Secreted Frizzled-Related Protein 1 Regulates Adipose Tissue Expansion and Is Dysregulated in Severe Obesity

International Journal of Obesity (2010) 34, 1695–1705 & 2010 Macmillan Publishers Limited All rights reserved 0307-0565/10 www.nature.com/ijo ORIGINAL ARTICLE Secreted frizzled-related protein 1 regulates adipose tissue expansion and is dysregulated in severe obesity

C Lagathu1, C Christodoulides2,CYTan1, S Virtue1, M Laudes1, M Campbell1, K Ishikawa1, F Ortega3, FJ Tinahones4, J-M Ferna´ndez-Real3, M Oresˇicˇ5, JK Sethi1 and A Vidal-Puig1

1Department of Clinical Biochemistry, Institute of Metabolic ScienceFMetabolic Research Laboratories, Addenbrooke’s Hospital, University of Cambridge, Cambridge, UK; 2Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford, UK; 3Department of Diabetes, Endocrinology and Nutrition, Hospital of Girona Dr Josep Trueta, Biomedical Research Institute and CIBEROBN Fisiopatologı´a de la Obesidad y Nutricio´n CB06/03/010, Girona, Spain; 4Service of Endocrinology and Nutrition, Hospital Clinico Universitario Virgen de Victoria de Malaga, Malaga, Spain and 5VTT Technical Research Centre of Finland, Espoo, Finland

Aim: The Wnt/b-catenin signaling network offers potential targets to diagnose and uncouple obesity from its metabolic complications. In this study, we investigate the role of the Wnt antagonist, secreted frizzled-related protein 1 (SFRP1), in promoting adipogenesis in vitro and adipose tissue expansion in vivo. Methods: We use a combination of human and murine, in vivo and in vitro models of adipogenesis, adipose tissue expansion and obesity-related metabolic syndrome to profile the involvement of SFRP1. Results: SFRP1 is expressed in both murine and human mature adipocytes. The expression of SFRP1 is induced during in vitro adipogenesis, and SFRP1 is preferentially expressed in mature adipocytes in human adipose tissue. Constitutive ectopic expression of SFRP1 is proadipogenic and inhibits the Wnt/b-catenin signaling pathway. In vivo endogenous levels of adipose SFRP1 are regulated in line with proadipogenic states. However, in longitudinal studies of high-fat-diet-fed mice, we observed a dynamic temporal but biphasic regulation of endogenous SFRP1. In agreement with this profile, we observed that SFRP1 expression in human tissues peaks in patients with mild obesity and gradually falls in morbidly obese subjects. Conclusions: Our results suggest that SFRP1 is an endogenous modulator of Wnt/b-catenin signaling and participates in the paracrine regulation of human adipogenesis. The reduced adipose expression of SFRP1 in morbid obesity and its knock-on effect to prevent further adipose tissue expansion may contribute to the development of metabolic complications in these individuals. International Journal of Obesity (2010) 34, 1695–1705; doi:10.1038/ijo.2010.107; published online 1 June 2010

