histological sections and (E) clinical scoring of
773
mouse colons after treatment with TNBS reveals that colitis outcome is exacerbated in obese
774
mice (HFD-TNBS) compared to lean mice with (LFD-TNBS) or without (LFD-C) colitis and
775
obese mice without colitis (HFD-C). (F) Obese mice exhibited increased weight loss in response
776
to TNBS colitis (HFD-TNBS) compared to their lean counterparts (LFD-TNBS). (G) Survival
777
curve showing increased mortality in the HFD-TNBS compared to the LFD-TNBS group.
778
***p<0.001
779
780
Figure 3. Obesity exacerbates inflammatory cell infiltrate in the colon of mice with TNBS-
781
induced colitis. Real time PCR on total intestinal RNA showed that (A) mRNA levels of the
782
neutrophil marker proteinase 3 increase in the intestine of obese mice 48hrs after the induction of
783
TNBS colitis (HFD-TNBS) compared to lean mice with (LFD-TNBS) or without (LFD-C) colitis
784
and obese mice without colitis (HFD-C) while those of (B) the macrophage marker EMR1
785
decrease.
786
787
Figure 4. High fat diet-induced obesity affects cytokine expression in mouse intestine during
788
TNBS colitis. Real time PCR revealed that obese mice (HFD-TNBS) demonstrated increased (A)
789
IL-1β, (B) KC, (C) IL-6, and (F) IL-10 mRNA levels in the intestine 48hrs after the induction of
790
TNBS colitis compared to lean mice with (LFD-TNBS) or without (LFD-C) colitis and obese
791
mice without colitis (HFD-C). Contrary to the aforementioned data showing dramatic up-
792
regulation of cytokines, mRNA expression of (D) IFNγ and (E) IL-2 decreased significantly in
793
obese mice 48hrs after the induction of TNBS colitis (HFD-TNBS) compared to lean mice with
794
(LFD-TNBS) or without (LFD-C) colitis and obese mice without colitis (HFD-C) in which group
795
the mRNA levels of both cytokines increase. *p<0.05, **p<0.01, ***p<0.001 for LFD-TNBS vs.
796
HFD-TNBS; ##p<0.01, ###p<0.001 for LFD-C vs. HFD-C.
797
798
Figure 5. Obesity exacerbates inflammatory cell infiltrate within mesenteric fat depots of mice
799
with TNBS-induced colitis. H & E stained histological sections showed that, after 48 hrs, TNBS
800
colitis induced severe infiltration of inflammatory cells in mesenteric fat depots of (D) obese
801
mice compared to (B) lean mice with (LFD-TNBS) or (A) without (LFD-C) colitis and (C) obese
802
mice without colitis (HFD-C)
803
804
Figure 6. Total RNA was isolated from mesenteric fat depots and subjected to real time PCR
805
analysis. The (A) protein levels of MPO and (B) the mRNA levels of Proteinase 3 neutrophil
806
markers increased in the mesenteric adipose tissue of obese mice with colitis (HFD-TNBS)
807
compared to their non-obese counterparts (LFD-TNBS) or obese (HFD-C) and lean (LFD-C)
808
mice without colitis. (C) While EMR1 mRNA levels increase with obesity alone (HFD-C) they
809
are not affected by colitis either in lean (LFD-TNBS) or obese (HFD-TNBS) mice.
810
811
Figure 7. High fat diet-induced obesity increases proinflammatory cytokine expression in mouse
812
mesenteric adipose tissue during TNBS colitis. Total RNA was isolated from mouse mesenteric
813
fat depots and real time PCR showed that obese mice (HFD-TNBS) demonstrated increased (A)
814
IL-1β, (B) IL-6, (C) MCP-1, (D) TNFα, (E) KC, and (G) mRNA levels in the mesenteric fat
815
depot 48hrs after the induction of TNBS colitis compared to lean mice with (LFD-TNBS) or
816
without (LFD-C) colitis and obese mice without colitis (HFD-C). However, (F) IFNγ mRNA
817
levels decreased significantly in obese mice 48hrs after the induction of TNBS colitis (HFD-
818
TNBS) compared to lean mice with (LFD-TNBS) or without (LFD-C) colitis and obese mice
819
without colitis (HFD-C). *p<0.05, **p<0.01 for LFD-TNBS vs. HFD-TNBS; #p<0.05 for LFD-C
820
vs. HFD-C.
