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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1309
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 1226-1232
Copyright © 2005 by The Endocrine Society

Essential Role of Fibroblast Growth Factor Signaling in Preadipoctye Differentiation

Nayan G. Patel, Sudhesh Kumar and Margaret C. Eggo

Division of Medical Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom

Address all correspondence and requests for reprints to: Dr. Margaret C. Eggo, Division of Medical Sciences, The Medical School, University of Birmingham, Birmingham B15 2TT, United Kingdom. E-mail: M.C.Eggo{at}bham.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We have examined the expression and role of autocrine fibroblast growth factors (FGFs) in human preadipocytes through their differentiation in vitro. A high-molecular weight form of FGF-2 was initially strongly expressed, but 6–9 d after induction of differentiation, its expression decreased markedly. This coincided with the first appearance of visible lipid droplets within the cells. FGF-2 (18 kDa) was not found. FGF receptor (FGFR) 1 was detected as a single band of 125 kDa that also decreased with differentiation. Its decrease preceded that of FGF-2. Despite the decrease in cell-associated FGF-2 with differentiation, secreted FGF-2 was 2.5-fold higher in the differentiated preadipocytes. To determine whether FGF-2 had an autocrine role, FGFR signaling was inhibited using recombinant adenovirus expressing dominant negative FGFR1 (RAdDN-FGFR1) and a specific inhibitor of FGFR1 signaling, PD166866. Preadipocytes transduced with RAdDN-FGFR1 expressed a truncated, 79-kDa FGFR1. Differentiation, assessed by lipid droplet formation, was completely prevented by RAdDN-FGFR1 and by PD166866. The protein content in the cell layer and glucose uptake were significantly reduced by both agents. The insulin-sensitizing drug, rosiglitazone, did not prevent the actions of RAdDN-FGFR1 or PD166866. Controlling adipose tissue growth by limiting FGF actions may provide a means to combat obesity.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
OBESITY IS A major worldwide pandemic with a recent health survey finding 17% of men and 20% of women classed as obese in England and Wales (1), whereas the U.S. National Health and Nutrition Examination Survey (1999–2000) shows that 28% of American men and 34% of American women are obese (2). Means to control adipose tissue growth and function are, therefore, important. Adipogenesis requires the cooperative effects of several signaling pathways (3). Differentiation in vitro is dependent on exogenously added factors such as insulin, glucocorticoids, cAMP elevators, and thyroid hormone, but factors secreted by the adipocyte are known to influence adipocyte differentiation. These include TNF-{alpha} (4, 5), leptin (5, 6, 7), IL-6 (8), IGF-I (9), agouti (10), and prostaglandins (11). Because fibroblast growth factors (FGFs) can be expressed by many cell types and have autocrine effects, we have examined their role in preadipocyte differentiation.

FGFs are a large family of at least 23 members sharing a common sequence homology, which ranges from 13–71% shared amino acid identity and range in molecular mass from 17–34 kDa (12). The defining features of the FGF family include a strong affinity for heparin and heparin-like glycosaminoglycans of the extracellular matrix, as well as a central core of 140 amino acids that is highly homologous between different family members. In mammals, the members of the FGF family are differentially expressed in most tissues, but the pattern and timing of expression vary. Most FGFs (FGFs 3–8, 10, 15, 17–19, 21–23) have amino-terminal signal peptides and are readily secreted from cells. FGF 9, 16, and 20 lack this amino-terminal signal sequence but are still secreted (13, 14, 15), whereas FGF 11–14 lack these signal sequences and are thought to remain intracellular (16, 17, 18, 19). FGF-1 and FGF-2 are found on the cell surface and within the extracellular matrix but lack the classical signal sequence directing secretion. They may be released from damaged cells or by an exocytotic mechanism independent of the endoplasmic reticulum-Golgi pathway (12, 20).