Keywords: metabolic syndrome; adipose tissue; adipogenesis; Wnt signaling

Introduction vascular complications. Thus, in parallel with efforts to tackle the obesity problem, a more pragmatic approach Obesity is associated with a greater risk of developing suggests that efforts should aim to uncouple obesity from its chronic diseases, such as cardiovascular disease, type II cardiometabolic complications. diabetes, atherosclerosis and the so-called metabolic syn- Although seemingly counter intuitive, strategies that drome.1,2 Despite improvements in social awareness, poli- increase the capacity of adipose tissue to store lipid and, tical measures to promote changes in lifestyle and scientific therefore, make individuals more obese may confer meta- success in unraveling the mechanisms controlling energy bolic benefits. Allowing adipose tissue to store more lipid balance, the prevalence of obesity continues to escalate. may prevent secondary metabolic complications caused by Current predictions indicate that the epidemic of obesity lipid being deposited in nonadipose organs. The concept of will be followed by a second wave of devastating cardio- adipose tissue expansion being beneficial is supported by the apparent paradox that a paucity of adipose tissue results in Correspondence: Dr JK Sethi or Professor AJ Vidal-Puig, Department of Clinical lipodystrophy, and this also recapitulates many features of Biochemistry, Institute of Metabolic ScienceFMetabolic Research Labora- the metabolic syndrome. Taken together, it is likely that an tories, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 optimal range exists for total body adipose tissue mass, 0QQ, UK. beyond these boundaries of which metabolic dysregulation E-mail: [email protected] or [email protected] Received 31 January 2010; revised 22 April 2010; accepted 25 April 2010; develops. In this regard, the ability of adipose tissue to published online 1 June 2010 expand and match the storage needs of energy surplus may SFRP1 and adipogenesis C Lagathu et al 1696 be a key determinant of the susceptibility to develop Written informed consent was obtained from all subjects metabolic syndrome associated with obesity.1–4 Thus, under- before enrollment and the appropriate research ethics standing the signaling factors that control the titrated committees approved the studies. Four independent study process of adipose tissue expansion is fundamental for a populations were used: Group A (used in Figures 1a and b) rational approach to design effective therapies to prevent comprised of samples acquired from eight subjects under- and treat the metabolic syndrome.5 going elective open abdominal surgery at Addenbrooke’s The Wnt family of secreted growth factors act in a Hospital (six males and two females; age 66±10 years, body paracrine and/or autocrine manner and are known to mass index (BMI) 26.2±3.8 kg mÀ2). All subjects fasted for control adipocyte differentiation.6–9 Wnt signaling relies 6 h before the operation. None were taking medications on a sophisticated regulated network capable of providing known to affect adipose tissue mass or metabolism.12 Group context-dependent graded responses. Briefly, binding of B (used in Figure 5a) comprised of adipose tissue obtained specific Wnt proteins to Frizzled receptors transduces from subcutaneous depots during elective surgical proce- intracellular signals through either b-catenin-dependent or dure. Samples were collected from 31 female subjects with a b-catenin-independent pathways. Although both pathways BMI between 18 and 70 kg mÀ2, who were invited to may be active in preadipocytes,10 it is the former that has participate at the Endocrinology Service of the Hospital been best characterized and shown to potently inhibit Universitari de Girona Dr Josep Trueta (Girona, Spain) and at adipogenesis both in vitro and in vivo.8,9 In the b-catenin- the Hospital Clinico Universitario Virgen de Victoria de dependent pathway, receptor activation leads to stabilization Malaga (Ma´laga, Spain).21 Group C (used in Figure 5b) and accumulation of cytosolic b-catenin. b-catenin subse- comprised of needle biopsies of subcutaneous adipose tissues quently translocates to the nucleus, where it binds and obtained from 13 monozygotic twin pairs (eight male and activates the lymphoid enhancer-binding factor/T cell- five female pairs), discordant for weight identified through specific transcription factor (LEF/TCF) family of transcrip- the national population registry of Finland. One co-twin was tion factors. Wnt/TCF target genes include cyclin D1, Id2 and not obese (BMI o25 kg mÀ2) and the other one was obese c-myc, which inhibit adipogenesis.11,12 (BMI 430 kg mÀ2). The recruitment and selection process of Constitutive activation of Wnt/b-catenin signaling in subjects were carried out as previously published.22 For all preadipocytes inhibits differentiation by preventing the subjects, BMI cutoff was determined according to WHO BMI induction of C/EBPa and PPARg.13,14 Conversely, inactivation classification (http://apps.who.int/bmi/). of intracellular Wnt/b-catenin signaling releases the brake on adipogenesis.8,11,12,15 Adipogenesis may also be enhanced by extracellular Wnt antagonists, including secreted frizzled- Murine white adipose tissue expression studies related proteins (SFRPs, also known as secreted apoptosis- Animals were housed in a temperature-controlled room with related proteins or SARPs).16,17 At least five structurally similar 12-h light/dark cycles. Food and water were available SFRPs have been identified and are characterized by a ad libitum unless noted. For analysis of the fed state, animals cysteine-rich domain, which resembles the Wnt ligand- were killed immediately following a dark cycle. Induction of binding domain found on Frizzled receptors.17 It is this the fasted state involves a withdrawal of feed for 12 h, domain that is required to provide modulator activities for whereas the refeeding state involves the reintroduction of Wnt ligands.18,19 Consistent with this, exogenous treatment food for 12 h following a fast. Both the murine diet studies with recombinant SFRP1 and SFRP2 can disrupt Wnt/b- (high-fat diet (HFD) and thiazolidinedione supplemented) catenin signaling and promote adipocyte differentiation.15 were performed as previously described.23 All animal proto- Furthermore, the Sfrp1 knockout mice also show a reduction cols used in this study were approved by the UK Home Office in percent body fat,20 consistent with unopposed antiadipo- and the University of Cambridge. genic Wnt/b-catenin signaling. However, there is limited evidence to support a role for endogenous SFRP1 in the physiological and/or pathological development of human Human white adipose tissue fractionation and expression obesity and the metabolic syndrome. In this study, we report studies on SFRP1 expression profile studies in humans and mice and Human adipocyte and preadipocyte isolations were per- in vitro functional assays to examine the role and regulation of formed on samples obtained from Group A subjects, as SFRP1 during adipogenesis and in the development of human previously described.12,23 Briefly, adipose tissue biopsies and mouse obesity. were placed in phosphate-buffered saline (Sigma-Aldrich, Dorset, England) and processed within 30 min. Samples were finely diced and digested in collagenase solution (Hank’s Materials and methods balanced salt solution containing 3 mg mlÀ1 type II collagenase (Sigma-Aldrich) and 1.5% bovine serum albumin) at Subjects 37 1C for 1 h. Subsequently, the digest was filtered through a The characteristics of the populations studied are summar- stainless steel mesh and centrifuged at 400 g for 5 min to ized in the online appendix (Supplementary Table 1). separate mature adipocytes from the stromavascular cells.

