Increased Adipose Tissue Expression of Hepcidin in Severe Obesity Is Independent From Diabetes and NASH
2006; Elsevier BV; Volume: 131; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2006.07.007
ISSN1528-0012
AutoresSoumeya Bekri, Philippe Gual, Rodolphe Anty, Nathalie Luciani, Monsef Dahman, Balasubramaniam Ramesh, Antonio Iannelli, A. Staccini‐Myx, Dominique Casanova, Imed Ben Amor, Marie‐Christine Saint‐Paul, Pierre–Michel Huet, J L Sadoul, Jean Gugenheim, Surjit Kaila Srai, Albert Tran, Y. Le Marchand‐Brustel,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoBackgrounds & Aims: Hepcidin is an acute-phase response peptide. We have investigated the possible involvement of hepcidin in massive obesity, a state of chronic low-grade inflammation. Three groups of severely obese patients with or without diabetes or nonalcoholic steatohepatitis were investigated. Methods: Hepcidin expression was studied in liver and adipose tissue of these patients. Hepcidin regulation was investigated in vitro by adipose tissue explant stimulation studies. Results: Hepcidin was expressed not only in the liver but also at the messenger RNA (mRNA) and the protein levels in adipose tissue. Moreover, mRNA expression was increased in adipose tissue of obese patients. The presence of diabetes or NASH did not modify the hepcidin expression levels in liver and adipose tissue. In adipose tissue, mRNA expression correlated with indexes of inflammation, interleukin-6, and C-reactive protein. Interleukin-6 also promoted in vitro hepcidin expression. A low transferrin saturation ratio was observed in 68% of the obese patients; moreover, 24% of these patients presented with anemia. The observed changes in iron status could be due to the role of hepcidin as a negative regulator of intestinal iron absorption and macrophage iron efflux. Interestingly, a feedback control mechanism on hepcidin expression related to low transferrin saturation occurred in the liver but not in the adipose tissue. Conclusions: Hepcidin is a proinflammatory adipokine and may play an important role in hypoferremia of inflammation in obese condition. Backgrounds & Aims: Hepcidin is an acute-phase response peptide. We have investigated the possible involvement of hepcidin in massive obesity, a state of chronic low-grade inflammation. Three groups of severely obese patients with or without diabetes or nonalcoholic steatohepatitis were investigated. Methods: Hepcidin expression was studied in liver and adipose tissue of these patients. Hepcidin regulation was investigated in vitro by adipose tissue explant stimulation studies. Results: Hepcidin was expressed not only in the liver but also at the messenger RNA (mRNA) and the protein levels in adipose tissue. Moreover, mRNA expression was increased in adipose tissue of obese patients. The presence of diabetes or NASH did not modify the hepcidin expression levels in liver and adipose tissue. In adipose tissue, mRNA expression correlated with indexes of inflammation, interleukin-6, and C-reactive protein. Interleukin-6 also promoted in vitro hepcidin expression. A low transferrin saturation ratio was observed in 68% of the obese patients; moreover, 24% of these patients presented with anemia. The observed changes in iron status could be due to the role of hepcidin as a negative regulator of intestinal iron absorption and macrophage iron efflux. Interestingly, a feedback control mechanism on hepcidin expression related to low transferrin saturation occurred in the liver but not in the adipose tissue. Conclusions: Hepcidin is a proinflammatory adipokine and may play an important role in hypoferremia of inflammation in obese condition. CME Quiz on page 946. CME Quiz on page 946. Obesity is a pathologic condition tightly linked to increased mortality and associated with a severe chronic morbidity from such diverse causes as systemic arterial hypertension, diabetes, and liver pathology, particularly nonalcoholic steatohepatitis (NASH). These conditions are associated with a state of chronic low-grade systemic inflammation evidenced by increased plasma concentrations of acute-phase proteins and cytokines such as C-reactive protein (CRP),1Yudkin J.S. Stehouwer C.D. Emeis J.J. Coppack S.W. 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Plasminogen activator inhibitor 1, transforming growth factor-β1, and BMI are closely associated in human adipose tissue during morbid obesity.Diabetes. 2000; 49: 1374-1380Crossref PubMed Scopus (302) Google Scholar, 5Lundgren C.H. Brown S.L. Nordt T.K. Sobel B.E. Fujii S. Elaboration of type-1 plasminogen activator inhibitor from adipocytes A potential pathogenetic link between obesity and cardiovascular disease.Circulation. 1996; 93: 106-110Crossref PubMed Scopus (211) Google Scholar It is now recognized that adipose tissue (AT) contributes to the production of proinflammatory molecules, also named adipokines, and that the increase in their production participates in the metabolic syndrome (for review, see Trayhurn and Wood6Trayhurn P. Wood I.S. Adipokines: inflammation and the pleiotropic role of white adipose tissue.Br J Nutr. 2004; 92: 347-355Crossref PubMed Scopus (1714) Google Scholar). Numerous recent studies have linked systemic inflammation to obesity-related insulin resistance.7Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr, A.W. Obesity is associated with macrophage accumulation in adipose tissue.J Clin Invest. 2003; 112: 1796-1808Crossref PubMed Scopus (7430) Google Scholar, 8Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance.J Clin Invest. 2003; 112: 1821-1830Crossref PubMed Scopus (5170) Google Scholar, 9Grimble R.F. Inflammatory status and insulin resistance.Curr Opin Clin Nutr Metab Care. 2002; 5: 551-559Crossref PubMed Scopus (339) Google Scholar Hepcidin, a key regulator of iron homeostasis, is also known to be increased in generalized inflammatory disorders. Hepcidin is a small antimicrobial peptide,10Park C.H. Valore E.V. Waring A.J. Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver.J Biol Chem. 2001; 276: 7806-7810Crossref PubMed Scopus (1747) Google Scholar which inhibits iron absorption by enterocytes, iron release from macrophages, and iron transport across the placenta.11Fleming R.E. Sly W.S. Hepcidin: a putative iron-regulatory hormone relevant to hereditary hemochromatosis and the anemia of chronic disease.Proc Natl Acad Sci U S A. 2001; 98: 8160-8162Crossref PubMed Scopus (260) Google Scholar The role of hepcidin was very recently shown to be related to its regulation of the iron transporter, ferroportin.12Knutson M.D. Oukka M. Koss L.M. Aydemir F. Wessling-Resnick M. Iron release from macrophages after erythrophagocytosis is up-regulated by ferroportin 1 overexpression and down-regulated by hepcidin.Proc Natl Acad Sci U S A. 2005; 102: 1324-1328Crossref PubMed Scopus (381) Google Scholar, 13Nemeth E. Tuttle M.S. Powelson J. Vaughn M.B. Donovan A. Ward D.M. Ganz T. Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3604) Google Scholar, 14Yeh K.Y. Yeh M. Glass J. Hepcidin regulation of ferroportin 1 expression in the liver and intestine of the rat.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G385-G394Crossref PubMed Scopus (95) Google Scholar An effect on another iron transporter, divalent metal ion transporter-1 (DMT1) was also proposed.15Laftah A.H. Ramesh B. Simpson R.J. Solanky N. Bahram S. Schumann K. Debnam E.S. Srai S.K. Effect of hepcidin on intestinal iron absorption in mice.Blood. 2004; 103: 3940-3944Crossref PubMed Scopus (185) Google Scholar, 16Yamaji S. Sharp P. Ramesh B. Srai S.K. Inhibition of iron transport across human intestinal epithelial cells by hepcidin.Blood. 2004; 104: 2178-2180Crossref PubMed Scopus (120) Google Scholar Hepatic hepcidin production is modulated by 3 major pathways: iron homeostasis, hypoxia, and inflammatory stimuli.17Pigeon C. Ilyin G. Courselaud B. Leroyer P. Turlin B. Brissot P. Loreal O. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J Biol Chem. 2001; 276: 7811-7819Crossref PubMed Scopus (1415) Google Scholar, 18Nicolas G. Chauvet C. Viatte L. Danan J.L. Bigard X. Devaux I. Beaumont C. Kahn A. Vaulont S. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.J Clin Invest. 2002; 110: 1037-1044Crossref PubMed Scopus (1346) Google Scholar, 19Nemeth E. Valore E.V. Territo M. Schiller G. Lichtenstein A. Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.Blood. 2003; 101: 2461-2463Crossref PubMed Scopus (1182) Google Scholar The increase in hepcidin expression in inflammatory diseases results in hypoferremia because of a combination of a decrease in duodenal iron absorption and an increase in iron sequestration in the macrophages. This hypoferremia is responsible for subsequent anemia of inflammation, a common clinical manifestation of chronic diseases.18Nicolas G. Chauvet C. Viatte L. Danan J.L. Bigard X. Devaux I. Beaumont C. Kahn A. Vaulont S. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.J Clin Invest. 2002; 110: 1037-1044Crossref PubMed Scopus (1346) Google Scholar, 19Nemeth E. Valore E.V. Territo M. Schiller G. Lichtenstein A. Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.Blood. 2003; 101: 2461-2463Crossref PubMed Scopus (1182) Google Scholar Acute inflammatory stimuli such as lipopolysaccharide (LPS) or turpentine have been shown to induce hepcidin expression in cultured cell lines and in a murine model mainly through the mediation of IL-6.20Lee P. Peng H. Gelbart T. Beutler E. The IL-6- and lipopolysaccharide-induced transcription of hepcidin in HFE-, transferrin receptor 2-, and β 2-microglobulin-deficient hepatocytes.Proc Natl Acad Sci U S A. 2004; 101: 9263-9265Crossref PubMed Scopus (185) Google Scholar, 21Nemeth E. Rivera S. Gabayan V. Keller C. Taudorf S. Pedersen B.K. Ganz T. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin.J Clin Invest. 2004; 113: 1271-1276Crossref PubMed Scopus (1987) Google Scholar, 22Roy C.N. Custodio A.O. de Graaf J. Schneider S. Akpan I. Montross L.K. Sanchez M. Gaudino A. Hentze M.W. Andrews N.C. Muckenthaler M.U. An Hfe-dependent pathway mediates hyposideremia in response to lipopolysaccharide-induced inflammation in mice.Nat Genet. 2004; 36: 481-485Crossref PubMed Scopus (103) Google Scholar Indeed, the increase in hepcidin expression by inflammatory stimuli in cultured hepatocytes is abolished in the presence of anti-IL-6 antibodies; furthermore, in turpentine-treated IL-6 knockout mice, hepcidin expression is suppressed, and no hypoferremia is observed. A complementary study was performed in human volunteers that demonstrated that IL-6 infusion induces an elevation of urinary hepcidin and hypoferremia.21Nemeth E. Rivera S. Gabayan V. Keller C. Taudorf S. Pedersen B.K. Ganz T. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin.J Clin Invest. 2004; 113: 1271-1276Crossref PubMed Scopus (1987) Google Scholar Furthermore, it was previously observed that IL-6 induces an anemia in cancer patients.23Nieken J. Mulder N.H. Buter J. Vellenga E. Limburg P.C. Piers D.A. de Vries E.G. Recombinant human interleukin-6 induces a rapid and reversible anemia in cancer patients.Blood. 1995; 86: 900-905Crossref PubMed Google Scholar Therefore, hepcidin is likely to mediate the anemia of inflammation (for review, see Means24Means Jr, R.T. Hepcidin and anaemia.Blood Rev. 2004; 18: 219-225Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The aim of this study was to investigate the effect of obesity as a low-grade inflammatory state on hepcidin expression, to characterize the adipose tissue and liver involvement in this expression in obese patient, and to assess the potential additive effect of 2 other inflammatory states: diabetes and NASH. The consequences of hepcidin expression on iron status of the patients were then investigated. This study involved 3 groups of obese patients: obese (n = 8), obese with diabetes (n = 7) and obese with NASH (n = 10). These patients were selected for bariatric surgery as a treatment for their obesity. Before surgery, blood samples were obtained. Patients’ clinical and biologic characteristics are presented in Table 1. The 3 groups were comparable for body mass index (BMI). The diabetic group had higher age, HbA1c, and glucose levels. Transaminase values were significantly elevated in the NASH group. These patients presented with no biologic features of hemochromatosis and no C282Y and H63D mutations on the HFE gene. During surgery, a surgical liver biopsy specimen and 2 biopsy specimens of adipose tissues (visceral adipose tissue [VAT] and subcutaneous adipose tissue [SCAT]) were collected. All tissues for molecular studies were immediately frozen in liquid nitrogen and stored at −80°C. Liver biopsy specimen analyses revealed that 1 patient presented with no steatosis, 14 patients had steatosis without inflammation, and 10 patients had steatohepatitis.25Brunt E.M. Grading and staging the histopathological lesions of chronic hepatitis: the Knodell histology activity index and beyond.Hepatology. 2000; 31: 241-246Crossref PubMed Scopus (405) Google Scholar Perls’ staining was performed on liver sections and scored from 0+ to 4+ in hepatocytes and Kupffer cells, as previously described.26Scheuer P. Lefkowitch J. Liver biopsy interpretation. Saunders, Philadelphia1994Google Scholar The experimental protocol was performed according to French legislation regarding Ethic and Human Research (Huriet-Serusclat law, DGS 2003/0395) and approved by the local ethical committee. Informed consent was obtained from all the patients involved in this study.Table 1Clinical Characteristics of the SubjectsCharacteristics (unit) [Normal range]Lean controls Median (95% confidence interval)Obese Median (95% confidence interval)Obese with diabetes Median (95% confidence interval)Obese with NASH Median (95% confidence interval)n98710Females/males9/07/15/25/5Age (yr)29 (25–38)37.5 (30.7–43.2)47 (39.3–48.8)34.5 (29.9–41.3)BMI (kg/m2)21 (20.5–21.5)46.2 (40.0–49.4)42.3 (37.8–49.6)44.8 (40.8–48.9)HbA1c (%) [<6.5]4.7 (4.6–5)5.2 (4.6–5.4)8.7 (6.6–10.4)5.4 (5.1–5.9)Blood glucose (mmol/L) [<5.8]4.8 (4.5–5)4.6 (4.4–6)12.6 (7.5–15.3)5.4 (4.4–6.1)Blood insulin (mIU/L) [3–15]7 (5–8)10.0 (5.0–10.2)16.0 (6.6–18.0)19.5 (12.9–24.7)ASAT (IU/L) [10–31]24 (21–29)20.0 (17.9–27.0)23.0 (17.1–27.7)30.0 (26.0–47.0)ALAT (IU/L) [10–34]14 (11–23)28.5 (15.8–39.1)24.0 (18.4–45.2)46.5 (39.0–82.5)CRP (mg/L) [<1]1 (1–3.3)8.7 (3.9–16.1)10.0 (7.2–13.6)9.0 (4.3–14.3)NOTE. All blood analyses were performed on morning fasting samples.BMI, Body mass index; HbA1c; Hemoglobin A1c; ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase; CRP, C-reactive protein. Open table in a new tab NOTE. All blood analyses were performed on morning fasting samples. BMI, Body mass index; HbA1c; Hemoglobin A1c; ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase; CRP, C-reactive protein. The subcutaneous adipose tissue from 9 lean females (Table 1) undergoing lipectomy for cosmetic purpose was studied as control adipose tissue. Total RNA from 6 liver samples were purchased from Stratagene (La Jolla, CA) (female, 34 years old, normal adjacent tissue to stromal sarcoma), Clontech (Mountain View, CA) (white male, 51 years old, sudden death), and Biochain (Hayward, CA) (4 males; 24, 26, 28, and 30 years old; sudden deaths). These tissues were confirmed to be histologically normal, without fatty liver disease or other abnormalities by the Stratagene and Biochain companies. No clinical and biologic data were available for these individuals; only samples with low levels of CRP mRNA were used, as indicative of the absence of inflammatory process.27Anty R. Bekri S. Luciani N. Saint-Paul M. Dahman M. Iannelli A. Ben Amor I. Staccini-Myx A. Huet P. Gugenheim J. Sadoul J. Le Marchand-Brustel Y. Tran A. Gual P. The inflammatory C-reactive protein is increased in both liver and adipose tissue in severely obese patients independently from metabolic syndrome, type 2 diabetes, and NASH.Am J Gastroenterol. 