Volume 132, Issue 2, Pages 601-614 (February 2007) Adiponectin Deficiency Protects Mice From Chemically Induced Colonic Inflammation  Raja Fayad, Maria Pini, Joseph A. Sennello, Robert J. Cabay, Lawrence Chan, Aimin Xu, Giamila Fantuzzi  Gastroenterology  Volume 132, Issue 2, Pages 601-614 (February 2007) DOI: 10.1053/j.gastro.2006.11.026 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Response of WT and APN KO mice to DSS-induced colitis. (A) Clinical scores of WT, APN KO, and APN KO mice receiving recombinant APN treated with DSS for 5 days and killed on day 10 (P < .001 for APN KO vs WT or KO + APN for each time point starting on day 3). (B) Histologic scores of WT, APN KO, and APN KO mice receiving recombinant APN treated with DSS for 5 days and killed on day 10 (P < .001 for APN KO vs WT or KO + APN). (C) Representative H&E staining of colonic mucosa and immunohistochemistry staining for BrdU in the colon. (D) Levels of MIP2 and (E) IL-6 in colon culture supernatants of WT, APN KO, and APN KO mice that had received recombinant APN (P < .005 for APN KO vs WT or KO + APN for both IL-6 and MIP2). Data are mean ± SEM. N = 7 mice per group. Gastroenterology 2007 132, 601-614DOI: (10.1053/j.gastro.2006.11.026) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Colonic protein and gene expression. (A) RT-PCR data of mRNA expression for APN, ADIPOR1, ADIPOR2, HB-EGF, bFGF, and GAPDH in the colon of control WT (lanes 1 and 2), DSS-treated WT (lanes 3–5), control APN KO (lanes 6 and 7), DSS-treated APN KO (lanes 8 and 9) mice, as well as in adipose tissue of control WT mice (lane 10). (B) Quantitative RT-PCR for APN in adipose and colonic tissue. (C) Adiponectin levels in colon culture supernatants of WT, APN KO, and APN KO mice receiving recombinant APN. (D) Representative immunohistochemistry for APN in colonic mucosa. Gastroenterology 2007 132, 601-614DOI: (10.1053/j.gastro.2006.11.026) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Induction of proinflammatory cytokines by APN in the colon. Levels of IL-6 and MIP2 in supernatants of colon cultures obtained from control and DSS-treated WT mice and cultured overnight in the absence or presence of APN at 10 μg/mL. P < .001 for WT mice treated with DSS and in vitro APN vs WT mice treated with DSS alone for both IL-6 and MIP2. Data are mean ± SEM. N = 3 mice per group. Gastroenterology 2007 132, 601-614DOI: (10.1053/j.gastro.2006.11.026) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Association of APN with bFGF and HB-EGF in mouse serum. Adiponectin was purified from albumin- and IgG-depleted mouse serum using an anti-mouse APN IgG as an affinity ligand. The upper panel shows a CBB staining of the eluted fractions. The middle and lower panels show Western blot analyses of the concentrated samples using an anti-bFGF or anti-HB-EGF antibody. The 35-kilodalton band in the upper panel was confirmed to be APN by both Western blot and peptide fingerprinting (data not shown). Gastroenterology 2007 132, 601-614DOI: (10.1053/j.gastro.2006.11.026) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 HB-EGF expression in colonic mucosa. Representative immunohistochemistry of colonic tissue obtained from control and DSS-treated mice stained for HB-EGF. Gastroenterology 2007 132, 601-614DOI: (10.1053/j.gastro.2006.11.026) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Effect of EGF-R inhibition on DSS-induced colitis. WT and KO mice were treated with DSS for 5 days and received daily IP injection of Tyrphostin or vehicle until killed on day 10. (A). Clinical score (P < .001 for KO + vehicle vs each of the other groups starting on day 5). (B) Histologic score on day 10 (P < .001 for KO DSS + vehicle vs each of the other groups). (C) IL-6 levels in colon culture supernatant (P < .02 for KO vs WT mice, irrespective of treatment). (D) MIP2 levels in colon culture supernatant (P < .03 for KO DSS + vehicle vs each of the other groups). (E) Representative BrdU immunohistochemistry of colonic tissue. Data are mean ± SEM. N = 7 mice per group. Gastroenterology 2007 132, 601-614DOI: (10.1053/j.gastro.2006.11.026) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 A proposed model for the activity of APN in modulating colonic inflammation. In the colon, APN acts as a proinflammatory molecule, possibly by binding its receptor and inducing mucosal production of proinflammatory cytokines. Moreover, APN associated with bFGF and HB-EGF, thus preventing them from binding their receptors. This results in the inability of bFGF to maintain epithelial integrity in the colon in response to inflammatory stimuli and in prevention of HB-EGF from down-regulating adhesion molecules, inhibiting NF-κB activation, and inducing proliferation of colonic epithelial cells. Gastroenterology 2007 132, 601-614DOI: (10.1053/j.gastro.2006.11.026) Copyright © 2007 AGA Institute Terms and Conditions