International Journal of Obesity SFRP1 and adipogenesis C Lagathu et al 1697

500 Human WAT 250 *** ** rRNA 400 200 ** rRNA 80 * 18S

300 18S 150 ** 60 200 40 100 mRNA/ * mRNA/ relative levels 100 20 50 Sfrp1 SFRP1 0 0 0 0243612 48 SFRP1 SVF MA 0243612 48 Hours post-induction Hours post-induction

300 *** ** 250 ** rRNA Hours post MDI 200 18S 0962448 144 192 150 100 * sFRP1 mRNA/ ** ** 50 ERK1/2

Sfrp1 0 0 48 96 144 192 Hours post induction

Figure 1 SFRP1 expression during human and mouse adipogenesis. (a) Human SFRP1 mRNA levels, normalized to 18S rRNA levels, were measured using real-time RT-PCR at the indicated hours of differentiation of primary human (Group A) SVF cultures. *Po0.05, **Po0.01, ***Po0.001 versus Time 0. (b) SFRP1 mRNA levels, normalized to 18S rRNA, were measured in stromavascular cells (SVF) and mature adipocytes (MA) from human subcutaneous white adipose tissue (WAT from Group A, n ¼ 8). **Po0.01. (c) Mouse Sfrp1 mRNA levels, normalized to 18S rRNA levels, were measured using real-time RT-PCR at the indicated hours of differentiation of primary mouse SVF cultures. *Po0.05, **Po0.01 versus Time 0. (d) Sfrp1 mRNA levels, normalized to 18S rRNA levels, were measured at the indicated hours post-induction (MDI) of 3T3-L1 preadipocyte differentiation. *Po0.05, **Po0.01, ***Po0.001 versus Time 0. (e) Whole-cell protein lysates were extracted at the indicated days of differentiation of 3T3 L1 cells and analyzed by immunoblotting. Representative immunoblots of Sfrp1 and ERK1/2 (loading control) are shown.

Primary preadipocyte cultures into the SnaBI site of the retroviral expression vector pBabe- For human preadipocyte isolation, human adipose tissue puro. Correct orientation was confirmed by at least three samples were obtained from Group A subjects. Murine different diagnostic digests and sequencing. 3T3-L1 cells primary preadipocytes were isolated from epididymal white were cultured, differentiated into adipocytes and stained for adipose tissue (WAT) of 6-week-old male C57B/6 mice, as Oil Red-O, as described previously.11 Briefly, 3T3-L1 pre- described previously.24 Human and murine primary cultures adipocytes were differentiated 2 days after confluence in were induced to differentiate 3 days after confluence by Dulbecco’s modified Eagle’s medium with 10% fortified adding Dulbecco’s modified Eagle’s medium/Hams F12 with bovine serum (Hyclone, Perbio Science, Northumberland, 33 mmol lÀ1 Biotin, 17 mmol lÀ1 pantothenic acid, 10 mgmlÀ1 UK) supplemented with 5 nmol lÀ1 insulin, 0.25 mmol lÀ1 apotransferrin, 0.2 nmol lÀ1 triiodothyronine, 100 nmol lÀ1 dexamethasone and 0.5 mmol lÀ1 IBMX (for full induction cortisol and 500 nmol lÀ1 insulin. For the first 3 days of cocktailFIBMX, dexametasone and insulin (MDI)). IBMX culture, 0.25 mmol lÀ1 1-methyl-3-isobutylxanthine (IBMX) was omitted from submaximal induction cocktail (DI). The was also added to the medium. Differentiation medium was medium was then supplemented only with insulin for 2 replaced after 3 days on the first occasion and thereafter at days. Differentiated cells were only used when at least 95% of every 2 days. the cells showed an adipocyte phenotype by accumulation of lipid droplets by day 8. Cells were analyzed by phase contrast microscopy. All retrovirally transfected 3T3-L1 cell lines were Generation, culture and differentiation of 3T3-L1 kept in puromycin-containing medium throughout culture preadipocyte cell lines and differentiation procedures. SFRP1 3T3-L1 cell lines (or control) were generated with the pBabe-Puro retroviral vector system, as previously described.11 The retroviral expression construct encoding RNA isolation and quantitative reverse transcription PCR human SFRP1 cDNA was cloned by excision from Human RNA preparation, reverse transcription and conditions for image clone no. 5265277 (NCBI BC036503) with XmaI and TaqMan real-time reverse transcription PCR were performed NotI digest, followed by Klenow fill-in and blunt-end ligation as previously described: refer Christodoulides et al.12 and

International Journal of Obesity SFRP1 and adipogenesis C Lagathu et al 1698 Lagathu et al.23 for Group A, Ortega et al.21 for Group B and and in the murine preadipocyte cell line, 3T3-L1 (Figure 1d). Pietilainen et al.22 for Group C. Primers and probes were SFRP1 protein levels closely followed the pattern of RNA either purchased from Perkin Elmer (Cambridge, UK) or expression (Figure 1e). These results also indicate that the designed using Primer Express software (Applied Biosystems, 3T3-L1 cell line model could be a useful tool to further Cheshire, UK) and sequences from the GenBank database. characterize the regulation and functional role of SFRP1 during adipogenesis. Protein extraction and western blotting Before protein analysis by western blotting, medium was SFRP1 overexpression promotes adipogenesis in 3T3-L1 removed and cell monolayers were washed with ice-cold preadipocytes through the inhibition of Wnt/b-catenin- phosphate-buffered saline and then frozen in liquid nitro- dependent signaling gen. These were thawed on ice and scraped into lysis buffer, To assess the functional consequences of increased SFRP1 as previously described23 at 4 1C. Proteins were electroblotted expression on the process of adipocyte differentiation, we onto nitrocellulose membrane (Amersham Biosciences, generated stable preadipocyte cell lines constitutively over- Piscataway, NJ, USA). Specific proteins were detected by expressing human SFRP1. SFRP1-expressing 3T3-L1 preadi- incubation with the appropriate primary and horseradish- pocytes displayed a 10-fold increase in SFRP1 mRNA peroxidase-conjugated secondary antibodies. Immune compared with EV-expressing 3T3-L1 cells (Figure 2a). After complexes were detected by enhanced chemiluminescence adipogenesis, significantly greater levels of SFRP1 expression (Amersham Biosciences). were maintained in day 8 adipocytes (Figure 2a). A similar expression pattern was reflected in the protein levels of SFRP1 (Figure 2b). To examine whether SFRP1 could modulate Wnt-TCF4/LEF reporter assay Wnt/b-catenin signaling, EV- and SFRP1-expressing For TCF4 reporter assay, SFRP1 and empty vector (EV) 3T3-L1 preadipocytes were induced to differentiate and cyto- cells were grown to confluence, and Topflash promoter– solic protein extracts were screened for b-catenin levels 12 reporter assays were performed as previously described. (Figure 2c). When compared with control EV preadipocytes, ectopic expression of human SFRP1 resulted in decreased Statistical analysis cytosolic b-catenin levels in 3T3-L1 preadipocytes. This Data from densitometrical analysis, luciferase assays and reduction was also consistent with reduced expression of quantitative real-time PCR are presented as mean±s.e.m. of Cyclin D1 (Ccnd1), a Wnt/b-catenin target gene during at least three independent experiments. Statistical signifi- adipogenesis (Figure 2d). Furthermore, in promoter assays, cance was determined with Student’s t-test (*Po0.05, SFRP1-expressing cells also displayed reduced TCF/LEF **Po0.01 and ***Po0.001). Analysis of variance was deter- activity (Figure 2e) and seemed to be less responsive to mined using single factor analysis of variance (ANOVA) on exogenous Wnt3a treatment (Figure 2e). Hence, SFRP1 SPSS 16.0 (SPSS(UK) Ltd, Surrey, UK). can inhibit Wnt/b-catenin-dependent signaling in stably transfected 3T3-L1 preadipocytes. To evaluate the impact of increased SFRP1 levels on Results adipogenic potential, lipid accumulation was assessed in EV- and SFRP1-overexpressing preadipocytes after adipo- SFRP1 gene expression increases during human and murine genic induction. As illustrated in Figure 3a, although SFRP1 adipogenesis 3T3-L1 cells and EV 3T3-L1 cells accumulated lipids to a Our previous microarray profiling studies of human pre- similar extent when stimulated with the full induction adipocytes identified numerous novel genes regulated early cocktail (MDI), submaximal stimulation (DI) revealed that during adipogenesis.25 These included SFRP1, a secreted Wnt SFRP1 3T3-L1 cells have greater adipogenic potential. This antagonist, which was induced threefold, 48 h after the onset was consistent with increased expression levels of the of differentiation. This observation was first validated by adipogenic/lipogenic markers aP2 (Fabp4) and PPARg (Pparg) measuring SFRP1 gene expression during differentiation of in DI-treated SFRP1 3T3-L1 cells (Figures 3b and c, respec- subcutaneous preadipocytes from human subjects using real- tively). Thus, constitutive expression of SFRP1 in 3T3-L1 time PCR (Figure 1a). The results of our study indicate that preadipocytes promotes adipogenesis and lipid accumula- SFRP1 mRNA can be detected in confluent preadipocytes and tion. This suggests that in vivo endogenous SFRP1 is a good that its expression increases after induction of adipocyte candidate to facilitate adipogenesis and that it is not simply differentiation, being fourfold higher at 48 h compared with induced as a consequence of differentiation. baseline. This is consistent with the greater relative expression found in mature human adipocytes relative to the stromavascular fraction (Figure 1b). The adipogenic profile is Sfrp1 is regulated in vivo by nutritional and metabolic status also not restricted to human adipogenesis, as Sfrp1 mRNA We next sought to determine whether Sfrp1 mRNA levels was also increased after induction of differentiation in both were physiologically regulated in vivo by nutritional status. mouse primary stromavascular fraction cultures (Figure 1c) Fasting and refeeding experimental conditions were selected

International Journal of Obesity SFRP1 and adipogenesis C Lagathu et al 1699 as opposing physiological paradigms with respect to acute (P ¼ 0.07). However, in response to 24-h refeeding, Sfrp1 fuel availability and requirement for storage in adipose mRNA levels were markedly increased beyond the levels tissue. We evaluated Sfrp1 mRNA expression in whole WAT found in the fed state (Figure 4a), suggesting that WAT SFRP1 and isolated mature adipocytes. In response to 24 h fasting, is regulated during changes in nutritional flux. The same there was a nonsignificant trend toward reduced Sfrp1 levels profile was observed in mature adipocytes isolated from a relative to whole adipose tissue collected from fed mice separate group of mice subjected to the same manipulation (Figure 4b). To further investigate the physiological regulation of Sfrp1 2000 in the context of sustained positive energy balance, we EV * analyzed the expression of Sfrp1 in adipose tissue of mice fed 1600 sFRP1 rRNA with a HFD. We observed that following short-term exposure ***

18S 1200 (3 days) to a HFD, Sfrp1 expression was significantly induced (Figure 4c). Intriguingly, with longer-term HFD exposure 800 (that is, 4 weeks and 6 months), adipose tissue expansion mRNA/ 400 plateaus (Figure 4e) and becomes increasingly dysfunctional as assessed by Glut4 expression (Figure 4d). During this time, Sfrp1 0 day0 day8 the relative levels of Sfrp1 progressively fall and by 6 months, they drop below the levels observed in lean chow-fed mice (Figure 4c).

EV sFRP1 EV sFRP1 To further test the correlation between fat deposition and endogenous SFRP1 expression, we used rosiglitazone sFRP1 (thiazolidinedione) treatment as a pharmacological approach to stimulate adipogenesis and lipid accumulation ERK1/2 in vivo. Mice fed with a thiazolidinedione supplemented diet showed increased expression of adipogenic markers in their 23 day0 day8 adipose tissue such as aP2. More importantly, this increase in adipose tissue expandability was associated with significantly increased Sfrp1 levels (Figure 4f).

EV sFRP1 EV sFRP1 EV sFRP1 EV sFRP1 Taken together, these observations suggest that in vivo, WAT Sfrp1 mRNA level is increased when there is a demand β -Catenin for fat storage and adipose expansion; a finding consistent with its in vitro role in promoting recruitment of new ERK1/2 adipocytes and adipocyte maturation. However, this increase in Sfrp1 seems not to be sustainable despite the maintenance day0 day2 day4 day8 of a positive caloric balance and fat storage demand. This suggests that SFRP1 may be a useful marker, and supports the 100 EV existence of an upper physiological limit to which adipose sFRP1 depot can expand and accumulate lipid. Interestingly,

rRNA 80 the fall in Sfrp1 levels at 6 months of HFD is associated with

18S 60 *** the development of metabolic complications. 40 * ** mRNA/ Figure 2 SFRP1 is an antagonist of the Wnt/b-catenin signaling in 3T3-L1 20 adipocytes. Preadipocytes were infected with a retrovirus carrying Human SFRP1 or vector alone (EV). (a) SFRP1 mRNA levels, normalized to 18S rRNA Ccnd1 0 levels, were measured using real-time RT-PCR at day 0 and day 8 of day0 day2 day4 day8 differentiation in SFRP1- and EV-expressing cells. *Po0.05, ***Po0.001 versus EV. (b) Whole-cell lysates were extracted at day 0 and day 8 of differentiation 500 * EV in SFRP1- and EV-expressing cells. Representative immunoblots of SFRP1 and ** ** sFRP1 ERK1/2 (loading control). (c) Whole-cell lysates were extracted at indicated 400 time points of SFRP1 and EV differentiated with MDI and analyzed by immunoblotting. Representative immunoblots of b-catenin and ERK1/2 300 (loading control). (d) CyclinD1 (Ccnd1) mRNA levels, normalized to 18S rRNA levels, was measured at the indicated hours of differentiation in SFRP1 200 and EV cells differentiated with DI. *Po0.05, **Po0.01,***Po0.001 versus EV. * (e) Topflash reporter activity in 3T3-L1 cells expressing SFRP1 or an empty TopFlash (A.U.) 100 vector (EV) treated with MDI ± Wnt3a (10 ng mlÀ1) for 48 h. Results are expressed as fold difference relative to EV. Results are presented as mean ± 0 s.e.m. of at least three independent experiments performed in triplicates. Control + Wnt3a *Po0.05, **Po0.01.

International Journal of Obesity SFRP1 and adipogenesis C Lagathu et al 1700 1200 NS EV 1000 sFRP1 *** 800 600 400 * 200 *

EV sFRP1 EV sFRP1 EV sFRP1 EV sFRP1 Oil-Red-O quantification 0 - M DI MDI -M DI MDI

4000 EV ** 6000 sFRP1 ** EV rRNA 3000

rRNA sFRP1 4500

18S *

2000 18S ** 3000 mRNA/

1000 ** mRNA/ 1500 Fabp4

0 Fabp4 0 02468 Day 0 Day 8 Day 8 Days post DI DI MDI

10000 ** ** 10000 * 8000 ** rRNA

rRNA 8000

18S 6000 **

18S 6000 4000 *

mRNA/ 4000 2000 mRNA/ 2000 Pparg * 0 Pparg 0 02468 Day 0 Day 8 Day 8 Days post DI DI MDI

Figure 3 SFRP1 promotes adipogenesis in 3T3-L1 preadipocytes. (a) SFRP1 and EV 3T3-L1 cells were differentiated or not (À) with submaximal conditions: IBMX alone (M) or dexametasone and insulin (DI) or with full differentiation cocktail (MDI). Cells were stained with Oil Red-O to visualize lipid droplets 8 days after induction. Staining was quantified at 540 nm and expressed as the percentage of EV (À). *Po0.05, ***Po0.001 versus EV. (b) Fabp4 and (c) Pparg and mRNA levels, normalized to 18S rRNA levels, were measured using real-time RT-PCR at the indicated hours of differentiation in SFRP1 (black circles and bars) and EV (white circles and bars) cells differentiated with DI. *Po0.05, **Po0.01 versus EV.

SFRP1 expression in human adipose tissue tends to rise in states of mild obesity, peaking at a BMI of We next investigated whether the results observed in rodents 30–35 kg mÀ2, and gradually falls back to baseline levels in could be extrapolated to obesity and metabolic dysregulation individuals with higher BMIs. Although the limited sample in humans. First, we profiled SFRP1 expression in adipose size in the 30–35 kg mÀ2 BMI category (n ¼ 3 subjects) tissue in a cohort of 31 nondiabetic white European female prevented ANOVA analysis from achieving statistical sig- subjects across a range of BMI (Supplementary Table 1). On nificance (P ¼ 0.059), a second independent cohort of the basis of our previous observations of murine Sfrp1 subjects with BMI between 25 and 30 kg mÀ2 further expression during diet-induced obesity (Figure 4c), we substantiated this observation (Figure 5b). Specifically, postulated that SFRP1 expression in human adipose tissue microarray expression analysis was performed on subcuta- would also vary in a manner that would be associated with neous adipose tissue from 13 pairs (8 male pairs and 6 female (a) the degree of adiposity (that is, BMI), (b) the remaining pairs) of monozygotic twins discordant for weight.22 The potential of adipose tissue for further expansion and/or average BMI was 25.17 kg mÀ2 (95% confidence interval (c) the metabolic status (insulin sensitivity). As shown in (CI): 24.5–25.9 kg mÀ2) and 30.57 kg mÀ2 (95% CI: 29.5– Figure 5a, the profile of SFRP1 expression in human WAT 31.5 kg mÀ2) for lean and obese twins, respectively

International Journal of Obesity SFRP1 and adipogenesis C Lagathu et al 1701 * 100 *** 300 ** *** 250 rRNA rRNA 200 18S 18S 50 150 P=0.07 mRNA/

mRNA/ 100 50 Sfrp1

Sfrp1 0 0 Fed Fed Fasted Refed Fasted Refed

200 ** 140 120 rRNA

rRNA 150 100 18S

18S NS 80 100 ** 60 ***

mRNA/ ** mRNA/ 50 40 20 Sfrp1 0 Slc2a4 0 Chow HFD Chow HFD Chow HFD Chow HFD Chow HFD Chow HFD 3 days 4 weeks 6 months 3 days 4 weeks 6 months

500 200 400 160 * rRNA 300 120 18S

200 80 mRNA/ 100 40 Sfrp1

Fat Pad weight (% of Chow) weight Pad Fat 0 0 Ctrl TZD 0 5 10 15 20 25 treated Weeks on high-fat diet

Figure 4 Sfrp1 is regulated by nutritional signals, genetic obesity and metabolic status in vivo. Sfrp1 mRNA levels, normalized to 18S rRNA, were measured in (a) whole epididymal adipose tissue and (b) mature adipocytes from 8-week-old male C57/Bl6 mice challenged under the following conditions: fed (n ¼ 8–9), fasted (24 h) (n ¼ 7–8) and refed (24 h) (n ¼ 7–8). *Po0.05, **Po0.01, ***Po0.001. (c) Sfrp1 mRNA and (d) Glut4 (Slc2a4) mRNA levels were measured in whole adipose tissue from C57/Bl6 mice fed either chow diet or high-fat diet for 3 days (acute), 4 weeks (short term) or 6 months (long term) post weaning (n ¼ 7–8). **Po0.01, ***Po0.001 versus chow-fed mice. (e) Changes in epididymal fat pad weights during HFD feeding relative to chow feeding (f) Expression data from 4-month-old C57/Bl6 mice fed with rodent chow supplemented with or without rosiglitazone (thiazolidinedione) for 3 weeks (n ¼ 9–10). *Po0.05 versus untreated mice.

0.6 800 ** 0.5

0.4 600

0.3 400 relative levels relative levels 0.2 200

SFRP1 0.1 SFRP1 0 0 Lean Obese <25 25-30 30-35 35-40 >40 Twin Twin BMI Category

Figure 5 Regulation of SFRP1 in human adipose tissue. (a) SFRP1 mRNA levels, normalized to cyclophilin A, in subcutaneous adipose tissue of 31 white European females (Group B). For all subjects, BMI cutoff follows WHO classification (BMIo25, n ¼ 6; BMI 25–30, n ¼ 5; BMI 30–35, n ¼ 3; BMI 35–40, n ¼ 5 and BMI440, n ¼ 12). Analysis of variance (ANOVA) did not reach statistical significance, P ¼ 0.059. (b) Microarray SFRP1 expression were measured in subcutaneous adipose tissue of 13 pairs of monozygotic twins discordant for weight (Group C). Paired t-test, **P ¼ 0.005.

International Journal of Obesity SFRP1 and adipogenesis C Lagathu et al 1702 (Po0.001). This degree of obesity falls in the region where Our results from mouse models indicate that Sfrp1 is the previous data would suggest a correlation between a rise upregulated in vivo during anabolic states, leading to fat in SFRP1 levels and BMI. Despite the mean difference in BMI deposition such as refeeding and activation of the proadipo- (between the lean and obese twin within each pair) being genic program by thiazolidinedione treatment. Induction of as little as 5.39 kg mÀ2, SFRP1 levels in WAT showed a Sfrp1 expression is also a feature of the early stages of HFD- significantly higher expression in the obese twins (P ¼ 0.005) induced obesity, particularly when fat accretion rate is at its (Figure 5b). These findings are consistent with the data peak and the rate of adipose expansion, with respect to total generated from our rodent models and support the hypoth- adipose mass, is at its highest. However, just as increase in fat esis that SFRP1 expression increases when BMI rises, but with mass expansion reaches a plateau despite continued positive sustained positive energy balance, and despite marked energy balance, the program of adipogenesis in vivo is adipose expansion, the adipose tissue is no longer able to unlikely to be sustained indefinitely. Intriguingly, it is under maintain a high level of expression and the levels of SFRP1 these circumstances that we observed a relative downregula- gradually fall. tion of Sfrp1 expression (after 6 months of exposure to HFD). These observations suggest that Sfrp1 exerts a modulatory role on Wnt signaling in immature adipocytes and in preadipocytes, which may ensure the timely recruitment Discussion and development of adipose tissue to accommodate a surplus of energy in the early phases of the evolution toward Our group, together with others, has previously shown that obesity. Moreover, the biphasic profile ending in marked the Wnt/b-catenin signaling pathway can regulate adipogen- downregulation of SFRP1 when the adipose tissue mass is no esis both in vitro and in vivo (reviewed in Prestwich and longer expanding (but interestingly is also more metaboli- Macdougald8 and Christodoulides et al.9). Many of these cally compromised) suggests that the subsequent down- studies have focused on genetic and/or pharmacological regulation of Sfrp1 with sustained caloric excess may reflect manipulation of Wnt/b-catenin signaling components in the fact that this adipose tissue has reached its physiological murine models. However, recently we have sought to extend limit of expansion. To date, only Dact1 has been reported to these investigations to better understand the role of exhibit such a profile, although slightly delayed. In contrast, endogenous Wnt signaling in adipose tissue plasticity in the expression of closely related Sfrp5 in the same tissues rodents and humans. Recently, we reported on a nutrition- continues to increase as nutritional surplus continues.23 ally regulated, preadipocyte gene, Dact1, which promotes Taken together, these data suggest that the biphasic adipogenesis through coordinated effects on gene expres- phenomenon is restricted to specific components of the sion. This results in the selective alteration of both Wnt/b-catenin signaling. intracellular and paracrine/autocrine components of the Despite the well-established role for Wnt/b-catenin signal- Wnt/b-catenin signaling pathway.23 Furthermore, the ex- ing during adipogenesis in vitro and in vivo, an important pression and activity of Wnt/b-catenin signaling network aspect that remains less well characterized is whether the seems to be context- and species dependent, indeed some same signals have a role in regulating human adipose tissue components such as Dkk1 appear to be expressed in human mass. We show that endogenous SFRP1 is also regulated in adipose tissue, but not in murine models.12 human adipose tissue and that it is predominantly expressed Here, we show that SFRP1 may also be an important in adipocytes. Furthermore, our cross sectional studies extracellular modulator of Wnt/b-catenin signaling and is suggest that SFRP1 expression may exhibit a biphasic profile itself regulated during adipogenesis and adiposity. We have in human adipose tissue, similar to that observed in rodents. shown that SFRP1 is dynamically regulated during both Specifically, human adipose SFRP1 expression rises in human and murine adipogenesis and also promotes adipo- individuals showing mild obesity (BMI between 25 kg mÀ2 cyte differentiation in vitro. A proadipogenic role for SFRP1 is and 30 kg mÀ2) and progressively falls thereafter during the consistent with the reduced adiposity of SFRP1-null mice.20 evolution toward morbid obesity. Clearly, complete valida- More importantly, we also present evidence that endogenous tion of this model in humans would require a prospective SFRP1 is acutely upregulated in response to refeeding and study, which is currently not feasible. short-term HFD in vivo in WAT. This places SFRP1 as a strong We have already shown that WNT signaling components candidate that contributes to the titrated adipose tissue found in adipose tissue are not the same in mice and men.12 response to short-term nutritional challenges. However, our Indeed, this may be pertinent here as human and murine data also supports the concept that the same homeostatic SFRP1 share only 55% identity at the amino acid level and, system that regulates adipose tissue expansion and function we find that unlike the data from our murine models of diet- is limited and can fail under chronic nutritional surplus, as induced obesity, the relative SFRP1 levels in WAT from found in morbid obesity. Beyond a set limit of adipose tissue morbidly obese individuals (BMI 440 kg mÀ2) do not fall mass, the capacity for further adipose tissue expansion is below that of lean individuals (BMI o25 kg mÀ2). However, a exhausted, which we believe results in the metabolic more careful interrogation reveals that both experiments complications associated with obesity. may not be directly comparable. In fact a key difference is