FGFs bind to specific, high-affinity receptor tyrosine kinases, FGF receptors (FGFRs) 1–4. They consist of three components, an extracellular section that is composed of three domains (Ig domains I, II, and III), a transmembrane section, and an intracellular domain that contains the tyrosine kinase. FGFRs are distributed in many tissues, and there is temporal and spatial expression of receptors. This allows many different FGFs to signal through FGFRs and elicit different effects. Binding of FGF to its receptor leads to receptor dimerization and tyrosine phosphorylation that activates target enzymes such as phospholipase C-{gamma}, FRS2, PKC, Src, Grb2, SHC, and Crk, leading to the activation of many signaling pathways (21, 22). Few studies have been carried out on the expression of FGFRs in adipose tissue, with only one study looking at the mRNA expression of FGFRs (23).

FGF-10 mRNA and protein have been found in mouse 3T3 cells and mouse preadipocytes, and blocking FGF-10 actions in these cells with adenovirus expressing dominant negative FGFR1 inhibited preadipocyte differentiation. Similarly, in FGF-10 knockout mice, adipogenesis was reduced (24). In contrast, early studies showed that exogenous FGF-2 inhibited differentiation of the immortalized mouse preadipocyte cell line TA1 (25). In humans, FGF-10 mRNA has been found in human adipose tissue along with transcripts for FGFs 1, 2, 7, 9, and 18. FGF-2 mRNA was reported to be absent from the stromal-vascular fraction (23) of human adipose tissue, yet preadipocytes from massively obese individuals were found to express more FGF-2 protein than normal individuals (26). Addition of exogenous FGF-2 to human preadipocytes resulted in a small suppression of glycerol-3-phosphate dehydrogenase, a marker of adipose differentiation (27). This effect was much less than that seen with epidermal growth factor. Whether the small effects were due to absence of FGFR or to saturation of the receptors with endogenous ligands was not addressed.

In view of the discrepancies in the literature on the expression and role of FGFs in human adipocyte differentiation, we have examined the expression of FGF-2 and FGFR1 in human preadipocytes throughout their differentiation. We chose to examine the FGF-2 isoform because it is the best characterized of the FGFs, it is reported to be synthesized by cells of the mesenchyme (28) from which preadipocytes are derived, and because it usually remains cell-associated, in keeping with an autocrine function. To determine the autocrine potential of adipocyte FGFs, we inhibited signaling through FGFRs by two methods. The compound PD166866 specifically inhibits FGFR1 signaling and, thus, would inhibit the effects of FGF-2. It is an ATP competitive inhibitor of FGFR1 and has been shown to inhibit FGFR1 in a number of cells including NIH 3T3 cells, L6 muscle cells, and human placenta by inhibiting tyrosine kinase activity of FGFR1, while having no effect on c-Src, platelet-derived growth factor receptor-ß, epidermal growth factor receptor, insulin receptor tyrosine kinases, protein kinase C, or mitogen-activated protein kinase (29). The second method used an adenovirus vector expressing a C terminus truncated form (dominant negative) of FGFR1 (DN-FGFR1). A similar construct has been shown to inhibit signaling through all FGFR isoforms (30).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Abdominal adipose tissue (sc) was obtained from female subjects [age 49.3 ± 9.9 yr (mean ± SEM); body mass index 27.30 ± 6.42 kg/m2] undergoing elective surgery in accordance with the guidelines of the South Birmingham ethics committee. None of the subjects had diabetes or severe systemic illness, and none was taking medications known to influence adipose tissue mass, distribution, or metabolism.

Isolation and culture of human preadipocytes

Preadipocytes were isolated by a variation of the method of Rodbell (31). Adipose tissue was digested with 1 mg/ml type I collagenase (Worthington, Freehold, NJ) in Hank’s balanced salt solution (HBSS) (Invitrogen Ltd., Paisley, United Kingdom), for 1 h at 37 C and shaken at 100 cycles/min. The disrupted tissue was filtered through a double-layered cotton mesh, and isolated cells were washed with HBSS and centrifuged at 250 x g for 5 min to give a pellet containing preadipocytes. The cell pellet was resuspended in erythrocyte lysis buffer [154 mmol/liter NH4Cl, 5.7 mmol/liter K2HPO4, and 0.1 mmol/liter EDTA (pH 7.0), Sigma, Poole, United Kingdom] for 10 min and centrifuged at 250 x g for 5 min to remove erythrocyte contamination. The resulting pellet was washed in HBSS and centrifuged at 250 x g for 5 min and resuspended in DMEM/Ham’s F-12 medium (Invitrogen Ltd.) supplemented with 15% bovine fetal calf serum (First Link Ltd., Brierley Hill, United Kingdom). Approximately 105 cells were plated in 12-well (4.5 cm2) tissue culture dishes and grown until confluent (106 cells per well). All media used were supplemented with 100 U/ml penicillin G (Sigma) and 0.1 mg/ml streptomycin sulfate (Sigma).

