Danièle Lacasa

Fibrosis in Human Adipose Tissue: Composition, Distribution, and Link With Lipid Metabolism and Fat Mass Loss

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 omental WAT (oWAT) fibrosis in obesity and its relationship with metabolic alterations and surgery-induced weight loss. RESEARCH DESIGN AND METHODS Surgical biopsies for scWAT and oWAT were obtained in 65 obese (BMI 48.2 ± 0.8 kg/m2) and 9 lean subjects (BMI 22.8 ± 0.7 kg/m2). Obese subjects who were candidates for bariatric surgery were clinically characterized before, 3, 6, and 12 months after surgery, including fat mass evaluation by dual energy X-ray absorptiometry. WAT fibrosis was quantified and characterized using quantitative PCR, microscopic observation, and immunohistochemistry. RESULTS Fibrosis amount, distribution and collagen types (I, III, and VI) present distinct characteristics in lean and obese subjects and with WAT depots localization (subcutaneous or omental). Obese subjects had more total fibrosis in oWAT and had more pericellular fibrosis around adipocytes than lean subjects in both depots. Macrophages and mastocytes were highly represented in fibrotic bundles in oWAT, whereas scWAT was more frequently characterized by hypocellular fibrosis. The oWAT fibrosis negatively correlated with omental adipocyte diameters (R = −0.30, P = 0.02), and with triglyceride levels (R = −0.42, P < 0.01), and positively with apoA1 (R = 0.25, P = 0.05). Importantly, scWAT fibrosis correlated negatively with fat mass loss measured at the three time points after surgery. CONCLUSIONS Our data suggest differential clinical consequences of fibrosis in human WAT. In oWAT, fibrosis could contribute to limit adipocyte hypertrophy and is associated with a better lipid profile, whereas scWAT fibrosis may hamper fat mass loss induced by surgery.

Adipose tissue transcriptomic signature highlights the pathological relevance of extracellular matrix in human obesity

BackgroundInvestigations performed in mice and humans have acknowledged obesity as a low-grade inflammatory disease. Several molecular mechanisms have been convincingly shown to be involved in activating inflammatory processes and altering cell composition in white adipose tissue (WAT). However, the overall importance of these alterations, and their long-term impact on the metabolic functions of the WAT and on its morphology, remain unclear.ResultsHere, we analyzed the transcriptomic signature of the subcutaneous WAT in obese human subjects, in stable weight conditions and after weight loss following bariatric surgery. An original integrative functional genomics approach was applied to quantify relations between relevant structural and functional themes annotating differentially expressed genes in order to construct a comprehensive map of transcriptional interactions defining the obese WAT. These analyses highlighted a significant up-regulation of genes and biological themes related to extracellular matrix (ECM) constituents, including members of the integrin family, and suggested that these elements could play a major mediating role in a chain of interactions that connect local inflammatory phenomena to the alteration of WAT metabolic functions in obese subjects. Tissue and cellular investigations, driven by the analysis of transcriptional interactions, revealed an increased amount of interstitial fibrosis in obese WAT, associated with an infiltration of different types of inflammatory cells, and suggest that phenotypic alterations of human pre-adipocytes, induced by a pro-inflammatory environment, may lead to an excessive synthesis of ECM components.ConclusionThis study opens new perspectives in understanding the biology of human WAT and its pathologic changes indicative of tissue deterioration associated with the development of obesity.

Activin A Plays a Critical Role in Proliferation and Differentiation of Human Adipose Progenitors.