821
822
Figure 8. High fat diet-induced obesity lowers mRNA expression of adiponectin in the fat and of
823
adiponectin receptors 1 and 2 in the intestine during TNBS colitis in mice. We performed real
824
time PCR on adipose tissue and intestine total RNA and observed (A) increased levels
825
adiponectin mRNA in adipose tissue of lean mice with colitis (LFD-TNBS) compared to lean
826
controls (LFD-C) and significantly decreased levels in obese mice 48hrs after the induction of
827
TNBS colitis (HFD-TNBS) compared to lean mice with (LFD-TNBS) or without (LFD-C) colitis
828
and obese mice without colitis (HFD-C). (B) AdipoqR1 and (C) R2 mRNA levels decrease
829
significantly in the adipose tissue of obese mice 48hrs after the induction of TNBS colitis (HFD-
830
TNBS) only compared to lean mice with colitis (LFD-TNBS). (D) AdipoqR1 and (E) R2
831
decreased significantly in the intestine of obese mice 48hrs after the induction of TNBS colitis
832
(HFD-TNBS) compared to lean mice with (LFD-TNBS) or without (LFD-C) colitis and obese
833
mice without colitis (HFD-C). # p<0.05 vs LFD-C; *p<0.05, **p<0.01, ***p<0.001
834
835
Figure 9. Intracolonic AdipoR1 knock down exacerbates colitis in mice. (A) Schematic
836
representation of AdipoR1 knockdown followed by induction of TNBS colitis in mice. Mice that
837
received intracolonic injections of anti-AdipoR1 duplexes showed (B) increased weight loss, (C)
838
increased macroscopic damage, as well as elevated (D) colitis severity, (E) mucosal damage, and
839
(F) crypt formation compared to mice treated with scrambled control nucleotides. *p<0.05,
840
**p<0.01 for scrambled-TNBS vs. sham; #p<0.05 for scrambled-TNBS vs. AdipoR1-TNBS.
841
842
Figure 10. Conditioned media isolated from control, obese and IBD patient (n=6-9) preadipocyte
843
cultures induce differential mRNA expression responses of AdipoR1 in human colonic epithelial
844
NCM460 cells. (A) Human colonic epithelial NCM460 cells were exposed to conditioned media
845
from mesenteric fat depots of control UC and CD patients and AdipoR1 mRNA expression
846
decreased significantly in comparison to controls. (B) NCM460 colonocytes were treated with
847
conditioned media from preadipocytes of control obese and IBD patients for 24hrs and total
848
RNA was collected. Real time PCR revealed that AdipoR1 mRNA levels are significantly
849
decreased in human NCM460 colonocytes after exposure to media from UC patient
850
preadipocytes while there is also a strong trend towards decrease when media from CD and
851
obese patient preadipocytes are employed. (C) Conditioned media from IBD preadipocytes
852
decrease phosphor IGF-1R levels while (D) conditioned media from fat tissues from IBD
853
patients decrease PPARγ mRNA levels in NCM460 cells compared to media from control
854
patients. (E) Treatment of NCM460 colonocytes with IGF-1 increased AdipoR1 mRNA levels.
855
*p<0.05, **p<0.01
856
857
Figure 11. AdipoR1 levels increase in human colonic biopsies during IBD and adiponectin
858
reduces cytokine mRNA expression in human NCM460 colonocytes. (A) Immunohistochemistry
859
of human colonic sections show elevated levels of AdipoR1 during IBD compared to non-IBD
860
controls. Human colonic epithelial NCM460 cells were exposed to 10ng/ml adiponectin for 24
861
hrs in the medium and the mRNA levels were determined using a multiplex assay (42-plex). (B-
862
G) IL-2, IL-5, IL-2, IL-17, IL-23, and TGFβ2 mRNA levels were significantly reduced in
863
adiponectin-treated NCM460 colonocytes compared to untreated controls while (H) the mRNA
864
levels of VEGFA were significantly increased. *p<0.05, **p<0.01.
865
866
Figure 12. Mesenteric fat depots removed from UC and CD patients demonstrate differential
867
mediator release from each other and from depots isolated from healthy controls (n=8-14).
868
Multiplex analysis of an 11 human adipokine-containing panel revealed that (A, D, F) mesenteric
869
fat isolated from UC and CD patients secrete higher levels of adiponectin, IL-1β, and TNFα,
870
respectively, compared to controls. (B) Mesenteric fat depots from UC patients secrete higher
871
levels of leptin and (E) those of CD patients secrete higher levels of IL-8 compared to controls.
872
(G) Mesenteric fat depots from CD patients secrete lower levels of resistin compared to controls.
