Fibroblast Growth Factor 1 a Key Regulator of Human Adipogenesis

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Fibroblast Growth Factor 1 A Key Regulator of Human Adipogenesis Louise Hutley,1,2 Wenda Shurety,1 Felicity Newell,1 Ross McGeary,3 Nicole Pelton,1 Jennifer Grant,1 Adrian Herington,4 Donald Cameron,5 Jon Whitehead,1 and Johannes Prins1,2

Obesity, with its related problems, is recognized as the fastest growing disease epidemic facing the world, yet we still have limited insight into the regulation of t is established that prevention or treatment of adipose tissue mass in humans. We have previously obesity plays a significant role in reducing rates of shown that adipose-derived microvascular endothelial cardiovascular disease, type 2 diabetes, and ortho- cells (MVECs) secrete a factor(s) that increases prolif- pedic disease, all of which impact considerably on eration of human preadipocytes. We now demonstrate I health care and social costs. To develop more sustainable that coculture of human preadipocytes with MVECs significantly increases preadipocyte differentiation, ev- therapies for the treatment of obesity, a better understand- idenced by dramatically increased triacylglycerol accu- ing of human adipose tissue growth is essential. mulation and glycerol-3-phosphate dehydrogenase Expansion of adipose tissue in obesity is preceded by activity compared with controls. Subsequent analysis the development of new capillaries (1). These new capil- identified fibroblast growth factor (FGF)-1 as an adipo- laries are formed by the migration and replication of genic factor produced by MVECs. Expression of FGF-1 microvascular endothelial cells (MVECs) from established was demonstrated in MVECs but not in preadipocytes, blood vessels (2). Studies in fetal adipose tissue suggest while preadipocytes were shown to express FGF recep- both spatial and temporal relationships between adipogen- tors 1–4. The proliferative effect of MVECs on human esis and angiogenesis (3). Therefore, paracrine interaction preadipocytes was blocked using a neutralizing antibody specific for FGF-1. Pharmacological inhibition of between MVECs in the developing capillary network and FGF-1 signaling at multiple steps inhibits preadipocyte preadipocytes resident in the adipose tissue depots may replication and differentiation, supporting the key adi- play a role in the regulation of adipose tissue growth. We pogenic role of FGF-1. We also show that 3T3-L1 cells, a have previously reported that MVECs derived from human highly efficient murine model of adipogenesis, express adipose tissue secrete a factor(s) that stimulates prolifer- FGF-1 and, unlike human preadipocytes, display no ation in human subcutaneous and omental (intraabdomi- increased differentiation potential in response to exog- nal) preadipocytes (4). This proliferative effect of MVECs enous FGF-1. Conversely, FGF-1–treated human preadi- on human preadipocytes was significantly greater from pocytes proliferate rapidly and differentiate with high endothelial cells isolated from adipose tissue than from efficiency in a manner characteristic of 3T3-L1 cells. We dermally derived MVECs (4), suggesting that endothelial therefore suggest that FGF-1 is a key human adipogenic factor, and these data expand our understanding of cells from human adipose tissue have unique activities human fat tissue growth and have significant potential necessary for preadipocyte proliferation. for development of novel therapeutic strategies in the For an increase in adipose tissue mass to occur, prolif- prevention and management of human obesity. Diabetes eration of preadipocytes needs to be followed by differen- 53:3097–3106, 2004 tiation of these cells to the mature adipocyte phenotype (5). This is an energy-requiring, regulated process characterized in vitro by the sequential acquisition of protein expression patterns and morphological features (6). Intrin- From the 1Department of Diabetes and Endocrinology and University of Queensland Department of Medicine, Princess Alexandra Hospital, Wool- sic to the process is the development of the potential to loongabba, Australia; 2Adipogen, University of Queensland, St. Lucia, Austra- store energy in the form of triglyceride, development of lia; the 3Chemistry Department and School of Pharmacy, University of Queensland, Brisbane, Australia; the 4Centre for Molecular Biotechnology, the capacity for insulin-stimulated glucose uptake, and School of Life Sciences, Queensland University of Technology, Brisbane, production of characteristic proteins such as glycerol-3- Australia; and the 5Princess Alexandra Hospital Centres for Health Research, phosphate dehydrogenase (G3PDH) (7) and leptin (8,9). Princess Alexandra Hospital, Woolloongabba, Australia. Address correspondence and reprint requests to Dr. Louise Hutley and Prof. While the transcription factors peroxisome proliferator– Johannes Prins, Department of Diabetes and Endocrinology and University of activated receptor (PPAR)-␥ and CCAAT enhancer binding Queensland Department of Medicine, Princess Alexandra Hospital, Ipswich protein-␣ are known to be master regulators of this Road, Woolloongabba, Qld 4102, Australia. E-mail: [email protected] and [email protected]. differentiation process (10), factors affecting the earliest Received for publication 5 May 2004 and accepted in revised form 7 stages of adipocyte differentiation are yet to be defined. September 2004. J.P. is the Scientific Director of Adipogen. Furthermore, elucidation is needed of the factors regulat- ECGF, endothelial cell growth factor; FGF, fibroblast growth factor; FGFR, ing the mechanisms involved in cellular, metabolic, and FGF receptor; G3PDH, glycerol-3-phosphate dehydrogenase; MVEC, micro- regional differences among adipose tissue depots and how vascular endothelial cell; PPAR, peroxisome proliferator–activated receptor; TZD, thiazolidinedione. these factors may impact on the adipocyte hyperplasia © 2004 by the American Diabetes Association. characteristic of massive obesity.

