Volume 131, Issue 3, Pages 788-796 (September 2006) Increased Adipose Tissue Expression of Hepcidin in Severe Obesity Is Independent From Diabetes and NASH  Soumeya Bekri, Philippe Gual, Rodolphe Anty, Nathalie Luciani, Monsef Dahman, Bala Ramesh, Antonio Iannelli, Aline Staccini–Myx, Dominique Casanova, Imed Ben Amor, Marie–Christine Saint–Paul, Pierre–Michel Huet, Jean–Louis Sadoul, Jean Gugenheim, Surjit Kaila S. Srai, Albert Tran, Yannick Le Marchand–Brustel  Gastroenterology  Volume 131, Issue 3, Pages 788-796 (September 2006) DOI: 10.1053/j.gastro.2006.07.007 Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 1 Hepcidin expression in adipose tissue of obese patients. (A) Adipose tissue and liver biopsy specimens from 8 obese, 7 obese diabetic, and 10 obese patients with NASH were used to prepare total RNA according to experimental procedures. SCAT mRNA from 9 lean patients and 6 commercial mRNA from “normal” livers were used as controls. Levels of HAMP mRNA were measured by real-time PCR, normalized to RPLP0 mRNA using the “2−ΔΔCt” formula, and expressed in arbitrary units (mean ± SEM), with the control values taken as 1. *Denotes a statistical difference (P < .05) in obese groups compared with lean subjects, using the nonparametric Kruskal-Wallis test. (B) Five micrograms of the 2 hepcidin peptides (1, native urinary hepcidin; 2, synthetic hepcidin used to raise antibodies) were blotted with antihepcidin antibodies. Typical autoradiograph is shown. (C) Hepcidin protein is present in both liver and adipose tissue. Visceral adipose tissue sections (a–c) and frozen liver sections with steatosis (d–f) and without steatosis (g–i) were subjected to immunohistochemical analysis as described in the Materials and Methods section. Sections were incubated with PBS 1% BSA alone (left panels), with the rabbit antihepcidin antibody (middle panels), or with the antibody preincubated overnight at 4°C with the immunizing peptide (c and f) or anti-CD68 antibody (i). Immunohistochemical signals appear as brown staining, and nuclei are counterstained blue using Mayer Hemalum (original magnification, ×400). Arrows point to hepcidin signal; A, adipocytes; NAC, nonadipocyte cells. Gastroenterology 2006 131, 788-796DOI: (10.1053/j.gastro.2006.07.007) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 2 Hepcidin expression in subcutaneous adipose tissue is IL-6 related. Levels of HAMP, CRP, and IL-6 mRNA were measured by real-time PCR and normalized to RPLP0 mRNA (ΔCt) in 34 patients (9 lean controls and 25 obese patients). Positive correlations were found using the Spearman rank correlation test between (A) HAMP expression and the BMI of the patients (to better represent the correlation between HAMP expression and the BMI, −ΔCt was chosen [−ΔCt values are proportional to HAMP expression level]), (B) HAMP and CRP expressions, and (C) HAMP and IL-6 expressions. Open circles refer to controls, solid triangles to obese patients, solid diamonds to diabetic patients, and open diamonds to NASH patients. (D) HAMP expression is induced by LPS and IL-6 in adipose tissue explants. Adipose tissue explants from 3 patients were incubated with LPS (24 hours), IL-6 + its soluble receptor (sIL-6R), or TNF-α (48 hours). Adipose tissue fragments were frozen, and total RNA were extracted. The mRNA levels of HAMP and IL-6 were normalized to RPLP0 mRNA using the “2−ΔΔCt” formula and expressed as arbitrary units (mean ± SEM), with the control value obtained in the absence of cytokine taken as 1. *Denotes a statistical difference (P < .05) compared with control values, using the Student t test for paired data. Gastroenterology 2006 131, 788-796DOI: (10.1053/j.gastro.2006.07.007) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 3 Hepcidin expression is correlated to transferrin saturation in liver but not in adipose tissue in obese subjects. Levels of HAMP mRNA in liver (A) and SCAT (B) of 25 obese patients were measured by real-time PCR and normalized to RPLP0 mRNA (ΔCt HAMP = Ct HAMP − Ct RPLP0). Thus, an elevated ΔCt HAMP is associated to a low HAMP expression level in the sample. To represent better the correlation between HAMP expression and transferrin saturation, −ΔCt was chosen (−ΔCt values are proportional to HAMP expression level). Correlation analyses were determined using the Spearman rank correlation test. Solid triangles refer to obese patients, solid diamonds to diabetic patients, and open diamonds to NASH patients. Gastroenterology 2006 131, 788-796DOI: (10.1053/j.gastro.2006.07.007) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions