ORIGINAL ARTICLE Fibrosis in Human Adipose Tissue: Composition, Distribution, and Link With Lipid Metabolism and Fat Mass Loss Adeline Divoux,1 Joan Tordjman,1 Danie`le Lacasa,2 Nicolas Veyrie,1,3 Danielle Hugol,1,4 Abdelhalim Aissat,1,3 Arnaud Basdevant,1,2 Miche`le Guerre-Millo,1 Christine Poitou,1,2 Jean-Daniel Zucker,1 Pierre Bedossa,1,5 and Karine Cle´ment1,2 OBJECTIVE—Fibrosis is a newly appreciated hallmark of the pathological alteration of human white adipose tissue (WAT). We investigated the composition of subcutaneous (scWAT) and hite adipose tissue (WAT) is the main energy omental WAT (oWAT) fibrosis in obesity and its relationship with repository in the body. It stores and mobi- metabolic alterations and surgery-induced weight loss. lizes, according to body demand, fatty acids RESEARCH DESIGN AND METHODS—Surgical biopsies for Wthat have been implicated in the development scWAT and oWAT were obtained in 65 obese (BMI 48.2 Ϯ 0.8 of insulin resistance. In turns, the phenotype and the kg/m2) and 9 lean subjects (BMI 22.8 Ϯ 0.7 kg/m2). Obese biology of WAT cellular components are altered by two subjects who were candidates for bariatric surgery were major processes: adipose cell hypertrophy and immune clinically characterized before, 3, 6, and 12 months after cells accumulation. Inflammation, reticulum endoplasmic surgery, including fat mass evaluation by dual energy X-ray stress, and hypoxia are part of the biologic alterations that absorptiometry. WAT fibrosis was quantified and character- ized using quantitative PCR, microscopic observation, and attract and retain inflammatory cells in WAT (1). Both immunohistochemistry. adipocytes and nonadipose cells of the stromal vascular fraction of WAT have been implicated in the secretion of RESULTS—Fibrosis amount, distribution and collagen types (I, inflammatory molecules and in the development of insulin III, and VI) present distinct characteristics in lean and obese resistance (2,3). WAT extracellular matrix (ECM) remod- subjects and with WAT depots localization (subcutaneous or omental). Obese subjects had more total fibrosis in oWAT and eling, which plays a pivotal role in adipogenesis (4) and had more pericellular fibrosis around adipocytes than lean tissue architecture (5), is crucial to accommodate obesity- subjects in both depots. Macrophages and mastocytes were induced cellular alterations (6). However, the persistence highly represented in fibrotic bundles in oWAT, whereas scWAT of an inflammation stimulus in WAT may be responsible was more frequently characterized by hypocellular fibrosis. The for an excessive synthesis of ECM components and sub- oWAT fibrosis negatively correlated with omental adipocyte sequent interstitial deposition of fibrotic material. Fibro- ϭϪ ϭ diameters (R 0.30, P 0.02), and with triglyceride levels sis, attributed to excessive deposition of ECM proteins, is (R ϭϪ0.42, P Ͻ 0.01), and positively with apoA1 (R ϭ 0.25, P ϭ 0.05). Importantly, scWAT fibrosis correlated negatively with fat a ubiquitous tissue response to an unresolved chronic mass loss measured at the three time points after surgery. inflammation (7). We recently highlighted this phenome- non in obese WAT (8), showing increased expression of CONCLUSIONS—Our data suggest differential clinical conse- genes encoding extracellular matrix components and dem- quences of fibrosis in human WAT. In oWAT, fibrosis could onstrating the presence of fibrosis (8,9). In a small number contribute to limit adipocyte hypertrophy and is associated with a better lipid profile, whereas scWAT fibrosis may hamper fat of subjects, we scored histologic WAT fibrous tissue using mass loss induced by surgery. Diabetes 59:2817–2825, 2010 a picrosirius red staining and observed an increased abundance of ECM in obese versus lean WAT (8). More- over, we demonstrated that human adipocyte precursors From the 1INSERM, U872, Nutriomique, Paris, France; Universite´ Pierre et secrete ECM components when challenged by macro- Marie Curie-Paris6, Centre de Recherche des Cordeliers, Paris, France; Universite´ Paris Descartes, Paris, France; the 2Assistance Publique-Hoˆ pi- phage secretions (10), emphasizing the prominence of taux de Paris, Nutrition and Endocrinology Department, Pitie´-Salpeˆtrie`re macrophage-preadipocyte interactions in WAT fibrosis de- Hospital, Paris, France; CRNH-Ile de France, Paris, France; the 3Assistance velopment or maintenance. Other inflammatory cells ac- Publique-Hoˆ pitaux de Paris, Surgery Department, Hoˆ tel-Dieu Hospital, Paris, France; the 4Assistance Publique-Hoˆ pitaux de Paris, Anatomopathol- cumulate in obese WAT, including T-lymphocytes and ogy Department, Hoˆ tel-Dieu