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. 2010 Oct;59(10):2513-21.
doi: 10.2337/db10-0013. Epub 2010 Jun 8.

Activin a plays a critical role in proliferation and differentiation of human adipose progenitors

Laure-Emmanuelle Zaragosi  1 , Brigitte Wdziekonski, Phi Villageois, Mayoura Keophiphath, Marie Maumus, Tamara Tchkonia, Virginie Bourlier, Tala Mohsen-Kanson, Annie Ladoux, Christian Elabd, Marcel Scheideler, Zlatko Trajanoski, Yasuhiro Takashima, Ez-Zoubir Amri, Daniele Lacasa, Coralie Sengenes, Gérard Ailhaud, Karine Clément, Anne Bouloumie, James L Kirkland, Christian Dani
Affiliations

Activin a plays a critical role in proliferation and differentiation of human adipose progenitors

Laure-Emmanuelle Zaragosi et al. Diabetes. 2010 Oct.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Expression of INHBA/activin A by human adipose progenitors and in adipose tissues. A: Expression of the INHBA gene in undifferentiated (Und.) and 3 and 15 days after induction of adipocyte differentiation (Diff.) of hMADS2 cells. Expression was quantified by real-time PCR, and results are shown by taking as 100 the signal obtained in undifferentiated cells. Similar results were obtained with hMADS3 cells. B: Expression of INHBA gene during differentiation of human primary preadipocytes isolated from different fat depots. Preadipocytes from indicated fat depots of four subjects were cultured in parallel to confluence, when differentiation-inducing or control media were added for 30 days. Expression was quantified by real-time PCR, and results are shown by taking as 100 the signal obtained in undifferentiated cells from each fat depot. C: Expression of the INHBA gene in native progenitor cells, defined as CD34+/CD31− cells, and mature adipocytes from adult subcutaneous adipose tissue (AT). Expression of INHBA was quantified by real-time PCR (n = 18 progenitor cells; n = 5 mature adipocytes). Results are shown by taking as 100 the value obtained in the CD34+/CD31− cell population. Values are means ± SEM. **P < 0.01.
FIG. 2.
FIG. 2.
Effects of activin A (Act. A) supplementation and of activin A signaling pathway inhibition on hMADS cell number. A: hMADS3 cell number in 0.5% FCS medium in the absence or presence of 100 ng/ml activin A or 1 μg/ml neutralizing activin A antibodies (α-Act.A) for 5 days. Results are the average of counting of three culture wells (12-well plates). B: Proliferating hMADS3 cells were transfected with siSmad2 and treated or not with 100 ng/ml activin A 24 h later. Impact on cell number was analyzed 5 days later. Results are the average of three culture wells (12-well plates). Data are means ± SEM (n = 3). *Cell numbers significantly different in treated cells vs. controls.
FIG. 3.
FIG. 3.
Inhibition of hMADS cells adipogenesis by activin A. A and B: hMADS3 cells were induced to undergo adipocyte differentiation in the absence or presence of the indicated concentrations of activin A. Twelve days later, adipogenesis was assessed by Oil red O for lipid droplets staining (34) and by GPDH activity (35). C: hMADS3 cells were induced to undergo adipocyte differentiation and treated with 100 ng/ml activin A for the indicated time intervals. GPDH activity was determined at day 12. Results are means of three culture wells (24-well plates). Values are means ± SEM (N = 3). *Significant differences in GPDH activities in treated cells vs. controls. Similar results were obtained with hMADS2 cells and with hMADS7–B7 and -B9 clones. D: Effects of activin A on the expression of adipogenic genes. hMADS3 cells were induced to undergo differentiation in the absence or presence of 100 ng/ml activin A. RNAs were prepared 6 days after induction of differentiation and expression was investigated by semiquantitative PCR. E: Effects of activin A on the expression of C/EBPb-LAP and -LIP isoforms. hMADS3 cells were induced to undergo differentiation in the absence or presence of 100 ng/ml activin A. Proteins were prepared 6 days after induction of differentiation. LAP and LIP isoforms were examined by Western blot analysis using 25 μg total proteins per lane and anti-C/EBPβ antibodies. Similar results were obtained when proteins were prepared 3 days after induction of differentiation (supplementary Fig. S6). The approximate molecular weight of C/EBPβ isoforms is indicated. The 14 kDa C/EBPβ proteolytic degradation product was not detected. Tubulin was used as a loading control. Samples were run on the same gel. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 4.
FIG. 4.