Adenoviruses

Recombinant adenovirus (RAd) expressing the extracellular and transmembrane domain of FGFR1 but deficient in the kinase domain (RAdDN-FGFR1) was produced within our laboratory (32). RAd-expressing ß-galactosidase (RAd-ß-gal) was used as a control E1/E3 deleted adenovirus. RAd-ß-gal was obtained from the Department of Cancer Studies (University of Birmingham, United Kingdom).

Transduction of replication-defective adenovirus in human preadipocytes

Confluent preadipocytes were transduced with replication-defective (E1/E3 deleted) adenovirus at a multiplicity of infection (MOI) of 20 for 90 min in serum-free medium with gentle agitation. The virus-containing medium was removed and replaced with fresh medium.

Differentiation of human preadipocytes

Confluent preadipocytes (referred to as d 0 preadipocytes) were washed twice with HBSS and cultured in DMEM/Ham’s F-12 medium containing 100 nmol/liter insulin (Sigma), 100 nmol/liter dexamethasone (Sigma), and 0.2 nmol/liter triiodothyronine (Sigma), and for the first 4 d of culture, 0.25 mmol/liter 3-isobutyl-1-methylxanthine (Sigma) (4). This is referred to as normal differentiation medium. Some preadipocytes cultured in normal differentiation medium were supplemented with the insulin-sensitizing agent rosiglitazone (1 µmol/liter, GSK, Essex, United Kingdom). In some experiments, preadipocytes were treated with the FGFR1 inhibitor PD166866 (10–7 M) (supplied by Pfizer Inc., Groton, CT). This was replaced at each medium change. Cells were incubated in 5% CO2:95% air at 37 C, and differentiation medium was changed every 2–3 d until cells had accumulated visible lipid droplets.

Glucose uptake assay

Cells were incubated for 24 h at 37 C, 5% CO2:95% air, in normal differentiated medium (Sigma), supplemented with 2 µCi/ml D-[U-14C] glucose (specific activity 291 mCi/mmol, Amersham Biosciences, Bucks, United Kingdom) and the additions as noted in the figure legends. At the end of the incubation, cells were washed twice with HBSS, and the cell layer was dissolved in 2% sodium dodecyl sulfate (Sigma) and 62.5 mmol/liter Tris-HCl (pH 6.8, Sigma) and transferred to scintillation vials containing 4 ml Optiphase scintillation fluid (Fisher Scientific Ltd., Loughborough, United Kingdom). Incorporated 14C glucose radioactivity was determined on an LKB scintillation counter.

Protein assay

The cell layer was dissolved in sample buffer [2% sodium dodecyl sulfate and 62.5 mmol/liter Tris-HCl (pH 6.8)], and protein content was determined using a modified Lowry assay (Bio-Rad, Preston, United Kingdom).

FGF-2 ELISA

Cell-conditioned media were collected and centrifuged to remove intact cells or debris. The total amount of secreted FGF-2 was measured using an ELISA kit according to the manufacturer’s instructions (R&D Systems, Oxon, United Kingdom).

