INFLAMMATORY BOWEL DISEASE

Production of adiponectin, an anti-inflammatory protein, in mesenteric adipose tissue in Crohn’s disease

K Yamamoto¹, T Kiyohara¹, Y Murayama¹, S Kihara¹, Y Okamoto¹, T Funahashi¹, T Ito², R Nezu³, S Tsutsui¹, J-I Miyagawa¹, S Tamura¹, Y Matsuzawa⁴, I Shimomura¹, Y Shinomura¹

¹ Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Osaka, Japan
² Department of Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan
³ Department of Surgery, Osaka Rosai Hospital, Osaka, Japan
⁴ Department of Internal Medicine, Sumitomo Hospital, Osaka, Japan

Correspondence to:
Correspondence to:
Dr T Kiyohara
Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, 2-2 B-5, Yamadaoka, Suita, 565-0871, Japan; kiyohara{at}imed2.med.osaka-u.ac.jp

Revised version received 28 October 2004

Accepted 1 November 2004

ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Background and aims: A characteristic feature of Crohn’s disease (CD) is mesenteric adipose tissue hypertrophy. Mesenteric adipocytes or specific proteins secreted by them may play a role in the pathogenesis of CD. We recently identified adiponectin as an adipocyte specific protein with anti-inflammatory properties. Here we report on expression of adiponectin in mesenteric adipose tissue of CD patients.

Methods and results: Mesenteric adipose tissue specimens were obtained from patients with CD (n = 22), ulcerative colitis (UC) (n = 8) and, for controls, colon carcinoma patients (n = 28) who underwent intestinal resection. Adiponectin concentrations were determined by enzyme linked immunosorbent assay, and adiponectin mRNA levels were determined by real time quantitative reverse transcription-polymerase chain reaction. Tissue concentrations and release of adiponectin were significantly increased in hypertrophied mesenteric adipose tissue of CD patients compared with normal mesenteric adipose tissue of CD patients (p = 0.002, p = 0.040, respectively), UC patients (p = 0.002, p = 0.003), and controls (p<0.0001, p<0.0001). Adiponectin mRNA levels were significantly higher in hypertrophied mesenteric adipose tissue of CD patients than in paired normal mesenteric adipose tissue from the same subjects (p = 0.024). Adiponectin concentrations in hypertrophied mesenteric adipose tissue of CD patients with an internal fistula were significantly lower than those of CD patients without an internal fistula (p = 0.003).

Conclusions: Our results suggest that adipocytes in hypertrophied mesenteric adipose tissue produce and secrete significant amounts of adiponectin, which could be involved in the regulation of intestinal inflammation associated with CD.

Abbreviations: CD, Crohn’s disease; UC, ulcerative colitis; TNF-, tumour necrosis factor-; IL, interleukin; BMI, body mass index; ELISA, enzyme linked immunosorbent assay; RT-PCR, reverse transcription-polymerase chain reaction; cDNA, complementary DNA; CRP, C reactive protein; PPAR, peroxisome proliferator activated receptor

Keywords: Crohn’s disease; fat wrapping; mesenteric adipocyte; adiponectin

Crohn’s disease (CD) is a chronic inflammatory bowel disease of the gastrointestinal tract. As its aetiology is still unknown, CD is identified and diagnosed by the appearance of a set of clinical, endoscopic, histological, and macroscopic characteristics. Transmural inflammation is the histological hallmark of CD. Occasionally, inflammation extends beyond the intestinal wall, and an internal fistula or an intra-abdominal abscess is formed. The macroscopic features of the mesentery adjacent to the intestinal segments involved in CD often include hypertrophied adipose tissue such as fat wrapping and mesenteric thickening.^1–5 Fat wrapping, defined as hypertrophied adipose tissue extending from the mesenteric attachment and partially covering the intestinal circumference, is common in both the small and large intestine and is also considered a hallmark of CD.¹ While considerable variation exists in the presentation of the disease in surgical specimens, CD is often recognised solely by the appearance of these macroscopic lesions of the mesentery.⁴ Their aetiology and significance, however, remain unknown.

While it is well known that adipose tissue serves as an energy reservoir, recent advances in cell biology have shown that adipocytes produce and secrete several bioactive molecules which are collectively referred to as adipocytokines. We previously identified an adipocyte specific secretory protein, adiponectin, the gene transcript of which was most abundant in the expression profiles of human adipose tissue.⁶ Adiponectin is composed of 244 amino acid residues containing a short non-collagenous N terminal segment followed by a collagen-like sequence. It belongs to the family of proteins that include C1q and the collectins,^6,7 which play important roles in the innate humoral immune system.^8–10 We and others have shown that adiponectin modulates a wide array of biological functions. Adiponectin was demonstrated to be related to improved insulin resistance and prevention of atherosclerosis, fatty liver, and liver fibrosis.^11–19 In addition to the above actions, we reported that adiponectin has several anti-inflammatory effects. For example, adiponectin reduces monocyte attachment to cultured human aortic endothelial cells by inhibiting expression of vascular cell adhesion molecules, intercellular adhesion molecules, and E-selectin.²⁰ Adiponectin inhibits the phagocytic activity and production of tumour necrosis factor (TNF)- and interleukin (IL)-6 in cultured macrophages.^7,21 These actions may be mediated by suppression of nuclear factor B signalling and extracellular signal regulated kinase 1/2 activity.^21,22 Adiponectin knockout mice display high levels of TNF- mRNA in adipose tissue and high concentrations in plasma, which decrease by supplementation of plasma adiponectin.¹² Furthermore, we recently reported that adiponectin enhances the production of IL-10 and tissue inhibitor of metalloproteinase 1 in human cultured macrophages.²³ Thus adiponectin exhibits anti-inflammatory properties, especially in endothelial cells and macrophages.