International Journal of Obesity SFRP1 and adipogenesis C Lagathu et al 1703 that after 6 months of HFD, C57Bl6 mice become severely In obese insulin-resistant states, localized adipose tissue insulin resistant and have impaired glucose tolerance, inflammation occurs and may contribute to limiting further whereas the cross sectional human study recruited indivi- adipose tissue expansion. Indeed, we and others have shown duals with normal glucose tolerance. Given the impaired that proinflammatory cytokines found in insulin-resistant glucose tolerance of the 6-month HFD-fed mice, a better states can impair adipogenesis by converging with Wnt/b- comparison between the human and murine obese groups catenin signaling.11,26 There is also evidence of impaired may be made between the 4-week HFD-fed mice and the adipogenic capacity in human obesity.27–29 The elevated obese individuals (Figure 4c versus Figure 5a). Although a inflammatory cytokines found in insulin-resistant states of complete metabolic profile is not available for this human obesity may provide an explanation for the reduced levels of data set and only a small number of female subjects were SFRP1 found in the C57Bl6 mice fed with a HFD for 6 studied, it is clear that this human data cannot be used alone months. To confirm this relationship in human WAT, it to represent the general population. However, the findings would be necessary to have access to adipose tissue from from this first study are consistent with (a) the in vitro data, severely obese patients with untreated and/or uncontrolled (b) the murine data and (c) the human twin study data. diabetes. That being said, this does not preclude the Therefore, we believe that the observed trend is supportive of possibility that the apparently ‘normal’ levels of SFRP1 a role for SFRP1 in adipose tissue expansion. Taken together, observed in morbid obesity should be considered inappro- it provides a compelling basis for the focus of future studies to priately low for the degree of adipose expansion of these further analyze/explore these novel observations. individuals. In this respect, these inappropriate levels of

BMI Time in positive energy Balance Time on high calorific diet (HFD)

Nutritional/Fuel surplus

Adiposity/Obesity/Weight gain

β Glucose intolerence / Insulin resistance -Cell Failure/Diabetes

sFRP1

Titrated Adipocyte Adipocyte Adipose Recruitment hypertrophy inflammation Adipocyte (adipogenesis & (Macrophage Hypertorphy /hyperplasia) Hyperplasia recruitment)

Limit of expansion Impaired Adipose Tissue Max. storage capacity Function - Metabolic dysregulation

sFRP1

Adipocyte Preadipocyte Adipogenesis Maturation M1-Macrophage

Mature adipocyte Wnt10B/3A Immature adipocyte sFRP1

sFRP1

Figure 6 Postulated role of SFRP1 action in adipose tissue plasticity.

International Journal of Obesity SFRP1 and adipogenesis C Lagathu et al 1704 SFRP1 may reflect the biological limit to which these References individuals can expand their fat depot. Overall, our results suggest a model of adipose tissue 1 Despres JP, Lemieux I. Abdominal obesity and metabolic expansion characterized by upregulation of SFRP1 in early syndrome. Nature 2006; 444: 881–887. 2 Sethi JK, Vidal-Puig AJ. Thematic review series: adipocyte biology. stages of obesity. This elevation of SFRP1 could inhibit Wnt Adipose tissue function and plasticity orchestrate nutritional signaling and, therefore, facilitate the adipose tissue expan- adaptation. J Lipid Res 2007; 48: 1253–1262. sion, allowing nutrient storage demands to be met. Further 3 Virtue S, Vidal-Puig A. It’s not how fat you are, it’s what you do weight gain results in SFRP1 levels falling back to the same with it that counts. PLoS Biol 2008; 6: e237. 4 Tan CY, Vidal-Puig A. 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J Cell Sci 1999; 112 (Pt 21): Conflict of interest 3815–3820. 19 Galli LM, Barnes T, Cheng T, Acosta L, Anglade A, Willert K et al. Differential inhibition of Wnt-3a by Sfrp-1, Sfrp-2, and Sfrp-3. Dev The authors declare no conflict of interest. Dyn 2006; 235: 681–690. 20 Bodine PV, Zhao W, Kharode YP, Bex FJ, Lambert AJ, Goad MB et al. The Wnt antagonist secreted frizzled-related protein-1 is a Acknowledgements negative regulator of trabecular bone formation in adult mice. Mol Endocrinol 2004; 18: 1222–1237. 21 Ortega FJ, Mayas D, Moreno-Navarrete JM, Catalan V, Gomez- We thank the Addenbrooke’s hospital surgeons for perform- Ambrosi J, Esteve E et al. The gene expression of the main ing the adipose tissue biopsies. This research is funded by lipogenic enzymes is downregulated in visceral adipose tissue of the Biotechnology and Biological Science Research Council obese subjects. Obesity (Silver Spring) 2009; 18: 13–20. (BBSRC), the Medical Research Council (MRC) and the 22 Pietilainen KH, Naukkarinen J, Rissanen A, Saharinen J, Ellonen P, Keranen H et al. Global transcript profiles of fat in monozygotic European Union Sixth Framework Programme on Hepatic twins discordant for BMI: pathways behind acquired obesity. PLoS and Adipose tissue functions in the metabolic syndrome Med 2008; 5: e51. (EU-FP6 HEPADIP) (AV-P and JKS). We also acknowledge the 23 Lagathu C, Christodoulides C, Virtue S, Cawthorn WP, Franzin C, support received from ALFEDIAM and Marie Curie Post- Kimber WA et al. Dact1, a nutritionally regulated preadipocyte gene, controls adipogenesis by coordinating the Wnt/beta- doctoral fellowship (CL), MRC Doctoral Studentships (CC catenin signaling network. Diabetes 2009; 58: 609–619. and SV), MRC Career Establishment Award (AV-P) and BBSRC 24 Medina-Gomez G, Virtue S, Lelliott C, Boiani R, Campbell M, David Phillips Fellowship (JKS). Christodoulides C et al. The link between nutritional status and

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International Journal of Obesity