2006; (in press).PubMed Google Scholar Explants from abdominal subcutaneous adipose tissue were obtained from 7 women who were undergoing abdominoplasty (BMI 28 ± 3 kg/m2), and used either for the separation of adipose and nonadipose cells or for adipose tissue explant incubations as described. Total RNA from visceral and subcutaneous fat were prepared using the RNeasy total RNA kit (Qiagen, Valencia, CA). Total RNA from liver were prepared using the RNable total RNA extraction kit (Eurobio, Courtaboeuf, France). Complementary DNA (cDNA) was synthesized using the GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA) from 1 μg of total RNA in a final volume of 100 μL. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) was performed in duplicate with the ABI PRISM 7000 sequence Detection System and SYBRGreen dye (Applied Biosystems) according to the manufacturer’s protocol. Primers were designed using the Primer Express program (Applied Biosystems). The list of the primers is available upon request. Two reference genes were tested: the large P0 subunit of the acidic ribosomal phosphoprotein (RPLP0)28Gabrielsson B.G. Olofsson L.E. Sjogren A. Jernas M. Elander A. Lonn M. Rudemo M. Carlsson L.M. Evaluation of reference genes for studies of gene expression in human adipose tissue.Obes Res. 2005; 13: 649-652Crossref PubMed Scopus (95) Google Scholar and β 2 microglobulin (B2M). Because no significant difference was found in the cycle threshold (Ct) of RPLP0 for the same amount of mRNA in both tissues (see Table 2 and the Results section for comments), the mRNA levels of genes of interest (R) were expressed relative to levels of RPLP0, (ΔCt = CtR − CtRPLP0). The relative amount of R mRNA levels between 2 groups of patients is given by 2−ΔΔCt, where ΔΔCt = [ΔCt(R) of obese or diabetic patient] − [mean of ΔCt(R) of lean group]. Amplification of specific transcripts was confirmed by melting curve profiles generated at the end of the PCR program. Hepcidin (HAMP) and CRP amplicons were subcloned into pCR-Blunt vector using the zero Blunt PCR cloning kit (Invitrogen, Carlsbad, CA), and the specificity of these products was verified by DNA sequence analysis.Table 2Expression of Hepcidin (HAMP) and RPLP0 mRNA Levels in Livers, Visceral, and Subcutaneous Adipose Tissues of Severely Obese PatientsTissueRPLP0 (Ct)HAMP (Ct)Liver25.55 ± 0.1726.29 ± 0.30Visceral adipose tissue (VAT)24.65 ± 0.3231.30 ± 0.35Subcutaneous adipose tissue (SCAT)25.16 ± 0.4231.47 ± 0.20HAMP expression (ratios) of hepcidin expressionLiver/SCAT175 ± 51aLiver vs adipose tissues P < .05.Liver/VAT157 ± 47aLiver vs adipose tissues P < .05.VAT/SCAT1.81 ± 0.39bNS not significant for the visceral vs subcutaneous adipose tissue.HAMP, hepcidin gene.NOTE. Liver, subcutaneous, and visceral adipose tissue biopsy specimens were obtained from 25 obese patients. RPLP0 and HAMP mRNA expression levels were determined in the same amount of total RNA by real-time PCR and expressed as Ct (fluorescence cycle threshold) in the upper half of the Table or as ratios in the lower half. Values are expressed as the means of 25 values ± SEM. Statistical analyses were performed using the nonparametric Kruskal-Wallis test.a Liver vs adipose tissues P < .05.b NS not significant for the visceral vs subcutaneous adipose tissue.HAMP, hepcidin gene. Open table in a new tab NOTE. Liver, subcutaneous, and visceral adipose tissue biopsy specimens were obtained from 25 obese patients. RPLP0 and HAMP mRNA expression levels were determined in the same amount of total RNA by real-time PCR and expressed as Ct (fluorescence cycle threshold) in the upper half of the Table