To our knowledge, the role of adiponectin in the pathogenesis of CD has not been examined previously. The present study was designed to determine expression levels of adiponectin in hypertrophied mesenteric adipose tissue, and its relevance to the pathogenesis of CD.

MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The study protocol was approved by the Human Ethics Review Committee of Osaka University School of Medicine and a signed consent form was obtained from each subject. Mesenteric adipose tissue specimens were obtained from 22 patients with CD, eight with ulcerative colitis (UC), and 28 with colon cancer who underwent surgical resection. CD patients (16 men, six women; mean age 34.4 (1.8) years (range 24–56); mean body mass index (BMI) 18.8 (0.4) kg/m²) underwent ileal or colonic resection because of stenosis or an internal fistula. Three patients with CD were treated by steroids. The other patients were not on any specific medical treatment at the time of the study. Eleven patients with CD had pure ileal involvement, 10 had ileocolonic CD, and one had colonic CD. Ten patients with CD had an internal fistula. UC patients (five men, three women; mean age 49.4 (5.2) years (range 32–69); mean BMI 21.4 (0.8) kg/m²) underwent intestinal resection because of steroid dependency or lack of response to medication. Patients with colon cancer (19 men, nine women; mean age 67.3 (1.3) years (range 53–75); mean BMI 22.4 (0.6) kg/m²) were enrolled as the control group. All specimens were taken from mesenteric adipose tissue close to the intestinal wall. In 16 of 22 CD patients, paired samples were obtained from hypertrophied mesenteric adipose tissue adjacent to the involved intestine and apparently normal mesenteric adipose tissue contiguous with the healthy segment of the intestine. Resected specimens were immediately frozen in liquid nitrogen and stored –80°C until assayed. Some samples were fixed in 10% phosphate buffered formalin and embedded in paraffin for histopathological studies.

Haematoxylin and eosin staining was routinely performed. For quantitation of the size and number of adipocytes, sectional areas of adipose tissue were analysed with an image analysis system (Macscope version 2.59; Mitani, Fukui, Japan). Tissue samples were immunohistologically stained for CD3, CD20, and CD68 using the labelled streptavidin-biotin system (Dako, Glostrup, Denmark). Mouse monoclonal antihuman CD68, CD3, and CD20 antibodies were purchased from Nichirei (Tokyo, Japan). For immunohistological staining of adiponectin, paraffin sections were deparaffinised, dehydrated, blocked with normal goat serum (Vector Laboratories, Burlingame, California, USA) for 10 minutes at room temperature, and then incubated at 4°C overnight with 5 µg/ml of a mouse monoclonal antihuman adiponectin antibody, ANOC 9104.²² Sections were then rinsed with 0.05 M Tris HCl buffer (pH 7.6), and incubated for 30 minutes with a 1:200 dilution of Alexa Fluor 594 goat antimouse IgG (H+L) (Molecular Probes, Eugene, Oregon, USA) at room temperature. As a negative control, the primary antibody was replaced by normal mouse serum (Dako). Nuclear counterstaining was carried out with a 1:5000 dilution of 4', 6-diamidino-2-phenylindole (Molecular Probes) for five minutes at room temperature.

Mesenteric adipose tissue samples were homogenised in lysis buffer containing 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 10 mM ethylenediaminetetra acetic acid, 100 mM NaF, 1 mM phenylmethylsulfonylfluoride, 0.25 TIU/ml aprotinin, and 10 µg/ml leupeptin. Homogenates were incubated for 10 minutes at 4°C and then centrifuged at 16 000 g for 15 minutes at 4°C. The supernatants were collected and stored at –30°C until assayed. Adiponectin concentrations in homogenate supernatants of adipose tissues were determined by enzyme linked immunosorbent assay (ELISA), as described previously.²⁴ IL-6 concentrations were measured using ELISA kits (BioSource International, Camarillo, California, USA). Total protein concentration was then determined using BCA Protein Assay Reagent (Pierce, Rockford, Illinois, USA) according to the manufacturer’s instructions. The final concentrations were expressed in ng of adiponectin and pg of IL-6 per mg of total protein.

Short term cultures were prepared with surgical specimens to examine release of adiponectin from the mesenteric adipose tissue. Adipose tissue samples (250 mg, wet weight) were diced finely and incubated in 2 ml of culture medium at 37°C in a 95% O₂–5% CO₂ humidified incubator for 24 hours, as described previously.²⁵ Conditioned media were collected and stored at –30°C until used for assay. Adiponectin concentrations in conditioned media were then determined by ELISA.