DIABETES, VOL. 53, DECEMBER 2004 3097 FGF-1: KEY REGULATOR OF HUMAN ADIPOGENESIS

FIG. 1. Differentiation of human preadipocytes cocultured with adipose-derived MVECs. Differentiated human preadipocytes (subcutaneous) either cultured alone (A) or cocultured for 2 ;␮m 10 ؍ weeks with MVECs (B) (bar original magniﬁcation ؋100). G3PDH activity in subcutaneous (C) and omental (E) preadipocytes cultured either alone (preadipocytes [PA]) or in coculture with MVECs (preadipocytes plus -endothelial cells [PA؉EC]) (subcutane ,4 ؍ omental, n ;0.001 ؍ P ,6 ؍ ous, n -medium 1, serum-contain) (0.003 ؍ P ing preadipocyte growth medium; medium 2, medium 1 plus 10 ng/ml ␤-ECGF). Nile Red assay for nonpolar lipid in subcutaneous (D) and omental (F) cocultured preadipocytes (subcuta- ,3 ؍ omental, n ;0.006 ؍ P* ,3 ؍ neous, n .(0.001 ؍ P*

␤ We therefore further explored the effects of adipose- 0.15% NaHCO3 (wt/vol), with or without growth factors (all human): -endo- derived MVECs on human adipogenesis using coculture thelial cell growth factor (ECGF) (at 10 ng/ml) (Sigma), FGF-1, FGF-2, or IGF-1 (at 1 ng/ml) (R&D Systems). In some experiments, FGF inhibitors techniques and demonstrated that MVECs not only stimu- (anti–FGF-1 neutralizing antibody; R&D Systems) (10 ␮g/ml), the synthetic late proliferation of preadipocytes but also markedly in- ␮ ␮ peptide Ac-ValTyrMetSerProPhe-NH2 (10 g/ml), and suramin (20 mol/l) crease their differentiation potential. Subsequent work were used in the presence of FGF-1 to determine the effects on both identified a member of the fibroblast growth factor (FGF) proliferation and differentiation. Human dermal fibroblasts were isolated and family as a factor having profound effects on human cultured as previously described (13). These cells were used as controls in the preadipocyte proliferation and differentiation in vitro. coculture studies. MVECs plus preadipocytes. For coculture experiments, preadipocytes were seeded at low cell density with homogeneous MVECs (each cell type plated at RESEARCH DESIGN AND METHODS approximately one-third confluence at opposite ends of the culture dish) and Unless otherwise stated, all chemicals used were obtained from Sigma-Aldrich the mixed cultures allowed to grow together for 2–6 weeks in endothelial cell (Castle Hill, Australia). Goat anti-human polyclonal FGF-1 neutralizing anti- growth medium containing heparin and 10 ng/ml ␤-ECGF. Controls included body was purchased from R&D Systems (Minneapolis, MN). Mouse anti- preadipocytes plus human skin fibroblast cocultures and preadipocyte-only human monoclonal FGF-1 antibody (Sigma) was used in Western blotting cultures grown under the same conditions as the MVEC plus preadipocyte procedures. The thiazolidinedione (TZD) compound rosiglitazone was sup- cultures. plied by GlaxoSmithKline (Middlesex, U.K.). The FGF inhibitory peptide 3T3-L1 adipocytes. 3T3-L1 fibroblasts were grown to confluence in serum-

AcValTyrMetSerProPheNH2 was prepared by manual synthesis using Fmoc containing medium consisting of Dulbecco’s modified Eagle’s medium/HAM’s chemistry on Rink Amide MBHA resin. After washing and drying, the peptide F12 (1:1) (ICN Biomedical Australasia) containing 4.5 g/l glucose, 10% FCS, was cleaved using TFA/water/triisopropylsilane (95:2.5:2.5) and purified by 100 IU penicillin, 100 ␮g/ml streptomycin, and 2 mmol/l glutamine. In some reverse-phase high-performance liquid chromatography on a Vydac C-18 experiments FGF-1, FGF-2, or IGF-1 (all at 1 ng/ml) (R&D Systems) were column using aqueous acetronitrile containing 0.1% formic acid. included in the growth medium. Differentiation was initiated by addition of Paired omental (intra-abdominal) and subcutaneous adipose tissue biop- 250 nmol/l dexamethasone, 0.5 mmol/l of 1-methyl-3-isobutylxanthine, 1 sies were obtained from 17 male (average age 63.5 years [range 37–82]; ␮mol/l insulin, and 0.1 ␮mol/l rosiglitazone. After 2 days, cells were trans- average BMI 27.2 kg/m2 [21–39]) and 25 female (average age 51.2 years ferred to serum-containing medium containing 0.5 ␮mol/l insulin and refed [31–77]; average BMI 30 kg/m2 [20.5–47.9]) patients undergoing elective twice weekly. Cells were studied at 13–19 days postdifferentiation. To open-abdominal surgery. None of the patients had diabetes or severe systemic determine the role of endogenous FGF-1 in 3T3-L1 differentiation, cells were illness, and none were taking medications known to affect adipose tissue mass treated with anti–FGF-1 neutralizing antibody (10 ␮g/ml) for 1 week before or metabolism. The protocol was approved by the research ethics committees and throughout the differentiation period and effects assessed by G3PDH of the Princess Alexandra Hospital and the Queensland University of Tech- activity assay. nology. All patients gave their written informed consent. Adipose differentiation protocol. As previously reported, confluent prea- Isolation of stromal-vascular cells. Preadipocytes, adipocytes, and MVECs dipocytes at passage 2 were changed to a chemically defined serum-free were obtained from adipose tissue biopsies as previously reported (4,11,12). medium with or without rosiglitazone for induction of differentiation (4,6,12– Cell culture. Preadipocytes and MVECs were isolated, cultured, and charac- 14). In some experiments, growth factors were included in the differentiation terized as previously reported (4). Homogeneous cultures of both cell types medium at a concentration of 1 ng/ml. were grown for up to 8 weeks and maintained at 37°C with 5% CO2. Cells were Assessment of adipose differentiation. After 21 days in serum-free differ- used in experimental work at passages 2 and 3. For control and growth factor entiation medium, adipogenesis was assessed using a Nile Red assay for experiments, preadipocytes were grown in the same serum-containing (10%) triacylglycerol accumulation and an enzyme assay measuring G3PDH activity medium as the MVECs, containing 90 ␮g/␮l heparin, 0.014 mol/l HEPES, and (4,12,13). The Nile Red results are presented as optical density units at 480/540