or as ratios in the lower half. Values are expressed as the means of 25 values ± SEM. Statistical analyses were performed using the nonparametric Kruskal-Wallis test. A peptide corresponding to the 25 amino acid residues of the mature human form of hepcidin was chemically synthesized. Female New Zealand White rabbits were immunized with the peptide coupled to small keyhole limpet hemocyanin. The antibody was affinity purified (SulfoLink Coupling Gel; Perbio Cramlington, United Kingdom) and assessed by Western blotting using urinary and synthetic hepcidin peptides as previously described.15Laftah A.H. Ramesh B. Simpson R.J. Solanky N. Bahram S. Schumann K. Debnam E.S. Srai S.K. Effect of hepcidin on intestinal iron absorption in mice.Blood. 2004; 103: 3940-3944Crossref PubMed Scopus (185) Google Scholar Frozen liver or visceral adipose tissue sections obtained from severely obese patients were fixed in pure acetone. Tissue sections were blocked with bovine serum albumin (BSA). Endogenous biotins were blocked using the Biotin Blocking System (DakoCytomaton, Glostrup, Denmark). To determine the appropriate concentration of antihepcidin antibodies, serial dilutions were tested for 1 hour of incubation. The working dilution of antihepcidin antibody is 1:30 in phosphate-buffered saline (PBS), 1% BSA (final volume 100 μL). Slides were incubated with the polyclonal rabbit antihepcidin antibody, for 60 minutes, at room temperature, then with an appropriate biotinylated secondary antibody from the StreptABComplex/HRP Duet Mouse/Rabbit kit (DakoCytomaton) at a dilution of 1:100 in PBS, 1% BSA, for 60 minutes, at room temperature. Next, slides were incubated with the StreptABComplex Duet Reagent Set (DakoCytomaton) at a dilution of 1:100 in PBS, for 20 minutes, at room temperature, then peroxidase activity was detected by 3 amino-9 ethyl carbazole in N, N dimethyl formamide (Biogenex, Lyon, CA). Nuclear staining was obtained with Mayer Hemalun staining medium (Biolyon, Lyon, France). To verify the specificity of the antibody, the antihepcidin antibodies were preincubated with 0.8 μg/μL of hepcidin peptide overnight at 4°C before use. The monoclonal anti-CD68 antibody was purchased from DakoCytomaton. Five explants from abdominal subcutaneous tissues were used. Tissue samples (1 g) were cut into small pieces and rinsed in a washing buffer containing 120 mmol/L NaCl, 4 mmol/L KH2PO4, 1 mmol/L MgSO4, 750 μmol/L CaCl2, 10 mmol/L NaHCO3, and 30 mmol/L HEPES, pH 7.4. The samples were incubated at 37°C for 30 minutes in 15 mL of the previous buffer supplemented with 1% BSA, 280 μmol/L glucose, and 15 mg of bacterial collagenase. Adipocytes were then collected by filtration and flotation. The stroma vascular fraction (SVF) was collected by centrifugation for 15 minutes at 260g. Adipocytes and SVF were then frozen at −80°C, and RNA extraction was done as described above. Cultured human omental preadipocytes were obtained from omental adipose tissue of nonobese women (Biopredic, Rennes, France) and differentiated in adipocytes as described by www.zen-bio.com. Total RNA was prepared from fully differentiated adipocytes. Human myelomonocytic cells (THP1) were cultured as described29Schmid-Alliana A. Menou L. Manie S. Schmid-Antomarchi H. Millet M.A. Giuriato S. Ferrua B. Rossi B. Microtubule integrity regulates src-like and extracellular signal-regulated kinase activities in human pro-monocytic cells Importance for interleukin-1 production.J Biol Chem. 1998; 273: 3394-3400Crossref PubMed Scopus (64) Google Scholar and stimulated for 24 hours with phorbol ester (50 ng/mL). Three abdominal SCAT samples were cut into small pieces (1 mm3) under sterile conditions and rinsed in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen). Explants (1 g) were incubated for 24 or 48 hours at 37°C under 7% CO2 in 5 mL DMEM; 100 μg/mL gentamycin; and 2% BSA with IL-6 (50 ng/mL), sIL-6R (100 