Total RNA was extracted and reverse transcribed into complementary DNA (cDNA) as described previously.²⁴ Real time quantitative polymerase chain reaction (PCR) was carried out on cDNA as described previously.¹⁴ The sequences of the primers and the probe for adiponectin were as follows: adiponectin sense primer, 5'-AGG TTG GAT GGC GGG C-3'; adiponectin antisense primer, 5'-TTT CAC CGA TGT CTC CCT TAG G-3'; adiponectin probe, 5'-(FAM)-TGG CAG AGA TGG CAC CCC TGG-(TAMRA)-3'. The primers and probe for ß-actin were purchased from Applied Biosystems (Foster City, California, USA). The quantity of adiponectin was expressed as the ratio of adiponectin cDNA molecules to ß-actin cDNA molecules.

Data are expressed as mean (SEM). Differences among the four groups were studied using the Kruskal-Wallis test for global comparisons and the Mann-Whitney test for paired comparisons. Paired and unpaired t tests were used to compare data between two groups when they showed normal distribution and the Mann-Whitney test when they were asymmetrical. Spearman’s test was applied to test for correlations. A p value <0.05 denoted the presence of a statistically significant difference.

RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Histopathological examination showed a large number of inflammatory cells in hypertrophied mesenteric adipose tissue adjacent to the involved intestine of CD patients (fig 1B). In contrast, very few inflammatory cells were detected in normal mesenteric adipose tissue adjacent to the healthy intestine of CD patients, the intestines of UC patients, and the intestines of control subjects (fig 1A). Immunohistochemical staining using mouse monoclonal antibodies to CD68, CD3, and CD20 showed many CD68 positive (specific for monocytes/macrophages), CD3 positive (specific for T cells), and CD20 positive (specific for B cells) cells near the serosa in hypertrophied mesenteric adipose tissue of CD patients (fig 1C–E). The number of inflammatory cells decreased with increased distance from the serosa. The number of CD20 positive cells appeared to be less than other inflammatory cells. Quantitative analyses indicated that the size of adipocytes in hypertrophied mesenteric adipose tissue of CD patients (956 (21) µm²) was approximately one quarter the size of those in mesenteric adipose tissue from controls (4099 (174) µm²) (fig 1B, A). On the other hand, the number of adipocytes per unit area in hypertrophied mesenteric adipose tissue of CD patients was 3.5-fold that of adipocytes in the mesenteric adipose tissue from controls (603 v 173 per 900 000 µm²).

View larger version (108K): [in this window] [in a new window]   Figure 1  Haematoxylin-eosin (H&E) staining and immunohistochemical detection of CD68, CD3, and CD20 in hypertrophied mesenteric adipose tissue contiguous with involved intestinal segments of a representative patient with Crohn’s disease (CD). Mesenteric adipose tissue in a normal control subject (A) and hypertrophied mesenteric adipose tissue of a CD patient (B) were stained with H&E. Note that the size of adipocytes in hypertrophied mesenteric adipose tissue is approximately one quarter those of controls (A, B). CD68, CD3, and CD20 expressing cells were stained dark brown (C–E). Many CD68, CD3, and CD20 positive cells were present near the serosa in hypertrophied mesenteric adipose tissue. CD68 positive cells formed a non-caseous epithelioid cell granuloma (C). Nuclear counterstaining: Mayer’s haematoxylin. Original magnification x200.

 

Adiponectin immunoreactivity appeared as a red fluorescence rim at the periphery of the cytoplasm of adipocytes in the mesenteric adipose tissue of controls and CD patients (fig 2B, D). In addition, sections incubated with normal mouse serum instead of mouse antihuman adiponectin antibody (fig 2A, C) or mouse antihuman adiponectin antibody preabsorbed with sufficient adiponectin showed no positive staining reaction (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 2  Immunohistochemical staining of adiponectin in the mesenteric adipose tissue of a representative control and Crohn’s disease (CD) patient. The mesenteric adipose tissue of the control (B) and the hypertrophied mesenteric adipose tissue of a CD patient (D) reacted with antiadiponectin antibody. No reaction product was detected against non-immune serum in the mesenteric adipose tissue of the control (A) and hypertrophied mesenteric adipose tissue of the CD patient (C). Adiponectin staining appeared as a red fluorescence rim in the periphery of the cytoplasm of adipocytes (B, D). Nuclear counterstaining: 4', 6-diamidino-2-phenylindole. Original magnification: x200.

 

The mean adiponectin concentration in hypertrophied mesenteric adipose tissue of CD patients (404.7 (31.9) ng/mg) was significantly higher than that in normal mesenteric adipose tissue of controls (202.5 (17.7) ng/mg, p<0.0001), UC patients (176.1 (37.9) ng/mg, p = 0.002), or CD patients (249.3 (29.1) ng/mg, p = 0.002) (fig 3A). Moreover, adiponectin concentrations in hypertrophied mesenteric adipose tissues (407.4 (37.5) ng/mg) were also significantly higher than those in paired normal mesenteric adipose tissues (249.3 (29.1) ng/mg) from the same CD patients (p<0.001) (fig 3B).