3098 DIABETES, VOL. 53, DECEMBER 2004 L. HUTLEY AND ASSOCIATES

FIG. 2. Effect of growth factors on human preadipocyte differentiation, as measured by G3PDH activity. A: Six weeks with or without ␤-ECGF and in the absence or presence of coculture with MVECs before ;5 ؍ omental, n ;6 ؍ differentiation (subcutaneous, n *P > 0.1 for both subcutaneous and omental) (medium 1, serum-containing preadipocyte growth medium; medium 2, medium 1 plus 10 ng/ml ␤-ECGF). B: FGF-1 and -2 are potent adipogenic agents (FGF-1 ؍ more than FGF-2 in subcutaneous preadipocytes, P whereas no significant effect was ,(3 ؍ n ,0.003 demonstrated for IGF-1. C: The adipogenic effects of FGF-1 occurred subsequent to exposure of preadipocytes during proliferation. This effect was further enhanced when treatment with FGF-1 was main- 0.004 ؍ P ,3 ؍ tained throughout differentiation (n vs. proliferation only). nm, and G3PDH results are given as activity in milliunits per milligram of The homogenate was passed through a 21-gauge needle 10 times and a protein (Act mU/mg Pr). 22-gauge needle 4 times. The samples were cleared by centrifugation at 1,000g Preadipocyte proliferation assay. Following various treatments, prolifera- for 10 min at 4°C. Protein determination was performed using bicinchoninic tion of preadipocytes was assessed using an MTS proliferation assay (Pro- acid reagent (Pierce, Rockville, IL). Proteins (15 ␮g) were resolved on mega, Madison, WI) as described previously (4). SDS-polyacrylamide gels and transferred to Immobilon-P PVDF membranes Electrophoresis and Western blot analysis. FGF-1 expression was deter- (Millipore, Bedford, MA). Membranes were blocked with 2% (wt/vol) BSA/ mined in whole-cell protein isolated from human preadipocytes treated or not PBST overnight at 4°C, incubated with mouse anti-human monoclonal FGF-1 treated with FGF-1, human adipose–derived MVECs, 3T3-L1 preadipocytes antibody (Sigma), and probed with horseradish peroxidase–linked secondary (all these cell types were maintained in 10% serum-containing medium before antibodies (Amersham) for1hatroom temperature. Proteins were detected protein extraction), and isolated human adipocytes. Cells were harvested in by enhanced chemiluminescence substrate (Amersham). buffer containing 20 mmol/l HEPES, 1 mmol/l EDTA, phosphatase inhibitors (2 RT-PCR. Total RNA was obtained from both human subcutaneous adipose mmol/l Na3VO4, 1 mmol/l Na4P2O7, and 10 mmol/l NaF), and protease tissue and preadipocytes (at confluence and following 21 days of differentia- inhibitors (Complete Mini; Roche Diagnostics, Mannheim, Germany). Whole- tion). Total RNA was obtained using the TRIzol reagent extraction procedure cell lysates (15 ␮g) were resolved by SDS-polyacrylamide gel (8%) electro- (GIBCO/BRL) according to the manufacturer’s instructions. RNA was further phoresis, transferred to Immobilon-P PVDF membrane (Millipore, Bedford, purified using an RNAeasy column (Qiagen) as described by the manufacturer. MA), and probed with mouse anti-human monoclonal FGF-1 antibody An on-column DNase digest was performed as part of the purification (Sigma). Proteins were detected by enhanced chemiluminsence substrate protocol. Total RNA (1 mg) was reverse transcribed using random hexamers (Amersham, Little Chalfont, U.K.). and reverse transcription reagents from Applied Biosystems (Victoria). The

DIABETES, VOL. 53, DECEMBER 2004 3099 FGF-1: KEY REGULATOR OF HUMAN ADIPOGENESIS

FIG. 3. Inhibition of preadipocyte proliferation by neutralizing antibody specific for FGF-1. MTS assay demonstrates that proliferation of human preadipocytes in response to both FGF-1 (1 ng/ ml) and factors present in medium conditioned by adipose-derived MVECs (from both subcutaneous [S] and omental [O] sites) is completely abro- gated in the presence of neutralizing antibody and * ,4 ؍ Ab) specific for FGF-1 (10 ␮g/ml) (n) Specificity of the inhibitory effect .(0.05> ؍ P** of the FGF-1 antibody was demonstrated using a control IgG that had no effect on preadipocyte proliferation in these studies (data not shown). Results are presented as an increase in absor- bance units (490 nm) over controls. absence of contaminating genomic DNA was verified by including samples to medium (medium 1) or the same medium with added which no reverse transcriptase was added (no reverse transcriptase controls). ␤-ECGF (medium 2; ␤-ECGF is required for maintenance For PCR, no template controls confirmed the absence of PCR contamination. Fibroblast growth factor receptor (FGFR)-1 to -4 primer sequences were those of endothelial cells in the coculture conditions). This was used by Tartaglia, Fragale, and Battaglia (15). Thermal cycling conditions the case for preadipocytes from both subcutaneous were as follows: first denaturing step of 94°C for 8 min; 35 cycles of 94°C for (G3PDH, Fig. 1C; triacylglycerol, Fig. 1D) and omental 45 s, 60°C for 30 s, and 72°C for 30 s; and a final extension step of 72°C for 10 (G3PDH, Fig. 1E; triacylglycerol, Fig. 1F) adipose tissue min. FGFR cDNA (in pcDNA3.1) was a kind gift from Pamela Maher (Scripps Research Institute, La Jolla, CA) and was used as a positive control for each depots. While the rise in G3PDH activity in subcutaneous reaction. preadipocytes was greater than that seen in cells from Statistics. Data were analyzed using repeated-measures ANOVA for differ- omental tissue, the degree of differentiation of omental ences across experimental groups. Student’s t test was used to evaluate the cells in coculture was significant. To our knowledge, this significance of the difference in mean values between different treatments. Data are expressed as means Ϯ SE. represents the first time quantifiable and consistent differentiation that has been demonstrated in intraabdominal RESULTS preadipocytes following prolonged culture in the presence Adipocyte differentiation. Human MVECs and preadi- of serum. After treatment with the same differentiation pocytes were isolated, cultured, and characterized as medium as human preadipocytes, G3PDH activity was previously described (4). Once phenotype was established, negligible in human skin fibroblasts cocultured with adi- these two cell types were used in coculture experiments to pose-derived MVECs (data not shown). study involvement of MVECs in human adipogenesis. FGF-1 in human adipogenesis. To determine whether At confluence following a 2-week coculture period, all the differentiation potential of human preadipocytes could cultures were changed to serum-free medium containing be further enhanced, coculture with MVECs was extended known inducers of adipocyte differentiation, including the to a period of 6 weeks before induction of differentiation. PPAR-␥ ligand rosiglitazone. In preadipocyte-only cul- Following 21 days in differentiation medium, after the tures, in accordance with our previous observations using coculture period, both G3PDH activity and triacylglycerol standard human differentiation techniques (13), lipid ac- accumulation were once again significantly increased in cumulation was apparent in a small number of cells by day cocultured preadipocytes over that demonstrated in prea- 7–10, with 5–25% of cells acquiring lipid by 21 days (Fig. dipocytes previously maintained as homogeneous cultures 1A). In marked contrast, triacylglycerol accumulation was in regular serum-containing growth medium (medium 1) apparent as early as day 3–5 in the majority of cocultured (G3PDH, Fig. 2A). Surprisingly, however, both parameters preadipocytes, with 70–95% of cells accumulating large of differentiation were also elevated to the same high amounts of triacylglycerol by day 21 (Fig. 1B). Evidence levels as those measured in the cocultured cells in prea- for the specificity of these effects for human preadipocytes dipocyte-only cultures maintained for 6 weeks in serum- came from observations showing that human skin fibro- containing medium supplemented with ␤-ECGF (medium blasts cultured either alone or in coculture with MVECs, 2) (Fig. 2A). These results suggest that ␤-ECGF is acting as under identical conditions as the preadipocytes, did not an adipogenic agent under these conditions. The presence accumulate lipid when treated with differentiation me- of heparin in medium 2 was required for maximal ␤-ECGF dium (data not shown). activity, but heparin, in the absence of growth factor, had To confirm that the lipid-containing morphology of no effect on differentiation of human preadipocytes (data these cells was consistent with triacylglycerol accumula- not shown). tion and biochemical differentiation, Nile Red assay for ␤-ECGF is a member of the FGF family and is a nonpolar lipid and G3PDH activity assays were performed. precursor molecule of FGF-1 (16). To further investigate The results demonstrate that preadipocytes from the the role of growth factors in this system, differentiation of MVEC plus preadipocyte coculture had significantly in- preadipocytes was carried out following a 6-week prolif- creased G3PDH activity and triacylglycerol accumulation eration period with or without FGF-1, FGF-2, or IGF-1 (at following 21 days in differentiation medium over preadi- 1 ng/ml). Results demonstrated that pretreatment with pocytes cultured alone in both regular serum-containing FGF-1 and FGF-2 but not IGF-1 resulted in significant

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FIG. 4. An activator of PPAR-␥ during differentiation is required for maximal promotion of G3PDH activity in response to FGF-1 during growth. While G3PDH activity demonstrates a relatively small but significant increase in the differentiation potential of FGF- 1–treated human preadipocytes in the absence of ,3 ؍ TZD (with no depot specificity of this effect) (n #P < 0.01 by ANOVA), the maximal adipogenic effect of FGF-1 was only seen when cells were differenti- ,3 ؍ ated in the presence of the TZD rosiglitazone (n * and **P < 0.05). There was depot specificity in the TZD effects, with subcutaneous (S) greater than omental (O). increases in G3PDH activity in human preadipocytes, struct assays, which showed no endogenous PPAR re- relative to controls, with FGF-1 having the greatest effect sponse element binding or activation of PPAR-␥ (data not in subcutaneous preadipocytes (Fig. 2B). The differentia- shown) in response to FGF-1 treatment, and TZD-induced tion potential of these cells could be further enhanced if PPAR-␥ activity was not further enhanced in the presence exposed to FGF-1 during both proliferation and differen- of FGF-1 (data not shown). tiation (Fig. 2C). Intriguingly, preadipocytes treated with FGF-1 expression in human adipose tissue. Using a FGF-1 only during the differentiation period exhibited a combination of Western blot analysis and immunofluores- trend to a decrease in both G3PDH activity (Fig. 2C) and cence, robust expression of FGF-1 was demonstrated in lipid accumulation following differentiation compared adipose-derived MVECs and 3T3-L1 cells. In constrast, with cells grown in the absence of FGF-1. The time course expression in human preadipocytes, maintained under the of action of FGF-1 suggests that its effect is to prime same conditions, was low to undetectable (although this preadipocytes for subsequent differentiation rather than to does not rule out the possibility of expression at very low act as a direct inducer of differentiation. levels). Expression in isolated human adipocytes was Preadipocyte proliferation. The proliferation rate of more variable than in preadipocytes but, if detectable, was human preadipocytes is increased in response to FGF-1, always lower than that seen in either 3T3-L1 cells or and this effect can be blocked by addition of a commer- human MVECs (Fig. 5). Gabrielsson et al. (18) demon- cially available neutralizing antibody raised against FGF-1 strated FGF-1 expression in the stromovascular fraction of (Fig. 3). To establish more directly whether FGF-1 may human adipose tissue, which contains both MVECs and represent an adipogenic factor produced by MVECs, we preadipocytes. Our findings suggest that the FGF-1 expres- used the same neutralizing anti–FGF-1 antibody to block sion in this adipose tissue fraction is predominantly con- any endogenous FGF-1 actions in MVEC-conditioned me- tributed to by the MVEC component. dium. Proliferation assays were carried out and, in agree- FGFR expression in human adipose tissue. FGFR ment with our previous results (4), preadipocyte expression in human samples was determined using RT- proliferation was enhanced in response to medium that PCR (Fig. 6). In subcutaneous adipose tissue, expression had been conditioned by 48-h exposure to adipose-derived of FGFR-1 and -2 was detected. Similar expression pat- MVECs from both subcutaneous and omental sites (Fig. 3). terns were demonstrated in differentiated human preadi- Inclusion of the FGF-1 neutralizing antibody at 10 ␮g/ml to pocytes, with R1 and R2 being present, whereas little, if the MVEC-conditioned medium resulted in almost total any, expression of R3 and R4 was detected. In contrast, all abrogation of these proliferative effects (Fig. 3). four FGFRs were found to be expressed in nondifferenti- PPAR-␥ involvement in FGF-1–induced adipogenesis. ated human preadipocytes (Fig. 6). Western blotting and Significant, but relatively small, increases in G3PDH activ- immunofluorescence techniques also demonstrated ex- ity were demonstrated in FGF-1–treated preadipocytes pression of FGFR-1 to -4 in human nondifferentiated from both subcutaneous and omental adipose tissue dif- preadipocytes (data not shown). In 3T3-L1 cells, expres- ferentiated in the absence of PPAR-␥ ligand (Fig. 4). sion of all four receptors has been observed by RT-PCR or However, the maximal adipogenic effect of either cocul- Western blotting (data not shown). ture with MVECs or treatment with FGF-1 was only Effects of FGF inhibitors on human preadipocyte pro- observed when cells were differentiated in the presence of liferation and differentiation. To determine whether the the PPAR-␥ ligand rosiglitazone (a TZD) (Fig. 4). Differen- adipogenic effects of FGF-1 can be blocked in vitro, tiation in the presence of the TZD exhibited depot speci- preadipocytes were treated with a combination of FGF-1 ficity, in agreement with our previous results and those of and FGF inhibitors and effects on both proliferation and others (13,14,17), with the effect being greater in subcuta- differentiation assessed. The inhibitors used included the neous than omental preadipocytes (Fig. 4). The require- synthetic peptide Ac-ValTyrMetSerProPhe-NH2 (peptide ment for TZD for maximal differentiation suggests that 1), which specifically binds to, and blocks, FGFRs (19); neither the factor(s) produced by MVECs nor exogenous suramin (a polysulfonated binaphthyl urea that inhibits FGF-1 are acting as PPAR-␥ ligands. Further evidence for complex formation among FGF, heparin, and cognate this came from electromobility shift and reporter con- receptor) (20); and a neutralizing antibody specific for

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FIG. 5. FGF-1 expression in adipose cells. Western blot analysis was used to determine FGF-1 expression in whole- cell lysates from murine cell line 3T3-L1 (lane 1), adipose- derived MVECs (lane 2), subcutaneous (lane 3) and omental (lane 4) non–FGF-1–treated preadipocytes, subcutaneous (lanes 5) and omental (lanes 6) FGF-1–treated preadipocytes, and subcutaneous (lanes 7) and omental (lanes 8) isolated adipocytes. Strong expression is seen at the ex- pected size of 18 kDa in cell lysates from both 3T3-L1 cells and human adipose–derived MVECs. Expression of FGF-1 in isolated human adipocytes and preadipocytes, from both depots, is low to undectable under these experimental conditions. No difference in FGF-1 expression was observed between FGF-1–treated or –nontreated preadipocytes.

FGF-1 (21). All of these compounds are reported to inhibit underpin their capacity to differentiate more readily in the mitogenic activity of FGF-1 (19–21). All three com- vitro than human preadipocytes. pounds demonstrated significant inhibition of FGF-1 pro- Differentiation of human preadipocytes in the pres- liferative effects (Fig. 3, anti–FGF-1; Fig. 7A, peptide 1 and ence of serum. In contrast to 3T3-L1 cells, human prea- 7B, suramin), while suramin and anti–FGF-1 neutralizing dipocytes are slow growing and differentiate inefficiently antibody also demonstrated inhibition of differentiation in vitro. Human preadipocytes also have more defined and (Fig. 7C, suramin and 7D, anti–FGF-1). These data support specific requirements for induction of differentiation than the role of FGF-1 as an adipogenic agent and show that the 3T3-L1 cells, including an absolute requirement for serum- adipogenic effects can be blocked in vitro. free conditions (6). Following treatment with FGF-1, how- Effects of growth factors on 3T3-L1 proliferation and ever, we observe that human preadipocytes display rapid differentiation. Investigation of adipogenic effects of growth characteristics and a high capacity for differentia- growth factors was also carried out using the 3T3-L1 tion that are more similar to 3T3-L1 cells than to non–FGF- murine model of adipogenesis which, unlike human prea- 1–treated preadipocytes. To determine whether growth dipocytes, display rapid growth and high differentiation factor treatment of human preadipocytes renders them capacity. Exposure of 3T3-L1 fibroblasts to FGF-1, FGF-2, capable of differentiating in the presence of serum, cells or IGF-1, before differentiation, caused no significant were proliferated in the presence of FGF-1, FGF-2, or increases in either growth, G3PDH activity, or triacylglyc- IGF-1 and induced to differentiate in serum-containing erol accumulation (data not shown) compared with the medium. Using a 3T3-L1 differentiation protocol consisting already high levels of both parameters seen in the absence of serum-containing medium (10% vol/vol), insulin, and, of growth factors (controls). For the FGF-1 and -2 treat- for the first 3 days, glucocorticoid and IBMX, FGF-1– ments, this was in direct contrast to results in human treated preadipocytes from both subcutaneous and omen- preadipocytes. Western blot analysis demonstrated ex- tal sites developed both morphological (Fig. 8A) and pression of FGF-1 in 3T3-L1 fibroblasts but not in human biochemical (Fig. 8B) features of a differentiated pheno- preadipocytes (Fig. 5). Also, treatment of 3T3-L1 cells with type. The effect of FGF-2 treatment during growth also anti–FGF-1 neutralizing antibody for 1 week before and lead to an increased capacity for differentiation in the throughout differentiation resulted in a significant de- presence of serum, but this effect, although similar to crease in G3PDH activity following differentiation com- FGF-1 treatment for omental preadipocytes, was much pared with controls (Fig. 7E). Hence the expression of less in subcutaneous preadipocytes than that measured in FGF-1 by the embryonically derived 3T3-L1 cells may FGF-1–treated cells. Preadipocytes treated with IGF-1 were similar in differentiation capacity to controls, with no lipid accumulation and negligible G3PDH activity (Fig. 8B). In our experience, this is the first time preadipocyte differentiation has been observed in cells maintained for an extensive period in, and subsequently differentiated in, the presence of serum.

DISCUSSION Knowledge of the cellular mechanisms implicated in the overdevelopment of adipose tissue is a critical issue in the design of pharmaceutical strategies aimed at lessening nutritional obesity. We have previously reported that adipose-derived MVECs produce factors that have a mitogenic effect on human preadipocytes. We now demonstrate that these endothelial cells also secrete fac- FIG. 6. FGFR expression in adipose cells. RT-PCR analysis demon- tors that signiﬁcantly increase the differentiation potential strates expression of mRNAs for FGFR-1 to -4 in human nondifferentiated preadipocytes. No mRNA expression of FGFR-3 and -4 was of preadipocytes from both subcutaneous and omental detected in human whole adipose tissue (subcut) or differentiated adipose tissue depots (subcutaneous more than omental). preadipocytes. Lane 1: No template control; lane 2: pcDNA3.1 (FGFR-1 We present data that indicate that much of the adipogenic to -4, positive control); lane 3: human adipose tissue (subcut); lane 4: human nondifferentiated preadipocytes; lane 5: human differentiated effect of MVECs can be attributed to FGF-1 secretion. preadipocytes/adipocytes. FGF-1 is a member of a large family (currently Ͼ22

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FIG. 7. FGF-1 inhibition results in decreases in preadipocyte proliferation and/or differentiation. MTS assay demonstrates increases in proliferation rate of FGF-1–treated human preadipocytes versus non–FGF-1–treated controls (Ctrl) (A and B:**P < 0.05 for control vs. FGF-1). This FGF-1–induced growth is attenuated in the presence of the FGF ؍ P* ,6 ؍ inhibitors peptide 1 (Pep 1) (10 ␮g/ml) (n ,6 ؍ A) and suramin (Sur) (20 ␮mol/l) (n) (0.03 B). Data in A and B are presented as) (0.02 ؍ P* both raw data (490 nm) (which shows control value) and (see inserts) as increase/decrease in absor- bance units versus control. Differentiation of human preadipocytes in response to FGF-1 is also abro- gated in the presence of FGF inhibitors. G3PDH activity was assessed in differentiated preadipocytes following exposure, both before and during differentiation, to FGF-1 and the inhibitors suramin P < 0.001) (C) and specific* ,6 ؍ ␮mol/l) (n 20) neutralizing anti–FGF-1 antibody (anti-FGF-1) (10 P < 0.001) (D). E: Exposure of* ,6 ؍ ␮g/ml) (n non–FGF-1–treated 3T3-L1 preadipocytes to a specific anti–FGF-1 neutralizing antibody from 10 days before induction through day 12 of differentiation results in a decrease in G3PDH activity in differen- .(0.05 ؍ P* ,3 ؍ tiated cells versus controls (n (Inserts in A and B show results for the FGF-1 and FGF-1 plus inhibitor treatments [A, peptide 1; B, suramin], presented as the increase/decrease in ab- sorbance units versus non–FGF-1–treated cells.) identified) of related growth factors (22). All FGFs act via as, or promoting production of, a PPAR-␥ ligand. It is a dual receptor system comprising four high-affinity ty- possible, however, that FGF-1 treatment of preadipocytes rosine kinase receptors (FGFR-1 to -4) and a class of may be acting on the PPAR-␥ system in other ways. These low-affinity receptors (the heparan sulfate proteoglycans) could include increased expression of PPAR-␥, as well as (23,24). Multiple layers of regulation control the activity of members of the CCAAT enhancer binding protein family of FGFs, including tissue-specific expression of ligands and transcription factors, before induction of differentiation. receptors, modulation of binding specificity by alternative This would be consistent with the finding that FGF-1 splicing of FGFRs and by sequence differences between primes preadipocytes for TZD-induced differentiation. FGFs and FGFRs, and modulation of binding affinity and The adipogenic effects of MVECs and FGF-1 were specificity by heparan sulfate molecules with specific specific for cells obtained from the stromal-vascular com- patterns of sulfation (23,25–27). In addition to localization partment of adipose tissue because human skin fibroblasts in the plasma membrane, FGFRs are also expressed within maintained under identical conditions did not accumulate the nuclear envelope and matrix (28). Signal transduction cytoplasmic lipid and no G3PDH activity was measured in in response to FGFs occurs through receptor dimerization these cells. This is consistent with the premise that and complex formation with heparan sulfate proteogly- preadipocytes are a unique type of mesenchymal cell that cans (26). Subsequent phosphorylation at multiple sites on are distinctly different from dermal fibroblasts and capable the intracellular domain of the FGFR initiates recruitment of expressing a developmental commitment that, at termi- and/or phosphorylation of multiple downstream signal nal differentiation, results in a typical mature fat cell (5). transduction molecules and pathways (28,29). The proliferative effect of FGF-1 on human preadipo- This study demonstrates a novel role of FGF-1 as a key cytes was the same in cells from subcutaneous and human adipogenic molecule. Culture in the presence of intra-abdominal sites (data not shown). Human preadipo- FGF-1 from the time of preadipocyte isolation appears to cytes maintained in the presence of FGF-1 also retained prime the cells for differentiation. This priming effect is their differentiation capacity after prolonged culture in- made much more apparent if the cells are subsequently volving repeated passaging. FGF-1 also induced differen- differentiated in the presence of a PPAR-␥ ligand, the TZD tiation of preadipocytes to a similar degree irrespective of rosiglitazone. Our results suggest that FGF-1 is not acting depot, when differentiation was performed in the absence

DIABETES, VOL. 53, DECEMBER 2004 3103 FGF-1: KEY REGULATOR OF HUMAN ADIPOGENESIS

FIG. 8. In response to FGF-1 treatment, human preadipocytes readily differentiate in the presence of serum. A: Photomicrographs showing preadipocytes following 21 days in serum-containing differentiation medium. i, Non–FGF-1–treated preadipocytes; ii, FGF-1–treated subcutaneous preadipocytes; iii, FGF-1–treated omental preadi- ␮m; original magnification 10 ؍ pocytes (bar ؋100). B: G3PDH activity assay determined that treatment with both FGF-1 and -2 before differentiation renders human cells capable of undergoing differentiation in the presence of serum (FGF-1 more than FGF-2 in subcutaneous preadipocytes), whereas IGF-1 had no effect over control. of TZD. In contrast, and consistent with our previous role of FGF-1 in promotion of adipogenesis is demon- reports and those of others (13,14,30,31), differentiation strated by the inhibitory action of neutralizing anti–FGF-1 was greater in subcutaneous compared with omental antibodies on both human and 3T3-L1 differentiation. preadipocytes when a TZD was included. Taken together, The role of FGFs in adipogenesis is controversial. these observations suggest that subcutaneous and omental Although both positive and negative effects have been preadipocytes may share similar capacity to differentiate reported for different family members, including FGF-1 in the absence of TZDs and that subcutaneous preadipo- and -2 (34), in general these growth factors are reported to cytes are more responsive to the adipogenic effects of have a mitogenic effect on preadipocytes while having no TZDs than their omental counterparts. This difference may effect or even potently inhibiting their differentiation (35– contribute to the observed remodeling of adipose mass, 42). In contrast to previous studies, where exposure to with increased subcutaneous and decreased omental fat, FGF-1 was restricted to relatively short periods during in humans receiving TZDs (32,33). It is also noteworthy either growth or differentiation, we have cultured cells in that before this work, omental preadipocytes that have the presence of FGF-1 for sustained periods (1–2 months). been cultured in the presence of serum have proved In addition, previous studies have not addressed the refractory to differentiation in vitro (13,14). We have now effects of sustained culturing in the presence of FGF-1 shown that exposure of these cells to FGF-1 during during the growth period, before the induction of differ- proliferation results in marked differentiation, thus over- entiation. We suggest that these differences in protocol coming the serum-induced inhibition of differentiation. underlie the differences in experimental observations. Indeed, in FGF-1–treated omental preadipocytes, the lev- Consistent with this, when preadipocytes were exposed to els of G3PDH activity and intracellular lipid accumulation FGF-1 only during differentiation, as in earlier studies, we are comparable to those we have previously reported in observed no significant effect on either lipid accumulation subcutaneous preadipocytes (13). or G3PDH activity, with a trend toward a decrease in these The murine 3T3-L1 cell line is a widely used model markers of differentiation. Taken together, our results system for the study of differentiation. In contrast to suggest that sustained incubation with FGF-1 during the human preadipocytes, these cells differentiate with high growth period may “prime” the preadipocytes for subse- efficiency in the presence of serum and after high passage quent differentiation. FGF-2 treatment of human preadipo- number (typically up to 20 in our experience). We report cytes also resulted in increased adipocyte differentiation, that 3T3-L1 cells, unlike human preadipocytes, express suggesting that this feature may be common to members high levels of FGF-1 and that exogenous FGF-1 does not of the FGF family, although FGF-1 was consistently the further increase proliferation or differentiation of these more potent adipogenic agent, particularly in cells from cells. We also demonstrate that human preadipocytes subcutaneous depots. Further work is aimed at investiga- treated with FGF-1 resemble 3T3-L1 cells in terms of rapid tion of the role of a range of growth factors in human growth and very high capacity for differentiation. Taken adipogenesis, including that of FGF-2 and other members together, these results suggest that an important difference of the FGF family. between 3T3-L1 and human preadipocytes is higher endog- Evidence that members of the FGF family may play an enous expression of FGF-1 by the 3T3-L1 cells. The key in vivo role in adipogenesis is provided by studies demon-