ng/mL), tumor necrosis factor (TNF)-α (20 ng/mL), and LPS (10 μg/mL). Explants were then separated from the medium, frozen in liquid nitrogen, and stored at −80°C before RNA extraction. All chemical reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The fluorescence Ct was calculated to quantify the relative amount of gene expression. Statistical significance was determined using the nonparametric Kruskal-Wallis test with the ΔCt of the different groups of subjects. Correlations were analyzed using the Spearman rank correlation test with the ΔCt of different genes. P < .05 was considered as significant. The Student t test for paired data was used to analyze statistically the explant experiments, in which the mRNA levels obtained in basal and stimulated conditions were compared. HAMP expression was assessed in adipose tissue and liver in severely obese patients using RPLP0 as the reference gene. Indeed, RPLP0 expression was similar in liver and adipose tissues, allowing for a direct comparison of the level of expression of HAMP in these various tissues (Table 2). Unexpectedly, HAMP mRNA was detected in subcutaneous adipose tissue, and its expression was significantly enhanced in all obese conditions (Figure 1A). HAMP expression in SCAT was closely correlated with the BMI of the patients (Figure 2A). Hepatic HAMP mRNA levels in obese groups were not significantly different from those of control livers and appeared to be independent of the diabetic or NASH condition. It should be noted that HAMP mRNA expression was much higher in liver, its main site of production, than in SCAT and VAT; the level of HAMP expression did not significantly differ in both adipose tissue depots (Table 2).Figure 2Hepcidin 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.View Large Image Figure ViewerDownload (PPT) We then looked for hepcidin expression at the protein level using antihepcidin antibody. First, the specificity of our antibody was assessed by Western blotting against native and synthetic hepcidin peptides (Figure 1B). Immunohistochemical analysis of human adipose tissue showed the presence of hepcidin (Figure 1C, b) within both adipocytes and nonadipose cells. To determine the adipose tissue fraction involved in HAMP expression, SVF was separated from adipocytes. As shown in Table 3, HAMP mRNA was expressed by both fractions but at a significantly higher level in SVF. The validity of the cell separation was confirmed by the 21- ± 6-fold enrichment of the glucose transporter GLUT4 in the adipocyte fraction compared with SVF. Furthermore, HAMP mRNA expression was also found in human cultured adipocytes, and in THP1 monocytes stimulated by phorbol ester, and was thus considered as representative of macrophages (Table 3). As expected, this antibody was able to detect hepcidin on liver sections (Figure 1C, e and h). The hepcidin staining on liver section without steatosis (Figure 1C, h) along with the comparison with the macrophage staining (anti-CD68; Figure 1C, i) indicated that the signal was present in both hepatocytes and sinusoidal cells. The hepcidin staining appeared to be specific because the signal was almost completely inhibited when the hepcidin peptide was added to the antibody prior to immunostaining (Figures 1C, c and f).Table 3Expression of Hepcidin (HAMP) in Adipose Tissue Fractions From Nonmorbidly Obese Normal Women and in Various Cultured CellsTissue fractionHAMP expression (Δ Ct) Isolated adipocytes (5)10.97 ± 0.25 Stroma vascular fraction (SVF) (5)9.2 ± 0.6HAMP expression (ratio) Isolated adipocytes/SVF (5)0.38 ± 0.12aDifference in the HAMP expression between the 2 tissue fractions was statistically different with P = .035.Cultured cellsHAMP expression (Δ Ct) Cultured human adipocytes (6)13.1 ± 0.5 TPA stimulated THP1 (4)10.6 ± 0.5NOTE. Five explants from abdominal subcutaneous adipose tissue were obtai
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