View larger version (21K): [in this window] [in a new window]   Figure 3  Adiponectin concentrations in mesenteric adipose tissue. Adiponectin concentrations were determined by enzyme linked immunosorbent assay (ELISA) and values expressed in ng of adiponectin per mg of total homogenate protein. (A) Data are individual and mean values (horizontal bar) of adiponectin concentrations in normal mesenteric adipose tissues of controls (C), ulcerative colitis patients (UC), Crohn’s disease patients (CD-n), and hypertrophied mesenteric adipose tissues of CD patients (CD-ht). (B) Data are individual and mean values (horizontal bars) of adiponectin concentrations in normal mesenteric adipose tissues of CD patients (CD-n) and hypertrophied mesenteric adipose tissues of CD patients (CD-ht) from the same patients.

 

The amount of adiponectin released from hypertrophied mesenteric adipose tissue of CD patients (287.9 (36.0) ng/ml) was significantly higher than that from normal mesenteric adipose tissue of controls (76.0 (8.9) ng/ml, p<0.0001), UC patients (63.4 (19.0) ng/ml, p = 0.003), or CD patients (170.2 (31.9) ng/ml, p = 0.040) (fig 4). Furthermore, adiponectin release from normal mesenteric adipose tissue of CD patients was significantly higher than that from the mesenteric adipose tissue of control subjects (p = 0.030) or UC patients (p = 0.039) (fig 4).

View larger version (13K): [in this window] [in a new window]   Figure 4  Adiponectin release from the mesenteric adipose tissue in short term culture. Release of adiponectin from adipose tissue was determined by enzyme linked immunosorbent assay and values expressed in ng of adiponectin per ml of conditioned media. Data are individual and mean values (horizontal bars) of adiponectin release from normal mesenteric adipose tissues of controls (C), ulcerative colitis patients (UC), Crohn’s disease patients (CD-n), and hypertrophied mesenteric adipose tissues of CD patients (CD-ht).

 

Quantitative real time reverse transcription (RT)-PCR showed a significantly higher expression level of adiponectin mRNA in hypertrophied mesenteric adipose tissue of CD patients (15.1x10^–2 (2.1x10^–2) cDNA molecules) compared with that in paired normal mesenteric adipose tissue (8.9x10^–2 (1.1x10^–2) cDNA molecules) (p = 0.024) (fig 5).

View larger version (16K): [in this window] [in a new window]   Figure 5  Quantitative real time reverse transcription-polymerase chain reaction analysis of adiponectin mRNA expression in mesenteric adipose tissue. Data are individual and mean values (horizontal bars) of adiponectin cDNA molecules per molecule of ß-actin cDNA in normal mesenteric adipose tissues of Crohn’s disease patients (CD-n) and hypertrophied mesenteric adipose tissues of CD patients (CD-ht) from the same patients.

 

We next evaluated the relationship between adiponectin concentrations in hypertrophied mesenteric adipose tissue and laboratory data measured just prior to surgery in CD patients. There was no significant correlation between adiponectin concentrations in hypertrophied mesenteric adipose tissues and leucocyte count, haemoglobin level, or platelet count in CD patients (r = –0.13, p = 0.558; r = –0.15, p = 0.507; r = –0.18, p = 0.434, respectively) (fig 6A–C). On the other hand, adiponectin concentrations in hypertrophied mesenteric adipose tissues of CD patients correlated inversely with serum C reactive protein (CRP) levels (r = –0.51, p = 0.015) (fig 6D). There was no significant correlation between tissue adiponectin concentrations and serum CRP levels in control subjects or UC patients (r = –0.32, p = 0.097 and r = –0.37, p = 0.369, respectively).

View larger version (24K): [in this window] [in a new window]   Figure 6  Correlation between adiponectin concentrations in hypertrophied mesenteric adipose tissue of Crohn’s disease patients and leucocyte count (A), haemoglobin level (B), platelet count (C), and serum C reactive protein (CRP) levels (D). These laboratory data were determined just prior to surgery.

 

Based on the above finding, we evaluated the relationship between adiponectin and the proinflammatory cytokine IL-6, a major inducer of CRP. The concentration of IL-6 in hypertrophied mesenteric adipose tissues of CD patients (90.3 (19.1) pg/mg) tended to be higher than that in the mesenteric adipose tissues of control subjects (54.4 (7.6) pg/mg) but the difference was not statistically significant (p = 0.063) (fig 7A). In hypertrophied mesenteric adipose tissue of CD patients, IL-6 concentrations correlated inversely with adiponectin concentrations (r = –0.43, p = 0.045) (fig 7B).

View larger version (18K): [in this window] [in a new window]   Figure 7  (A) Interleukin 6 (IL-6) concentrations in the mesenteric adipose tissue of controls (C) and hypertrophied mesenteric adipose tissue of Crohn’s disease patients (CD-ht). IL-6 concentrations were determined by enzyme linked immunosorbent assay and values expressed in pg of IL-6 per mg of total homogenate protein. (B) Correlation between adiponectin and IL-6 concentrations in hypertrophied mesenteric adipose tissue of CD patients.