OBJECTIVE Growth of white adipose tissue takes place in normal development and in obesity. A pool of adipose progenitors is responsible for the formation of new adipocytes and for the potential of this tissue to expand in response to chronic energy overload. However, factors controlling self-renewal of human adipose progenitors are largely unknown. We investigated the expression profile and the role of activin A in this process. RESEARCH DESIGN AND METHODS Expression of INHBA/activin A was investigated in three types of human adipose progenitors. We then analyzed at the molecular level the function of activin A during human adipogenesis. We finally investigated the status of activin A in adipose tissues of lean and obese subjects and analyzed macrophage-induced regulation of its expression. RESULTS INHBA/activin A is expressed by adipose progenitors from various fat depots, and its expression dramatically decreases as progenitors differentiate into adipocytes. Activin A regulates the number of undifferentiated progenitors. Sustained activation or inhibition of the activin A pathway impairs or promotes, respectively, adipocyte differentiation via the C/EBPβ-LAP and Smad2 pathway in an autocrine/paracrine manner. Activin A is expressed at higher levels in adipose tissue of obese patients compared with the expression levels in lean subjects. Indeed, activin A levels in adipose progenitors are dramatically increased by factors secreted by macrophages derived from obese adipose tissue. CONCLUSIONS Altogether, our data show that activin A plays a significant role in human adipogenesis. We propose a model in which macrophages that are located in adipose tissue regulate adipose progenitor self-renewal through activin A.

Human adipocyte function is impacted by mechanical cues

Fibrosis is a hallmark of human white adipose tissue (WAT) during obesity‐induced chronic inflammation. The functional impact of increased interstitial fibrosis (peri‐adipocyte fibrosis) on adjacent adipocytes remains unknown. Here we developed a novel in vitro 3D culture system in which human adipocytes and decellularized material of adipose tissue (dMAT) from obese subjects are embedded in a peptide hydrogel. When cultured with dMAT, adipocytes showed decreased lipolysis and adipokine secretion and increased expression/production of cytokines (IL‐6, G‐CSF) and fibrotic mediators (LOXL2 and the matricellular proteins THSB2 and CTGF). Moreover, some alterations including lipolytic activity and fibro‐inflammation also occurred when the adipocyte/hydrogel culture was mechanically compressed. Notably, CTGF expression levels correlated with the amount of peri‐adipocyte fibrosis in WAT from obese individuals. Moreover, dMAT‐dependent CTGF promoter activity, which depends on β1‐integrin/cytoskeleton pathways, was enhanced in the presence of YAP, a mechanosensitive co‐activator of TEAD transcription factors. Mutation of TEAD binding sites abolished the dMAT‐induced promoter activity. In conclusion, fibrosis may negatively affect human adipocyte function via mechanosensitive molecules, in part stimulated by cell deformation. Published by John Wiley & Sons, Ltd

Roles of Chemokine Ligand-2 (CXCL2) and Neutrophils in Influencing Endothelial Cell Function and Inflammation of Human Adipose Tissue

The hypertrophied white adipose tissue (WAT) during human obesity produces inflammatory mediators, including cytokines (IL-6 and TNFα) and chemokines ([C-C motif] chemokine ligand 2 and IL-8). These inflammatory factors are preferentially produced by the nonadipose cells, particularly the adipose tissue infiltrating macrophages. We identified the chemokine (C-X-C motif) ligand 2 (CXCL2) by a transcriptomic approach. Because CXCL2 could represent a WAT-produced chemokine, we explored its role in obesity-associated inflammation. CXCL2 levels in serum and mRNA in WAT were higher in obese subjects compared with lean ones. CXCL2 secretions were higher in sc and visceral (vis) WAT from obese compared with lean subjects. In vis WAT, CXCL2 mRNA expression was higher in macrophages compared with other WAT cells and positively correlated with the inflammatory macrophage markers TNFα and IL-6. CXCL2 triggered the in vitro adhesion of the neutrophils, its selective cell targets, to endothelial cells (ECs) of vis WAT (vis WAT-ECs). Immunohistological analysis indicated that activated neutrophils were adherent to the endothelium of vis WAT from human obese subjects. Blood neutrophils from obese subjects released high levels of proinflammatory mediators (IL-8, chemokine motif ligand 2 [CCL2], matrix metalloproteinase [MMP] 9, and myeloperoxidase [MPO]). Visceral WAT-ECs, treated by neutrophil-conditioned media prepared from obese subjects, displayed an increase of the expression of inflammatory molecules associated with senescence and angiogenic capacities. To conclude, CXCL2, a WAT-produced chemokine being up-regulated in obesity, stimulates neutrophil adhesion to vis WAT-ECs. Activated neutrophils in obesity may influence vis WAT-ECs functions and contribute to WAT inflammation.