Western blot analysis

Cell layer proteins isolated from preadipocytes and differentiated preadipocytes were separated by SDS-PAGE using a 12.5% polyacrylamide gel and a 7.5% stack for the anti-FGF-2 primary antibody (Sigma) and a 10% polyacrylamide gel and a 7.5% stack for both the anti-C terminus FGFR1 primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-N terminus FGFR1 primary antibody (Santa Cruz Biotechnology). The cell layer was dissolved in sample buffer, reduced with 10% ß-mercaptoethanol (Sigma), and heated for 5 min at 95 C and run on a gel. Prestained molecular weight markers (Sigma) were used as standards. The separated proteins were transferred to a polyvinylidene difluoride Hybond membrane (Amersham Biosciences) by electroblotting at 425 mA for 3 h in a vertical transfer apparatus. The membrane was blocked by incubating in 10% nonfat milk in Tris-buffered saline-Tween 20 (TBS-T) [10 mmol/liter Tris-HCl (pH 7.5), 100 mmol/liter NaCl and 0.1% Tween 20, Sigma] for 1 h at room temperature to prevent nonspecific binding. Membranes were incubated in anti-C terminus FGFR1 primary antibody, anti-N terminus FGFR1 primary antibody, and anti-FGF-2 primary antibody, all at dilutions of 1:1000 in TBS-T with 10% nonfat milk for 3 h at room temperature. Membrane was washed three times in TBS-T and incubated with antirabbit IgG horseradish peroxidase secondary antibody (Santa Cruz Biotechnology) at a dilution of 1:10,000 in TBS-T for 1 h at room temperature. The antigens were detected by the enhanced chemiluminescence system (Insight Biotechnology Ltd., Middlesex, United Kingdom) after exposure to x-ray film (GRI Ltd., Essex, United Kingdom) for 15 min.

Statistics

All experiments in the study were performed using adipose tissue from at least three patients (n ≥ 3). At least three replicates per experiment were carried out. One-way ANOVA was used for data analysis in this study, and Dunnett’s post test was used to compare against control unless otherwise stated. Data are shown as mean ± SE of the mean (SEM). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Expression of FGF-2 in human differentiating preadipocytes

Preadipocytes were induced to differentiate over 12 d in differentiation medium with and without 1 µmol/liter rosiglitazone. Cell layer proteins were isolated daily from preadipocytes (d 0) to differentiated preadipocytes (d 12) and were analyzed by Western blotting. One hundred micrograms protein was loaded in each lane of the gel, and an antibody to FGF-2 was used. Figure 1AGo shows the time course of FGF-2 expression in preadipocytes differentiated in differentiation medium (d 0–12). A band of 24 kDa was detectable in d 0 preadipocytes and was present throughout the 12 d of differentiation. The level of FGF-2 expression began to fall at d 8 and continued to fall as lipid droplet formation began to occur. Figure 1BGo shows the time course of FGF-2 expression in preadipocytes differentiated in differentiation medium containing rosiglitazone (d 0–12). A band of 24 kDa was detectable in d 0 preadipocytes and was present throughout the 12 d of differentiation. FGF-2 expression fell at d 6 coincident with the earlier appearance of lipid droplets and FGF-2 expression continued to fall.



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  		FIG. 1. Western blot analysis of FGF-2 protein expression throughout preadipocyte differentiation. One hundred micrograms protein was loaded per lane. A, Preadipocytes differentiated over 12 d in differentiation medium. B, Preadipocytes differentiated over 12 d in differentiation medium containing rosiglitazone (1 µmol/liter).

 
Conditioned medium was collected from d 0 preadipocytes and d 12 differentiated preadipocytes, and the level of FGF-2 was measured using an ELISA kit. Day 12 differentiated preadipocytes secreted significantly more FGF-2 than d 0 preadipocytes (16.57 ± 0.48 pg/ml vs. 6.58 ± 0.09 pg/ml, n = 4, mean ± SEM, P < 0.0001).

Expression of FGFR1 in differentiating human preadipocytes

To quantify FGFR1 expression throughout differentiation, Western blotting was used. Preadipocytes were induced to differentiate over 9 d in normal differentiation medium. Cell layer proteins were isolated daily from preadipocytes throughout differentiation and were analyzed by Western blotting. One hundred micrograms protein was loaded in each lane of the gel, and an antibody to C terminus of FGFR1 was used. Figure 2AGo shows the time course of the C terminus of FGFR1 expression in preadipocytes differentiated in differentiation medium (d 0–9). A band of 125 kDa was detectable in d 0 preadipocytes and was present throughout the 9 d of differentiation. The level of FGFR1 expression, however, began to fall at d 5 and continued to fall.