3104 DIABETES, VOL. 53, DECEMBER 2004 L. HUTLEY AND ASSOCIATES strating de novo adipogenesis in mice at the site of Prins JB: Effects of rosiglitazone and linoleic acid on human preadipocyte implanted FGF-2–impregnated microspheres (43,44). Also, differentiation. Eur J Clin Invest 33:574–581, 2003 14. Adams M, Montague CT, Prins JB, Holder JC, Smith SA, Sanders L, Sewter a role for FGFs in human adipose tissue metabolism is CP, Digby JE, Lazar MA, Chatterjee VKK, O’Rahilly S: Activators of suggested by the observation that preadipocytes from peroxisome proliferator-activated receptor gamma have depot-specific massively obese individuals have much higher expression effects on human pre-adipocyte differentiation. J Clin Invest 100:3149– of FGF-2 than the same cells from lean counterparts (45). 3153, 1997 The fact that human adipose tissue expresses members of 15. Tartaglia M, Fragale A, Battaglia PA: A competitive PCR-based method to the FGF family, including their cognate receptors, pro- measure human fibroblast growth factor receptor 1–4 (FGFR 1–4) gene expression. DNA Cell Biol 20:367–379, 2001 vides further support for a metabolic role of these growth 16. Burgess WH, Mehlman T, Marshak DR, Fraser BA, Maciag T: Structural factors in human adipose tissue (18,40). evidence that endothelial cell growth factor beta is the precursor of both In summary, in this report we describe an important role endothelial cell growth factor alpha and acidic fibroblast growth factor. of adipose-derived MVECs in the paracrine regulation of Proc Natl Acad SciUSA83:7216–7220, 1986 human adipose tissue expansion and demonstrate a novel 17. Sewter CP, Blows F, Vidal-Puig A, O’Rahilly S: Regional differences in the response of human pre-adipocytes to PPAR␥ and RXR␣ agonists. Diabetes role of FGF-1 in human adipogenesis. Our data indicating 51:718–723, 2002 that disruption of FGF-1 signaling at multiple biochemical 18. Gabrielsson BG, Johansson JM, Jennische E, Jernas M, Itoh Y, Peltonen M, steps inhibits preadipocyte replication and/or differentia- Olbers T, Lonn L, Lonroth H, Sjostrom L, Carlsson B, Carlsson LM, Lonn M: tion support the key adipogenic role of FGF-1 and repre- Depot-specific expression of fibroblast growth factors in human adipose sent an important area for further research. Future studies tissue. Obes Res 10:608–616, 2002 19. Fan H, Duan Y, Zhou H, Li W, Li F, Guo L, Roeske RW: Selection of peptide will be aimed at establishing the molecular mechanisms by ligands binding to fibroblast growth factor receptor 1. IUBMB Life which FGF-1 exerts its adipogenic effects. These data 54:67–72, 2002 suggest that antagonists of FGF-1 or of FGFRs may 20. Lozano RM, Jimenez M, Santoro J, Rico M, Gimenez-Gallego G: Solution present new avenues for the development of novel phar- structure of acidic fibroblast growth factor bound to 1,3, 6-naphthalen- maceutical antiobesity strategies targeting adipose cell etrisulfonate: a minimal model for the anti-tumoral action of suramins and suradistas. J Mol Biol 281:899–915, 1998 number. 21. Rizzino A, Kazakoff P, Ruff E, Kuszynski C, Nebelsick J: Regulatory effects of cell density on the binding of transforming growth factor beta, epidermal growth factor, platelet-derived growth factor, and fibroblast growth ACKNOWLEDGMENTS factor. Cancer Res 48:4266–4271, 1988 This work was supported by the Diabetes Australia Re- 22. Ornitz DM, Itoh N: Fibroblast growth factors. Genome Biol 2:3005.1– search Trust, the National Heart Foundation of Australia, 3005.12, 2001 23. Steger K, Tetens F, Seitz J, Grothe C, Bergmann M: Localization of the Wellcome Trust, and the Princess Alexandra Hospital fibroblast growth factor 2 (FGF-2) protein and the receptors FGFR 1–4 in Foundation. L.H. holds a Smart State Fellowship. normal human seminiferous epithelium. Histochem Cell Biol 110:57–62, The authors give special thanks to the volunteers and 1998 surgeons at the Princess Alexandra Hospital for providing 24. Klint P, Claesson-Welsh L: Signal transduction by fibroblast growth factor receptors. Front Biosci 4:D165–D177, 1999 the tissue for study. 25. Choi DY, Toledo-Aral JJ, Lin HY, Ischenko I, Medina L, Safo P, Mandel G, Levinson SR, Halegoua S, Hayman MJ: Fibroblast growth factor receptor 3 induces gene expression primarily through Ras-independent signal trans- REFERENCES duction pathways. J Biol Chem 276:5116–5122, 2001 1. Varzaneh FE, Shillabeer G, Wong K-L, Lau DCW: Extracellular matrix 26. 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