Evidence for a regional-specific control of rat preadipocyte proliferation and differentiation by the androgenic status.

In the rat, castration induces a decreased weight of fat depots. One possible explanation for these alterations could be that the capacities of preadipocytes to proliferate and differentiate are reduced by castration. Considering the regional specification of adipose tissue metabolism, these capacities and their eventual modulation by the androgenic status were presently compared in cultured preadipocytes from rat subcutaneous (SC) and epididymal fat depots.In epididymal preadipocytes, castration induced an increase in their proliferative capacity and conversely, a decrease in their adipogenesis.In vivo treatment by testosterone reversed the proliferative alteration but not the defective adipogenesis caused by castration.In vitro, no direct effect of testosterone on the proliferative capacities of epididymal preadipocytes could be observed suggesting that testosterone acts indirectly or needs the presence of other cofactors, such as insulin, dexamethasone and growth hormone. Surprisingly, testosterone partly counteracted the inhibitory effect of growth hormone on preadipocyte differentiation.In contrast to these observations, SC preadipocytes were completely insensitive to the androgenic status in terms of proliferation and differentiation.This study showing site-specific effects of castration on preadipocyte proliferation and differentiation suggests that part of the decreased fatness induced by castration in the rat is related to the modulatory effect of androgenic status on adipogenesis in some deep fat depots.

Endothelial Cells From Visceral Adipose Tissue Disrupt Adipocyte Functions in a Three-Dimensional Setting: Partial Rescue by Angiopoietin-1

During obesity, chronic inflammation of human white adipose tissue (WAT) is associated with metabolic and vascular alterations. Endothelial cells from visceral WAT (VAT-ECs) exhibit a proinflammatory and senescent phenotype and could alter adipocyte functions. We aimed to determine the contribution of VAT-ECs to adipocyte dysfunction related to inflammation and to rescue these alterations by anti-inflammatory strategies. We developed an original three-dimensional setting allowing maintenance of unilocular adipocyte functions. Coculture experiments demonstrated that VAT-ECs provoked a decrease in the lipolytic activity, adipokine secretion, and insulin sensitivity of adipocytes from obese subjects, as well as an increased production of several inflammatory molecules. Interleukin (IL)-6 and IL-1β were identified as potential actors in these adipocyte alterations. The inflammatory burst was not observed in cocultured cells from lean subjects. Interestingly, pericytes, in functional interactions with ECs, exhibited a proinflammatory phenotype with diminished angiopoietin-1 (Ang-1) secretion in WAT from obese subjects. Using the anti-inflammatory Ang-1, we corrected some deleterious effects of WAT-ECs on adipocytes, improving lipolytic activity and insulin sensitivity and reducing the secretion of proinflammatory molecules. In conclusion, we identified a negative impact of VAT-ECs on adipocyte functions during human obesity. Therapeutic options targeting EC inflammation could prevent adipocyte alterations that contribute to obesity comorbidities.

Increased Basement Membrane Components in Adipose Tissue During Obesity: Links With TGFβ and Metabolic Phenotypes

CONTEXT Collagen accumulation around adipocytes and vessels (ie, pericellular fibrosis) is a hallmark of obese adipose tissue associated with altered metabolism. OBJECTIVE Our objective was to evaluate components of basement membrane (BM) in adipose tissue, including collagen IV, a major BM component, and its relationships with metabolic parameters and TGFβ isoforms. DESIGN AND SETTING We used immuno-techniques and gene expression approaches to detect BM components in subcutaneous and visceral adipose tissue samples. Adipocytes and endothelial cells were isolated from lean and obese adipose tissue. We also focused on the expression of COL4A1 correlated to metabolic variables in moderate obesity and, in severe obesity before and after bariatric surgery. Using in vitro analysis, we explored the impact of TGFβ isoforms on the expression of inflammatory and extracellular matrix genes in adipocytes and endothelial cells. RESULTS BM components were detected around adipocytes and endothelial cells, and were increased in obese adipocytes. COL4A1 expression was positively correlated with insulin-resistance indices in obese subjects and showed less reduction in severely obese subjects with poorer insulin-resistance outcomes 6 months after gastric bypass. COL4A1 expression also correlated with TGFβ1 and TGFβ3 gene expressions in subcutaneous adipose tissue. Stimulating isolated adipocytes and endothelial cells in vitro with these TGFβ isoforms showed an inflammatory and pro-fibrotic phenotype. However, TGFβ1 and TGFβ3 exposure only provoked COL4A1 overexpression in endothelial cells and not in adipocytes. CONCLUSION The disorganization of several BM components, including collagen IV, could contribute to pathological alterations of obese adipose tissue and cells.