 

To investigate the association of adiponectin production in mesenteric adipose tissue with the development of an internal fistula, we examined adiponectin concentrations in the mesenteric adipose tissues of CD patients with an internal fistula (eight men, two women; mean age 32.1 (1.9) years (range 24–43)) and without an internal fistula (eight men, four women; mean age 36.3 (2.9) years (range 25–56)). These two groups were matched for age, BMI (18.4 (0.8) v 19.0 (0.4) kg/m²), and disease duration (5.8 (1.7) v 7.5 (1.4) years). Adiponectin concentrations in hypertrophied mesenteric adipose tissues of CD patients with a fistula (312.4 (37.3) ng/mg) were significantly lower than those without a fistula (481.6 (34.2) ng/mg, p = 0.003) (fig 8). Furthermore, adiponectin concentrations in normal mesenteric adipose tissues of CD patients with a fistula (234.5 (28.8) ng/mg) also tended to be lower than those without a fistula (264.0 (52.4) ng/mg) although the difference was not statistically significant (p = 0.629).

View larger version (13K): [in this window] [in a new window]   Figure 8  Adiponectin concentrations in hypertrophied mesenteric adipose tissue of Crohn’s disease (CD) patients with or without internal fistula. Data are individual and mean values (horizontal bars) of adiponectin concentrations in hypertrophied mesenteric adipose tissues of CD patients without an internal fistula (CD-ht fistula (–)) and those of CD patients with an internal fistula (CD-ht fistula (+)).

 

Finally, we evaluated the correlation between BMI and adiponectin concentrations or its release. Adiponectin concentrations and the amount released in all specimens correlated inversely with BMI (r = –0.40, p<0.001 and r = –0.52, p<0.001, respectively) (fig 9A, B). However, there was no significant correlation between BMI and adiponectin concentration or its release in each subgroup; normal mesenteric adipose tissue of controls (r = –0.32, p = 0.099 and r = –0.31, p = 0.328, respectively), UC patients (r = –0.62, p = 0.102 and r = –0.25, p = 0.682, respectively), and CD patients (r = –0.13, p = 0.619 and r = –0.38, p = 0.318, respectively) and hypertrophied mesenteric adipose tissue of CD patients (r = –0.02, p = 0.934 and r = –0.25, p = 0.378, respectively).

View larger version (15K): [in this window] [in a new window]   Figure 9  Relationship between body mass index and tissue concentrations (A) or release (B) of adiponectin in controls, ulcerative colitis patients (UC), Crohn’s disease patients (CD-n), and hypertrophied mesenteric adipose tissue of CD patients (CD-ht).

 

DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The presence of hypertrophied mesenteric adipose tissue is one of the characteristic features of CD.¹ This change is important in terms of surgery for CD, and its recognition is crucial in determining the extent of resection and, from another point of view, in differentiating CD from other bowel diseases.⁴ Although the mesenteric fat hypertrophy in CD has long been recognised, its significance remains unknown. Adiponectin, an adipocyte specific secretory protein, was recently shown to play a role in the modulation of a wide array of biological functions. Recent studies have shown that adiponectin has anti-inflammatory effects, especially in endothelial cells and macrophages.^7,20–24 Thus its secretion by mesenteric adipocytes could negatively regulate some of the inflammatory reactions observed in CD. The present study demonstrated for the first time that adiponectin production is enhanced in hypertrophied mesenteric adipose tissue contiguous with the involved intestine of CD patients.

Only a few histopathological studies of the mesenteric adipose tissue in CD have been reported. Fibrosis, perivascular inflammation, and intimal and medial thickening of vessels have been observed in hypertrophied mesenteric adipose tissue adjacent to the involved intestine of CD patients.^26,27 In the present study, two additional histopathological features of hypertrophied mesenteric adipose tissue of CD patients were defined: significant infiltration of inflammatory cells and reduced size of adipocytes. Infiltrated cells were mainly CD68 positive and CD3 positive T lymphocytes. Massive infiltration of CD68 positive and CD3 positive cells suggests that the inflamed mucosa and its adjacent mesentery share a common inflammation in CD. These cells may disturb mesenteric functions, including blood and lymphatic flow, and hence contribute to mucosal damage. They may also interact with mesenteric adipocytes and induce hyperplasia. The morphological change in adipocytes is more interesting. As small adipocytes are capable of producing larger amounts of adiponectin compared with large adipocytes,²⁸ the reduced size of adipocytes in hypertrophied mesenteric adipose tissue of CD patients is consistent with the increase in adiponectin production reported herein. Desreumaux and colleagues²⁹ found significantly high expression levels of peroxisome proliferator activated receptor (PPAR) mRNA in hypertrophied mesenteric adipose tissue of CD patients compared with normal mesenteric adipose tissue of CD patients and control subjects. PPAR is a member of the nuclear hormone receptor superfamily, which is predominantly expressed in adipocytes.^30,31 PPAR is a key regulator of adipogenesis. As activation of PPAR leads to an increase in small adipocytes and upregulates their expression of adiponectin,^28,32–34 our results may be relevant to overexpression of PPAR. Infiltrated cells are a possible source of PPAR ligands such as 15-deoxy-^12–14-prostaglandin J₂.^35–37