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  		FIG. 2. Western blot analysis of full-length and truncated FGFR1 protein expression throughout preadipocyte differentiation. One hundred micrograms protein was loaded per lane. A, Preadipocytes differentiated over 9 d in differentiation medium and transduced with RAdDN-FGFR1. Cell layer protein was probed with an antibody to the C terminus of FGFR1 to determine expression of full-length FGRFR1. B, Preadipocytes transduced with RAdDN-FGFR1 and differentiated over 2 d in differentiation medium. Cell layer protein was probed with an antibody to the N terminus of FGFR1 to determine expression of truncated (DN) FGFR1.

 
Expression of virally transduced, truncated FGFR1 in preadipocytes

To show that truncated FGFR1 transduced with the adenovirus can be expressed in the preadipocytes, isolated preadipocytes were incubated with RAdDN-FGFR1 virus at a MOI of 20 plaque-forming units/cell and induced to differentiate in differentiation medium. Cell layer proteins were isolated daily from preadipocytes and RAdDN-FGFR1-transduced differentiated preadipocytes. Western blotting was used to show that cells were transduced with RAdDN-FGFR1 virus. One hundred micrograms protein was loaded in each lane of the gel, and an antibody to N terminus of FGFR1 was used to detect truncated FGFR1 as shown in Fig. 2BGo. Day 0 preadipocytes not transduced with RAdDN-FGFR1 virus showed no expression of the truncated form of FGFR1. Day 1 infected cells showed a low level of expression of N terminus of FGFR1 (79 kDa), but by d 2, there was strong expression of truncated FGFR1.

Effects of inhibition of FGFR signaling on protein content in differentiated preadipocytes using RAd expressing truncated FGFR1 and PD166866, specific inhibitors of FGFR signaling

Preadipocytes were transduced with RAdDN-FGFR1 virus at a MOI of 20 plaque-forming units/cell. Control cultures were transduced with RAd-ß-galactosidase virus at the same MOI. In a separate experiment, preadipocytes were treated with the FGFR1 inhibitor PD166866 (100 nmol/liter). Preadipocytes were induced to differentiate over 12 d in differentiation medium with and without 1 µmol/liter rosiglitazone. Light micrographs of 12-d-old RAd-ß-galactosidase-treated differentiated preadipocytes are shown in Fig. 3AGo. Large multilocular lipid droplets are seen. Figure 3BGo shows cells treated with RAdDN-FGFR1. There are fewer cells and no visible lipid droplets. Figure 3CGo shows cells treated with PD166866 throughout the 12-d incubation. Again, there is no evidence of lipid droplets. These inhibitory effects were found both in the presence and absence of rosiglitazone (data not shown).



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  		FIG. 3. The effect of RAdDN-FGFR1 and PD166866 on adipocyte differentiation. A, Light micrographs of 12-d culture of preadipocytes transduced with RAd-ß-galactosidase virus in differentiation medium; B, 12-d culture of preadipocytes transduced with RAdDN-FGFR1 virus in differentiation medium; C, 12-d culture of preadipocytes in differentiation medium treated with PD166866 (100 nmol/liter). Magnification x100.

 
To examine the protein content in the cell layer over 12 d of differentiation, preadipocytes were treated with RAd-ß-galactosidase virus, RAdDN-FGFR1 virus, and PD166866. They were induced to differentiate over 12 d in differentiation medium. PD166866 was maintained in the cells throughout the 12-d period. Cell layer proteins were isolated daily from preadipocytes (d 0) to differentiated preadipocytes (d 12), and the protein content was measured. Results of treated cells are given as percentage mean values ± SEM compared with their respective control d 0 preadipocytes (Fig. 4Go). There was a reduction in protein content as differentiation progressed in the RAd-ß-galactosidase cells, but this was comparable with that found in nontreated differentiating preadipocytes (data not shown). Treatment with PD166866 and particularly RAdDN-FGFR1 virus showed a marked and significant reduction in protein content throughout differentiation compared with the RAd-ß-galactosidase control cells. Addition of the insulin-sensitizing drug, rosiglitazone, did not prevent the reduction in protein content by either RAdDNFGFR1 (42 ± 6% reduction compared with d 12 RAd-ß-galactosidase control cells) or PD166866 (57 ± 4% reduction compared with d 12 RAd-ß-galactosidase control cells).