Suppression of antiadipogenic effects of activin A by C/EBPβ-LAP forced expression. A: hMADS3-EcoRec cells were infected with retroviral vectors expressing GFP, C/EBPβ-LAP, or C/EBPβ-LIP and induced to differentiate into adipocytes in the absence or presence of 100 ng/ml activin A. Adipogenesis was assessed 6 days later by GPDH activities. GPDH activity obtained in the absence of activin A for each transduced cells was taken as 100%. Results are means of three culture wells (24-well plates). Values are means ± SEM (n = 3). *Significant differences between treated versus untreated cells. B: Western blot analysis of FABP4 in the absence or presence of activin A. Samples were run on the same gel.
FIG. 5.
FIG. 5.
Effects of activin A pathway silencing on adipocyte differentiation of hMADS cells. A: hMADS3 cells were induced to undergo adipocyte differentiation in the presence of SB431542 (5 μmol/l) or neutralizing activin A antibodies (1 μg/ml). GPDH activities were quantified after 12 days. Results are the means of three culture wells (24-well plates). Values are means ± SEM (n = 3). *Significant differences of GPDH activities in treated cells vs. controls. B: hMADS3 cells were transfected with scrambled siRNA (si-Scr.), Inhba-siRNA, or Smad2-siRNA a day before they reached confluence. The day after, cells were induced to undergo adipocyte differentiation. GPDH activity of cell extracts 6 days after transfection. Results are means of three culture wells (24-well plates). Data are means ± SEM (n = 3). *Significant differences in GPDH activities in siInhba- and siSmad2-treated cells vs. controls.
FIG. 6.
FIG. 6.
Effects of dexamethasone (DEX) on the expression of INHBA/activin A in human adipose progenitors. A: Individual components of the adipogenic cocktail were added to confluent hMADS3 cells alone or in combination. Cells were harvested 3 days later for semiquantitative PCR analysis. 1, insulin (5 μg/ml) and 10 μg/ml transferrin; 2, insulin (5 μg/ml) and 10 μg/ml transferrin plus 1 μmol/l rosiglitazone; 3, insulin (5 μg/ml) and 10 μg/ml transferrin plus 0.2 nmol/l triiodothyronine; 4, insulin (5 μg/ml) and 10 μg/ml transferrin plus 1 μmol/l dexamethasone; 5, insulin (5 μg/ml) and 10 μg/ml transferrin plus 100 μmol/l isobutyl-methylxanthine; 6, insulin (5 μg/ml) and 10 μg/ml transferrin plus 1 μmol/l rosiglitazone, 0.2 nmol/l triiodothyronine, 100 μmol/l isobutyl-methylxanthine, and 1 μmol/l dexamethasone; 7, insulin (5 μg/ml) and 10 μg/ml transferrin plus 1 μmol/l rosiglitazone, 0.2 nmol/l triiodothyronine, and 100 μmol/l isobutyl-methylxanthine. B: Inhibition of activin A secretion by dexamethasone. hMADS3 cells were maintained in 5 μg/ml insulin and 10 μg/ml transferrin medium in the absence or presence of 1 μmol/l dexamethasone. Culture media were collected 3 or 6 days later, filtered on 0.2-μm membranes, and concentrated with Amicon ultra-15 columns (NMWL, three KDa; Millipore). Levels of activin A were analyzed by Western blot performed under nonreducing conditions because anti–activin A antibody selectively binds to the dimeric form of activin A. Secreted enolase one (ENO1) was used as a loading control (36).
FIG. 7.
FIG. 7.
Expression of INHBA/activin A in adipose tissues and regulation by factors secreted by adipose tissue–derived macrophages. A: Expression of INHBA in subcutaneous adipose tissue of lean (n = 7) and morbidly obese (n = 12) women. Expression was quantified by real-time PCR and CD34+/CD31−, and results are shown by taking as 1 the signal obtained in adipose tissue of lean subjects. B: Effects of conditioned medium from CD14+ adipose tissue macrophages on INHBA/activin A expression. INHBA expression in human preadipocytes treated for 10 days with ATM-conditioned media (ATM-CM) compared with control medium (n = 5). C: INHBA expression in CD34+/CD31− adipose progenitors treated for 24 h with ATM-conditioned media compared with control medium (n = 7). Expression was quantified by real-time PCR. Values are means ± SEM. ATMs were isolated from women (mean age 39.33 ± 4.37 years and mean BMI 26.21 ± 2.54 kg/m2). *Differ with P < 0.01. D: Undifferentiated hMADS2 cells were treated with factors secreted by ATM isolated from three biopsies (ATM-CM 1–3) or with control media (Ctr) for 24 h. The same volumes of conditioned media were maintained at 37°C in the absence of hMADS cells (ATM-CM alone). Culture media were collected and analyzed for expression of activin A as described in Fig. 6 B. Enolase 1 (ENO1) was used as a loading control. Enolase signals shown on the left part of gel combine enolase levels present in the ATM-CM and levels secreted by hMADS cells. ATMs were isolated from women (mean age 46.20 ± 6.80 years and mean BMI 25.48 ± 1.26 kg/m2).
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