A negative relationship between BMI and plasma adiponectin levels has been reported.³⁸ In the present study, adiponectin concentrations and its release in all specimens of mesenteric adipose tissues correlated inversely with BMI. The high adiponectin concentration and release in CD patients may be partly explained by the lower BMI compared with controls and UC patients. However, in the paired samples of CD patients, adiponectin concentrations and mRNA levels in hypertrophied mesenteric adipose tissues were significantly higher than in normal mesenteric adipose tissues. Furthermore, adiponectin concentration and release in hypertrophied mesenteric adipose tissue of CD patients were shifted upwards in the scattered plot diagrams with BMI compared with the other subgroups (fig 9A, B). These results suggest that increased adiponectin production in hypertrophied mesenteric adipose tissue of CD patients is not only attributed to the lower BMI but also to other factors, including PPAR ligands, as discussed above.

In the present study, adiponectin concentrations in hypertrophied mesenteric adipose tissue of CD patients correlated negatively with serum CRP levels. However, this inverse correlation was not significant in UC patients and controls. While the exact link between tissue adiponectin concentration and serum CRP level is not clear at present, this difference may be relevant to the characteristic involvement of mesenteric adipose tissue in the pathogenesis of CD. On the other hand, our study also demonstrated a negative correlation between adiponectin and IL-6 production in hypertrophied mesenteric adipose tissue of CD patients. This finding suggests that adiponectin inhibits inflammatory cytokine production in mesenteric adipose tissue. As IL-6 is a major inducer of CRP,^39,40 it may be the missing link between adiponectin and CRP.

In CD, internal fistulas develop almost exclusively at the mesenteric border. Our results showed significantly low adiponectin concentrations in hypertrophied mesenteric adipose tissues of CD patients with an internal fistula compared with those free of this complication. Such a decrease in adiponectin production may worsen the inflammation and increase the risk of internal fistula formation. As anti-TNF- therapy (infliximab) is effective in the treatment of internal fistulas in CD, this result may have relevance to the anti-TNF- effects of adiponectin.^7,12,21 The exact mechanisms that cause reduction of adiponectin concentrations in hypertrophied mesenteric adipose tissue of CD patients with a fistula remain elusive and further studies are needed to identify these mechanisms.

Adipocytes produce several adipocytokines, including proinflammatory cytokines. It has been reported that TNF- mRNA levels in hypertrophied mesenteric adipose tissue of CD patients tended to be higher than in normal mesenteric adipose tissue of CD patients but the difference between these locations was not statistically significant.²⁹ While mesenteric adipocytes may produce both TNF- and adiponectin, our results suggest that the latter is a significant adipocytokine derived from mesenteric adipocytes stimulated by transmural inflammation as a result of CD. Barbier and colleagues⁴¹ recently reported high mRNA levels of leptin in the mesenteric adipose tissue of patients with CD and UC. However, leptin levels in hypertrophied mesenteric adipose tissue of CD patients were lower than those in normal mesenteric adipose tissue of CD patients, and similar to those in the mesenteric adipose tissue of UC patients.⁴¹ Considered together, it is conceivable that the mechanisms that regulate leptin production are different to those of adiponectin and TNF-.

In conclusion, we have demonstrated in the present study that adipocytes in hypertrophied mesenteric adipose tissue of CD patients produce and secrete significant amounts of adiponectin. Mesenteric adipocytes may act, not only as energy storage cells, but also as immunoregulating cells in the case of intestinal inflammation via adiponectin production. The hypertrophied mesenteric adipose tissue of CD patients could serve as a barrier that prevents the spread of inflammation into the intra-abdominal space. Dysregulation of adiponectin production in mesenteric adipose tissue may play a role in the pathogenesis of intestinal disorders, including CD.

ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Sachiyo Tanaka and Fumie Katsube.

FOOTNOTES

Conflict of interest: None declared.

REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1. Sheehan AL, Warren BF, Gear MW, et al. Fat-wrapping in Crohn’s disease: pathological basis and relevance to surgical practice. Br J Surg 1992;79:955–8.[Medline]
2. Smedh K, Olaison G, Nystrom PO, et al. Intraoperative enteroscopy in Crohn’s disease. Br J Surg 1993;80:897–900.[Medline]
3. Weakley FL, Turnbull RB. Recognition of regional ileitis in the operating room. Dis Colon Rectum 1971;14:17–23.[CrossRef][Medline]
4. Fazio VW, Jones IT. Standard surgical treatment of Crohn’s disease of the small intestine and ileocolitis. In: Lee ECG, Nolan DJ, eds. Surgery of inflammatory bowel disorders. Edinburgh: Churchill Livingstone, 1987:147–56.
5. Borley NR, Mortensen NJ, Jewell DP, et al. The relationship between inflammatory and serosal connective tissue changes in ileal Crohn’s disease: evidence for a possible causative link. J Pathol 2000;190:196–202.[CrossRef][Medline]
6. Maeda K, Okubo K, Shimomura I, et al. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (Adipose Most abundant Gene transcript 1). Biochem Biophys Res Commun 1996;221:286–9.[CrossRef][Medline]
7. Yokota T, Oritani K, Takahashi I, et al. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood 2000;96:1723–32.[Abstract/Free Full Text]
8. Holmskov U, Malhotra R, Sim RB, et al. Collectins: collagenous C-type lectins of the innate immune defense system. Immunol Today 1994;15:67–74.[CrossRef][Medline]
9. Epstein J, Eichbaum Q, Sheriff S, et al. The collectins in innate immunity. Curr Opin Immunol 1996;8:29–35.[CrossRef][Medline]
10. Lu J . Collectins: collectors of microorganisms for the innate immune system. Bioessays 1997;19:509–18.[CrossRef][Medline]
11. Yamauchi T, Kamon J, Waki H, et al. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 2001;7:941–6.[CrossRef][Medline]
12. Maeda N, Shimomura I, Kishida K, et al. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nat Med 2002;8:731–7.[CrossRef][Medline]
13. Weyer C, Funahashi T, Tanaka S, et al. Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab 2001;86:1930–5.[Abstract/Free Full Text]
14. Okamoto Y, Kihara S, Ouchi N, et al. Adiponectin reduces atherosclerosis in apolipoprotein E-deficient mice. Circulation 2002;106:2767–70.[Abstract/Free Full Text]
15. Yamauchi T, Kamon J, Waki H, et al. Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis. J Biol Chem 2003;278:2461–8.[Abstract/Free Full Text]
16. Ouchi N, Kihara S, Arita Y, et al. Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages. Circulation 2001;103:1057–63.[Abstract/Free Full Text]
17. Arita Y, Kihara S, Ouchi N, et al. Adipocyte-derived plasma protein adiponectin acts as a platelet-derived growth factor-BB-binding protein and regulates growth factor-induced common postreceptor signal in vascular smooth muscle cell. Circulation 2002;105:2893–8.[Abstract/Free Full Text]
18. Xu A, Wang Y, Keshaw H, et al. The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice. J Clin Invest 2003;112:91–100.[CrossRef][Medline]
19. Kamada Y, Tamura S, Kiso S, et al. Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin. Gastroenterology 2003;125:1796–807.[CrossRef][Medline]
20. Ouchi N, Kihara S, Arita Y, et al. Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin. Circulation 1999;100:2473–6.[Abstract/Free Full Text]
21. Wulster-Radcliffe MC, Ajuwon KM, Wang J, et al. Adiponectin differentially regulates cytokines in porcine macrophages. Biochem Biophys Res Commun 2004;316:924–9.[CrossRef][Medline]
22. Ouchi N, Kihara S, Arita Y, et al. Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-kappaB signaling through a cAMP-dependent pathway. Circulation 2000;102:1296–301.[Abstract/Free Full Text]
23. Kumada M, Kihara S, Ouchi N, et al. Adiponectin specifically increased tissue inhibitor of metalloproteinase-1 through interleukin-10 expression in human macrophages. Circulation 2004;109:2046–9.[Abstract/Free Full Text]
24. Ouchi N, Kihara S, Funahashi T, et al. Reciprocal association of C-reactive protein with adiponectin in blood stream and adipose tissue. Circulation 2003;107:671–4.[Abstract/Free Full Text]
25. Sewter CP, Digby JE, Blows F, et al. Regulation of tumour necrosis factor-alpha release from human adipose tissue in vitro. J Endocrinol 1999;163:33–8.[Abstract]
26. Herlinger H, Furth EE, Rubesin SE. Fibrofatty proliferation of the mesentery in Crohn disease. Abdom Imaging 1998;23:446–8.[CrossRef][Medline]
27. Knutson H, Lunderquist A, Lunderquist A. Vascular changes in Crohn’s disease. Am J Roentgenol Radium Ther Nucl Med 1968;103:380–5.[Medline]
28. Yamauchi T, Kamon J, Waki H, et al. The mechanisms by which both heterozygous peroxisome proliferator-activated receptor gamma (PPARgamma) deficiency and PPARgamma agonist improve insulin resistance. J Biol Chem 2001;276:41245–54.[Abstract/Free Full Text]
29. Desreumaux P, Ernst O, Geboes K, et al. Inflammatory alterations in mesenteric adipose tissue in Crohn’s disease. Gastroenterology 1999;117:73–81.[CrossRef][Medline]
30. Brun RP, Kim JB, Hu E, et al. Peroxisome proliferator-activated receptor gamma and the control of adipogenesis. Curr Opin Lipidol 1997;8:212–18.[Medline]
31. Spiegelman BM, Flier JS. Adipogenesis and obesity: rounding out the big picture. Cell 1996;87:377–89.[CrossRef][Medline]
32. Okuno A, Tamemoto H, Tobe K, et al. Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats. J Clin Invest 1998;101:1354–61.[Medline]
33. Maeda N, Takahashi M, Funahashi T, et al. PPARgamma ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein. Diabetes 2001;50:2094–9.[Abstract/Free Full Text]
34. Combs TP, Wagner JA, Berger J, et al. Induction of adipocyte complement-related protein of 30 kilodaltons by PPARgamma agonists: a potential mechanism of insulin sensitization. Endocrinology 2002;143:998–1007.[Abstract/Free Full Text]
35. Shibata T, Kondo M, Osawa T, et al. 15-deoxy-delta 12, 14-prostaglandin J2. A prostaglandin D2 metabolite generated during inflammatory processes. J Biol Chem 2002;277:10459–66.[Abstract/Free Full Text]
36. Forman BM, Tontonoz P, Chen J, et al. 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell 1995;83:803–12.[CrossRef][Medline]
37. Kliewer SA, Lenhard JM, Willson TM, et al. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation. Cell 1995;83:813–19.[CrossRef][Medline]
38. Arita Y, Kihara S, Ouchi N, et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 1999;257:79–83.[CrossRef][Medline]
39. Heinrich PC, Castell JV, Andus T. Interleukin-6 and the acute phase response. Biochem J 1990;265:621–36.[Medline]
40. Bataille R, Klein B. C-reactive protein levels as a direct indicator of interleukin-6 levels in humans in vivo. Arthritis Rheum 1992;35:982–4.[Medline]
41. Barbier M, Vidal H, Desreumaux P, et al. Overexpression of leptin mRNA in mesenteric adipose tissue in inflammatory bowel diseases. Gastroenterol Clin Biol 2003;27:987–91.[Medline]