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  		FIG. 4. Protein content in differentiated preadipocytes transduced with RAd-ß-galactosidase, RAdDN-FGFR1, and PD166866 over 12 d. Protein content was measured daily as described in Subjects and Methods. Results are given as the percentage mean value compared with d 0 preadipocytes ± SEM of three independent samples (n = 3).

 
Effects of inhibition of FGFR signaling on glucose uptake in differentiated preadipocytes using RAd expressing truncated FGFR1 and PD166866, specific inhibitors of FGFR signaling

The time course of 14C-glucose uptake into cells over 12 d of differentiation with and without RAd-ß-galactosidase virus, RAdDN-FGFR1 virus, and PD166866 was examined. The uptake of 14C-glucose was measured daily from preadipocytes (d 0) to differentiated preadipocytes (d 12) and corrected for protein content. Results of treated cells are given as percentage mean values ± SEM compared with respective control d 0 preadipocytes (Fig. 5Go). Cells treated with RAd-ß-galactosidase virus showed a time-dependent increase in uptake of 14C-glucose as differentiation progressed. This was not markedly different from that seen in cells that had not been treated with RAd-ß-galactosidase virus (data not shown). Cells treated with RAdDN-FGFR1 or PD166866 showed a significant, time-dependent reduction in the uptake of 14C-glucose from d 7 onwards compared with the RAd-ß-galactosidase control cells. The increased uptake seen with differentiation was abolished by this treatment. In cells treated with RAdDN-FGFR1, 14C-glucose uptake was reduced to 9% of d 0 control, whereas PD166866-treated cells showed a reduction in 14C-glucose to 47% of d 0 control. Addition of the insulin-sensitizing drug, rosiglitazone, did not prevent the reduction in 14C-glucose uptake by either RAdDNFGFR1 (38 ± 5% reduction compared with d 12 RAd-ß-galactosidase control cells) or PD166866 (68 ± 6% reduction compared with d 12 RAd-ß-galactosidase control cells).



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  		FIG. 5. 14C-Glucose uptake in differentiated preadipocytes transduced with RAd-ß-galactosidase, RAdDN-FGFR1, and PD166866 over 12 d. 14C-Glucose uptake was measured daily as described in Subjects and Methods. Results are given as the percentage mean value compared with d 0 preadipocytes and corrected for protein content ± SEM of three independent samples (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In human sc preadipocytes and differentiating preadipocytes, we found strong expression of FGF-2 of the high-molecular mass 24-kDa form but not the 18-kDa form. All forms of FGF-2 are mitogenic, but the high-molecular weight forms of FGF-2 are thought to be nuclear (33) and are postulated to induce anchorage independent growth and growth in low serum. Whether it serves this function in adipocytes is unknown, although the preadipocyte cultures are maintained and survive in serum-free medium. The 18-kDa form of FGF-2 is found mainly in the cytosol and is known to increase cell motility and proliferation through interaction with its cell surface receptors (34).

We have shown that as preadipocytes were induced to differentiate, cellular levels of FGF-2 dropped coincidental with the appearance of lipid droplets. Secretion of FGF-2, however, was found to be higher in differentiated preadipocytes than preadipocytes. Whether this is due to release from membrane-bound stores or due to de novo synthesis of a secretable form of FGF-2 remains to be determined. The reduction in cell-associated FGF-2 with differentiation was also seen when using preadipocytes differentiated in rosiglitazone. These showed an earlier decrease in FGF-2 levels coincidental with the earlier appearance of lipid droplets in these cells (3). Studies looking at the expression of FGF-2 mRNA in adipose tissue have given conflicting data (26, 27, 28, 29, 36). Our study looking at protein expression clearly shows that FGF-2 is expressed in preadipocytes and is regulated through differentiation.