Hospital, Paris, France; and the 5Assistance mast cells (11), and might also participate in the orches- Publique-Hoˆ pitaux de Paris, Beaujon Hospital, Pathology Department, tration of fibrosis deposition. Clichy, France, and Centre de Recherche Bichat-Beaujon, INSERM U773, Clichy, France. The nature and consequences of ECM modification in Corresponding authors: Karine Cle´ment, [email protected], and WAT have been investigated in mice. In db/db obese mice, Joan Tordjman, [email protected]. various types of collagens are overexpressed in epididy- Received 26 April 2010 and accepted 30 July 2010. Published ahead of print at http://diabetes.diabetesjournals.org on 16 August 2010. DOI: 10.2337/ mal WAT (12). The predominantly expressed collagen db10-0585. mRNAs in epididymal WAT encode types I, IV, and VI. In A.D. and J.T. equally contributed to this study. mice deleted for the col6a1 gene, the lack of collagen VI © 2010 by the American Diabetes Association. Readers may use this article as associates with increased adipocyte size, both in response long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by to a high-fat diet and on the obese ob/ob genetic back- -nc-nd/3.0/ for details. ground (11). A similar phenotype of adipose cell hypertro- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance phy has been reported in mice deleted for secreted acidic with 18 U.S.C. Section 1734 solely to indicate this fact. cysteine-rich glycoprotein (SPARC), a matricellular glyco- diabetes.diabetesjournals.org DIABETES, VOL. 59, NOVEMBER 2010 2817 HUMAN ADIPOSE TISSUE FIBROSIS protein implicated in the synthesis of ECM components Immunohistochemical analysis of adipose tissue. Omental (oWAT) and (13). A mirror phenotype results from gene invalidation of subcutaneous (scWAT) biopsies were obtained during gastric or abdominal surgery in each individual. A portion of each WAT biopsy was immediately the collagenase MT1-matrix metalloproteinase (MT1- transferred into liquid nitrogen before RNA analysis. The other part was fixed MMP), which leads to the formation of a rigid network of overnight at 4°C in 4% paraformaldehyde, and processed for standard paraffin collagen fibrils and reduced lipid accumulation in the embedding. Sections of 5 ␮m were stained as described below and observed adipocytes (14). These observations suggest that in- under a Zeiss 20 Axiostar Plus microscope (Zeiss, Germany). Digital images creased ECM deposition in WAT may contribute to re- were captured by a camera (triCCD, Sony, France). Adipocyte diameters and strain adipocyte expansion in obesity. macrophage number were measured as described (18). We used cell-specific stains targeted to macrophages (HAM56, Dakocytomation, Trappes, France) In humans, the nature of WAT fibrosis and its clinical and to M1 or M2 macrophage subtypes, respectively, CD40 (R&D Systems, relevance are poorly documented and have been ad- Minneapolis, MN) and CD206 (R&D-Systems, Minneapolis, MN), CD3 (Neo- dressed mostly by gene expression evaluation. Subcutane- marker Microm, Francheville, France) for T-lymphocytes, tryptase (Dakocy- ous white adipose tissue (scWAT) collagen VI expression tomation, Trappes, France) for mast cells. An antibody binding to the ␣3 chain is increased in Asian Indian subjects compared with of the human collagen VI was used (Abcam, Paris, France). Method specificity Caucasians, in relation to their higher level of susceptibil- tests were performed by omission of primary antibodies and use of preim- mune serum. ity to insulin resistance (12). Pasarica et al. (15) showed Quantification and characterization of fibrotic depots. Slides of WAT that the expression of collagen VI in human WAT is biopsies were stained with picrosirius red. Fibrosis analysis was performed by upregulated after 8 weeks of overfeeding, concomitant histomorphometry using an Alphelys platform (Histolab Software, Plaisir, with increased inflammatory gene expression. These two France) with content color thresholds. The quantification of total fibrosis was studies suggest that increased collagen VI deposition expressed as the ratio of fibrous tissue area stained with picrosirius red/total could be a hallmark of WAT deregulation in obesity. tissue surface, as described by Henegar et al. (8). The same procedure was used for quantification of pericellular fibrosis, expressed as the ratio of the Here, we aimed to characterize more precisely fibrotic sum of stained areas/the sum of field surfaces measured in 10 random fields at depots in lean and obese subjects and the pathologic ϫ10 magnification for each biopsy. relevance of fibrosis in scWAT and omental white adipose Polarized light microscopic observation of