873
*p<0.05, **p<0.01, #p<0.1
874
Figure 1
LF
D
-C
S
B
N
-T
D
F
L
H
FD
D
LF
FD
-C
-T
N
-C
B
S
FD
0
B
H
5
S
10
-T
N
B
**
LF
D
100
50
Arbitrary mRNA Units
Proteinase 3
H
-T
N
B
S
A
1000
500
H
FD
-C
Arbitrary mRNA Units
Figure 3
EMR1
40
30
20
10
*
0
D
LF
LF
D
N
-T
0
H
FD
-C
10
FD
E
30
20
**
0
##
D
LF
F
IL-2
-C
LF
D
N
-T
B
S
FD
-C
B
S
FD
-T
N
H
S
-C
-T
N
B
LF
D
###
Arbitrary mRNA Units
300
200
H
-C
S
B
S
FD
-T
N
LF
D
H
H
-T
N
B
*
-C
*
6000
4000
2000
FD
20
C
H
40
IL-6
Arbitrary mRNA Units
40
B
S
80
-T
N
IFNJ
-C
##
100
80
60
40
20
0
FD
S
N
FD
H
-T
LF
D
B
H
D
FD
S
B
S
60
-C
-T
N
B
0
-C
###
LF
D
20
-C
30
D
*
LF
40
Arbitrary mRNA Units
50
Arbitrary mRNA Units
S
600
400
200
LF
D
B
FD
BS
-T
N
B
IL-1ß
H
-C
H
N
D
-C
-T
LF
D
10
FD
LF
Arbitrary mRNA Units
A
H
Arbitrary mRNA Units
Figure 4
KC
1500
1200
400
200
**
30
20
10
0
IL-10
20
***
15
10
5
0
H
FD
N
-T
BS
Figure 5
-C
D
LF
C
0
40
H
FD
-T
N
B
S
200
H
FD
-T
N
B
S
950
900
400
350
300
250
200
H
FD
-C
400
H
FD
-C
MPO
LF
D
-T
N
B
S
**
Arbitrary mRNA Units
Proteinase 3
LF
D
-T
N
B
S
LF
D
-C
Pg/ml
A
LF
D
-C
600
H
FD
-T
N
B
S
H
FD
-C
B
LF
D
-T
N
B
S
Arbitrary mRNA Units
Figure 6
**
100
150
50
0
EMR1
60
**
20
0
80
**
60
#
20
0
IFNJ
5
-C
D
LF
S
B
N
-T
D
F
H
15
10
*
0
LF
D
-C
H
FD
-T
N
B
S
300
2
0
-C
D
LF
G
100
E
IL-10
S
B
N
-T
D
LF
S
B
N
-T
D
LF
1500
KC
1250
1000
500
**
30
20
10
#
0
150
-C
FD
H
S
B
N
-T
FD
H
**
100
50
0
H
FD
-T
N
B
S
4
H
FD
-C
6
Arbitrary mRNA Units
C
H
FD
-T
N
B
S
TNFD
**
H
FD
-C
40
IL-6
LF
D
-T
N
B
S
100
S
B
N
-T
D
LF
Arbitrary mRNA Units
0
H
FD
-C
10
400
300
200
100
LF
D
-C
F
B
Arbitrary mRNA Units
-C
D
LF
LF
D
-C
#
Arbitrary mRNA Units
**
H
FD
-T
N
B
S
D
H
FD
-C
20
H
FD
-C
30
H
FD
-T
N
B
S
H
FD
-C
400
300
200
100
40
LF
D
-T
N
B
S
Arbitrary mRNA Units
IL-1ß
LF
D
-T
N
B
S
Arbitrary mRNA Units
-C
D
LF
LF
D
-T
N
B
S
Arbitrary mRNA Units
Figure 7
A
MCP-1
200
150
**
#
50
0
LF
0.5
0.0
H
FD
N
-T
BS
LF
D
E
-C
D
HF
Intestine
AdipoR2
40
30
20
10
***
0
-C
D-
TN
BS
-C
BS
80
FD
TN
C
C
H
D-
D-
0
HF
LF
LF
1
Arbitrary mRNA Units
2
BS
**
BS
*
N
1.0
TN
#
-T
1.5
D-
4
FD
2.0
C
AdipoR1
H
6
BS
5
2.5
TN
TN
AdipoR1
HF
3
D-
L
FD
BS
D-
0
HF
Arbitrary mRNA Units
B
LF
D
C
BS
***
D-
N
20
LF
-T
-C
AdipoQ
Arbitrary mRNA Units
D
FD
40
C
BS
HF
H
#
D-
TN
C
BS
80
HF
D-
D-
TN
-C
60
LF
D-
D
A
Arbitrary mRNA Units
LF
LF
Arbitrary mRNA Units
Figure 8
Adipose
AdipoR2
#
60
40
**
20
0