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

Relevant Article

Creeping fat in Crohn’s disease: travelling in a creeper lane of research?

A Schäffler and H Herfarth
Gut 2005 54: 742-744. [Extract] [Full Text] [PDF]

This article has been cited by other articles:

• Hyland, N. P., Chambers, A. P., Keenan, C. M., Pittman, Q. J., Sharkey, K. A. (2009). 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 [Abstract] [Full Text]  
• Schaffler, A., Scholmerich, J. (2009). The role of adiponectin in inflammatory gastrointestinal diseases. Gut 58: 317-322 [Full Text]  
• Pini, M., Gove, M. E., Fayad, R., Cabay, R. J., Fantuzzi, G. (2009). Adiponectin deficiency does not affect development and progression of spontaneous colitis in IL-10 knockout mice. Am. J. Physiol. Gastrointest. Liver Physiol. 296: G382-G387 [Abstract] [Full Text]  
• Aprahamian, T., Bonegio, R. G., Richez, C., Yasuda, K., Chiang, L.-K., Sato, K., Walsh, K., Rifkin, I. R. (2009). The Peroxisome Proliferator-Activated Receptor {gamma} Agonist Rosiglitazone Ameliorates Murine Lupus by Induction of Adiponectin. J. Immunol. 182: 340-346 [Abstract] [Full Text]  
• Drew, J. E., Farquharson, A. J., Padidar, S., Duthie, G. G., Mercer, J. G., Arthur, J. R., Morrice, P. C., Barrera, L. N. (2007). Insulin, leptin, and adiponectin receptors in colon: regulation relative to differing body adiposity independent of diet and in response to dimethylhydrazine. Am. J. Physiol. Gastrointest. Liver Physiol. 293: G682-G691 [Abstract] [Full Text]  
• Peyrin-Biroulet, L., Chamaillard, M., Gonzalez, F., Beclin, E., Decourcelle, C., Antunes, L., Gay, J., Neut, C., Colombel, J.-F., Desreumaux, P. (2007). Mesenteric fat in Crohn's disease: a pathogenetic hallmark or an innocent bystander?. Gut 56: 577-583 [Full Text]  
• Colombel, J F, Solem, C A, Sandborn, W J, Booya, F, Loftus, E V Jr, Harmsen, W S, Zinsmeister, A R, Bodily, K D, Fletcher, J G (2006). Quantitative measurement and visual assessment of ileal Crohn's disease activity by computed tomography enterography: correlation with endoscopic severity and C reactive protein. Gut 55: 1561-1567 [Abstract] [Full Text]  
• Thoeni, R. F., Cello, J. P. (2006). CT Imaging of Colitis. Radiology 240: 623-638 [Abstract] [Full Text]  
• Schaffler, A., Muller-Ladner, U., Scholmerich, J., Buchler, C. (2006). Role of Adipose Tissue as an Inflammatory Organ in Human Diseases. Endocr. Rev. 27: 449-467 [Abstract] [Full Text]  
• Martin, L. J, Woo, J. G, Geraghty, S. R, Altaye, M., Davidson, B. S, Banach, W., Dolan, L. M, Ruiz-Palacios, G. M, Morrow, A. L (2006). Adiponectin is present in human milk and is associated with maternal factors. Am. J. Clin. Nutr. 83: 1106-1111 [Abstract] [Full Text]  
• Schaffler, A, Herfarth, H (2005). Creeping fat in Crohn's disease: travelling in a creeper lane of research?. Gut 54: 742-744 [Full Text]