Site-specific control of rat preadipocyte adipose conversion by ovarian status: Possible involvement of CCAAT/enhancer-binding protein transcription factors.

The preadipocyte-adipocyte conversion process from two intraabdominal (parametrial and perirenal fat depots) is differently affected by ovarian status in the rat. We have tested the hypothesis that these sitespecific alterations of adipogenesis might be related to changes in the expression of the transcription factors c-myc and CCAAT/enhancer binding proteins (C/EBPα,-β, and-ζ) that regulate proliferation and differentiation. The increased proliferation rates observed in parametrial and perirenal preadipocytes after ovariectomy were not linked to variations in c-myc mRNA levels. Expression of the early marker of adipogenesis, lipoprotein lipase (LPL), remained insensitive to the ovarian status in early differentiated parametrial and perirenal preadipocytes. By contrast, LPL expression increased in early differentiated sc preadipocytes from ovariectomized rats, an, effect that was completely reversed by in vivo estradiol and progesterone treatment. Expression of C/EBPβ protein was unaffected by ovarian status whatever the anatomic origin of the preadipocytes. By contrast, the levels of p42 and p30 isoforms of C/EBPα were specifically decreased in parametrial preadipocytes, an alteration that was completely corrected by in vivo administration of estradiol and progesterone. C/EBPζ, a dominant inhibitor of C/EBPα and β, exhibited a strong site-specific expression since C/EBPζ content was fivefold higher in sc preadipocytes than in deep intraabdominal cells whatever the ovarian status. Furthermore, ovariectomy selectively decreased C/EBPζ levels in sc cells. In conclusion, our study suggests that some of the site-specific effects of ovariecaltered expressions of C/EBPα and ζ, both of which are important transcriptional regulators of fat cell differentiation and metabolism.

Semaphorin 3C is a novel adipokine linked to extracellular matrix composition

Aims/hypothesisAlterations in white adipose tissue (WAT) function, including changes in protein (adipokine) secretion and extracellular matrix (ECM) composition, promote an insulin-resistant state. We set out to identify novel adipokines regulated by body fat mass in human subcutaneous WAT with potential roles in adipose function.MethodsAdipose transcriptome data and secretome profiles from conditions with increased/decreased WAT mass were combined. WAT donors were predominantly women. In vitro effects were assessed using recombinant protein. Results were confirmed by quantitative PCR/ELISA, metabolic assays and immunochemistry in human WAT and adipocytes.ResultsWe identified a hitherto uncharacterised adipokine, semaphorin 3C (SEMA3C), the expression of which correlated significantly with body weight, insulin resistance (HOMA of insulin resistance [HOMAIR], and the rate constant for the insulin tolerance test [KITT]) and adipose tissue morphology (hypertrophy vs hyperplasia). SEMA3C was primarily found in mature adipocytes and had no direct effect on human adipocyte differentiation, lipolysis, glucose transport or the expression of β-oxidation genes. This could in part be explained by the significant downregulation of its cognate receptors during adipogenesis. In contrast, in pre-adipocytes, SEMA3C increased the production/secretion of several ECM components (fibronectin, elastin and collagen I) and matricellular factors (connective tissue growth factor, IL6 and transforming growth factor-β1). Furthermore, the expression of SEMA3C in human WAT correlated positively with the degree of fibrosis in WAT.Conclusions/interpretationSEMA3C is a novel adipokine regulated by weight changes. The correlation with WAT hypertrophy and fibrosis in vivo, as well as its effects on ECM production in human pre-adipocytes in vitro, together suggest that SEMA3C constitutes an adipocyte-derived paracrine signal that influences ECM composition and may play a pathophysiological role in human WAT.