Because FGF-2 secretion is higher in differentiated preadipocytes than preadipocytes, it may function as a paracrine factor in adipose growth. Adipose tissue is able to grow and regress throughout adulthood, and vascular remodeling and angiogenesis are required for this to occur (35). FGF is angiogenic (36) and may be secreted by the adipocyte to influence the surrounding vasculature and epithelium to undergo remodeling during adipocyte growth (37). During differentiation of adipocytes, preadipocytes undergo a morphology change to accumulate lipid, and FGF-2 secretion may accommodate this change by influencing the surrounding vasculature and epithelium.

FGFR1 is strongly expressed in human preadipocytes, with levels of FGFR1 declining as differentiation progresses and preceding the fall in FGF-2. The reduction in FGFR1 and FGF-2 expression as differentiation progresses may be due to the anti-adipogenic effects of FGF-2. A number of studies have shown FGF-2 to be antiadipogenic in both human and murine models of adipogenesis (25, 26, 27, 38). The antiadipogenic effects of FGF-2 may be reduced by decreasing expression of FGF-2 and its receptor FGFR1 as differentiation progresses and with the appearance of lipid droplets. However, expression of both proteins remains detectable even when preadipocytes are fully differentiated.

When FGFR1 signaling was blocked with the specific inhibitor of FGFR1, PD166866, or with a RAd to transduce a dominant negative construct of FGFR1, there were large reductions in both protein content and adipogenesis. The reduction in protein content with transduction with RAdDN-FGFR1 virus could be due to toxic effects caused by the virus, but transduction with RAd-ß-galactosidase virus at the same MOI did not have the same effect on protein content or adipogenesis. We, therefore, conclude that the effects observed with RAdDN-FGFR1 virus transduction are due to effects on FGF signaling. The addition of rosiglitazone to the differentiation medium had no effect on the reductions in protein content and adipogenesis observed by inhibiting FGF signaling. Because rosiglitazone is unable to prevent any of the effects of blocking FGF signaling, this suggests that there are no common pathways activated by peroxisome proliferator-activated receptor-{gamma} and FGF signaling. Additionally, it indicates that the differentiating effects of rosiglitazone are dependent on FGF signaling. That the inhibitory effects on protein content and differentiation were greater when RAdDN-FGFR1 was used rather than PD166866 may be because the DNFGFR1 construct, unlike PD166866, inhibits signaling through all FGFRs (30). This suggests that isoforms of FGF that do not act through FGFR1 may also be important in regulating adipocyte protein content and function. One study has shown the expression of mRNAs for FGF 1, 7, 9, 18, and FGF 2 and 10 in human adipose tissue (23). We confirmed FGF-10 mRNA by RT-PCR in human preadipocytes (data not shown). Protein expression in the human adipocytes of other FGF isoforms will have to be confirmed.

Limiting obesity by controlling angiogenesis has been attempted by using antiangiogenic compounds, e.g. TNP-470 and angiostatin (37, 39, 40). Our studies suggest that limiting FGF action in adipose tissue will have the additional benefit of inhibiting adipocyte differentiation and angiogenesis. This should lead to more effective ways of controlling obesity.


   Acknowledgments
 
We thank all the theater staff at the Priory Hospital (Edgbaston, UK) who aided in these studies. We thank Mr. Levick for kindly providing consented samples from his plastic surgery operations at the Priory Hospital. We thank Pfizer Inc. for providing PD166866.


   Footnotes
 
This work was supported by the Medical Research Council CASE award (Grant RURC 6439) and by GlaxoSmithKline United Kingdom.

Present address for S.K.: Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom.

First Published Online November 2, 2004

Abbreviations: FGF, Fibroblast growth factor; FGFR, FGF receptor; HBSS, Hank’s balanced salt solution; MOI, multiplicity of infection; RAd, recombinant adenovirus; TBS-T, Tris-buffered saline-Tween 20.

Received July 7, 2004.

Accepted October 25, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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