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,
* Centre for Diabetes and Endocrine Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia;
Diabetes Biology, R&D, Novo Nordisk, Måløv, Denmark; and
Department of Clinical Sciences, Diabetes and Endocrinology, Lund University, University Hospital MAS, Malmö, Sweden
1Correspondence: Centres for Health Research, Level 2, Bldg. 35, Princess Alexandra Hospital, Ipswich Rd, Woolloongabba 4102, Australia. E-mail: jprins{at}cder.soms.uq.edu.au
ABSTRACT
We recently established that fibroblast growth factor (FGF)-1 promotes adipogenesis of primary human preadipocytes (phPA). In the current report, we have characterized the adipogenic effects of FGF-1 in phPA and also in a human PA strain derived from an individual with Simpson-Golabi-Behmel syndrome (SGBS PA), which exhibit an intrinsic capacity to differentiate with high efficiency. In further studies, we compared these models with the well-characterized murine 3T3-L1 preadipocyte cell line (3T3-L1 PA). FGF-1 up-regulated the adipogenic program in phPA, with increased expression of peroxisome proliferator-activated receptor-
in confluent PA prior to induction of differentiation and increased expression of adipocyte markers during differentiation. Moreover, phPA differentiated in the presence of FGF-1 were more insulin responsive and secreted increased levels of adiponectin. FGF-1 treatment of SGBS PA further enhanced differentiation. For the most part, the adipogenic program in phPA paralleled that observed in 3T3-L1 PA; however, we found no evidence of mitotic clonal expansion in the phPA. Finally, we investigated a role for extracellular regulated kinase 1/2 (ERK 1/2) in adipogenesis of phPA. FGF-1 induced robust phosphorylation of ERK1/2 in early differentiation and inhibition of ERK1/2 activity significantly reduced phPA differentiation. These data suggest that FGF-1 treated phPA represent a valuable in vitro model for the study of adipogenesis and insulin action and indicate that ERK1/2 activation is necessary for human adipogenesis in the absence of mitotic clonal expansion.—Newell, F. S., Su, H., Tornqvist, H., Whitehead, J. P., Prins, J. B., Hutley, L. J. Characterization of the transcriptional and functional effects of fibroblast growth factor-1 on human preadipocyte differentiation
Key Words: obesity • adipogenesis • mitosis
OBESITY HAS DEVELOPED into a worldwide epidemic highlighting the necessity for basic research into the mechanisms that govern adipose tissue growth. The accumulation of adipose tissue mass is the result of both an increase in the triglyceride content of mature adipocytes and the proliferation and differentiation of the adipocyte precursors, preadipocytes (PA) (1)
. Insight into adipocyte differentiation has been enhanced by the establishment of protocols for PA isolation, culture, replication, and differentiation in vitro.
The process of PA differentiation has been well characterized in in vitro murine models such as 3T3-L1 preadipocytes (3T3-L1 PA). Following commitment to the preadipocyte lineage, precursor cells become growth arrested at confluence. PA differentiation can then be induced by treating cells with a hormonal cocktail containing inducers, including insulin, glucocorticoid, and isobutyl methylxanthine (MIX), in the presence or absence of serum. In murine models of differentiation, an increase in cell number, known as mitotic clonal expansion (MCE) is observed following growth arrest and induction of differentiation. Although somewhat controversial, many groups have reported that MCE is necessary for subsequent PA differentiation in these cell lines (2
3
4
5)
. In the early hours of differentiation, activity of the transcription factors CCAAT/enhancer binding protein beta-(C/EBPß) and 
are important, with expression of both transcription factors transiently increasing (6)
. Importantly, C/EBPß undergoes sequential phosphorylation by extracellular signal-regulated kinase 1 and 2 (ERK1/2) followed by glycogen synthetase kinase-ß (GSK3-ß) (7)
. Phosphorylation of C/EBPß by ERK1/2 is required for MCE, DNA-binding activity and terminal differentiation (3)
. The transcription factors C/EBP
and peroxisome proliferator-activated receptor- (PPAR) are expressed later in the adipogenic program and play a central role in promoting PA differentiation (via induction of the expression of adipocyte genes) and mediating insulin sensitivity (8
, 9)
. Genes induced during differentiation include the lipid droplet associated protein perilipin, the glucose (Glc) transporter GLUT4, and adipokines such as adiponectin and leptin (10
11
12)
.
Although these processes are well understood in murine models of adipogenesis such as 3T3-L1 PA, human models are relatively poorly characterized. Such investigations have, in the past, been hampered by a lack of human PA cell lines. Although primary human preadipocytes (phPA) have a high capacity for differentiation if plated at confluence and induced to differentiate soon after isolation, experiments are limited by the finite number of cells that can be obtained at isolation. When phPA are passaged or subjected to prolonged exposure to serum, they are slow growing, difficult to differentiate, and have a limited life span. In recent years however, a number of discoveries have facilitated more in-depth study of human PA differentiation.
We have recently demonstrated that FGF-1 significantly promotes both the replication and differentiation of phPA (13)
. Long-term treatment with FGF-1 during proliferation alone is sufficient to induce higher levels of phPA differentiation, while FGF-1 treatment during both proliferation and differentiation results in the highest levels of differentiation (13)
. However, the molecular mechanisms by which FGF-1 increases the adipogenic potential of phPA are yet to be established. Several recent papers have also detailed the isolation of human PA cell strains with a high capacity for differentiation (14
15
16)
. Wabitsch et al. (2001) described the isolation of a PA cell strain from an infant with Simpson-Golabi-Behmel syndrome (SGBS), a rare disease with an overgrowth phenotype. In contrast to other human preadipocytes, these cells retain their capacity to differentiate even after 30 population doublings. Adipocytes derived from these PA are morphologically, functionally, and biochemically identical to cells isolated from other human donors and therefore SGBS PA represent a new model for the study of human adipogenesis.
In the present study, we sought to elucidate the mechanisms of FGF-1 adipogenic actions in hPA and also to determine whether FGF-1 has similar effects on a new model of human adipogenesis, the SGBS cell strain. We also wished to compare and contrast findings in the human preadipocyte models with those obtained in the well-characterized murine 3T3-L1 PA cell line. On the basis of results obtained in these studies, we investigated the requirement for ERK1/2 activity in adipogenesis of hPA.
MATERIALS AND METHODS
Cell culture
Primary human preadipocytes
Paired omental (intra-abdominal) and subcutaneous (s.c.) adipose tissue biopsies were obtained from 5 male [average age 50 yr (range 21–76)], average BMI 25.8 kg/m2 (range 19–33), and 12 female [average age 43 yr (range 25–71)], average BMI 30.1 kg/m2 (range 19–55) patients undergoing elective open-abdominal surgery. None of the patients had diabetes or severe systemic illness, and none were taking medications known to affect adipose tissue mass or metabolism. The protocol was approved by the Research Ethics Committees of the University of Queensland and the Princess Alexandra Hospital, and all patients gave their written informed consent. Human s.c. and omental primary human preadipocytes (phPA) were isolated and cultured, as described previously (17)
. Cells were used for experiments at passage two after 8 to 12 wk of culture in serum-containing medium. To induce preadipocyte differentiation, cells were grown to confluence and then cultured in the presence of a chemically defined serum-free medium (SFM), as described previously (18)
. Briefly, cells were cultured for 21 days in Dulbecco’s modified Eagle medium (DMEM)/F12 medium containing 100 IU penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 15 mM NaHCO3, 15 mM HEPES, 33 µM biotin, 17 µM pantothenic acid, 10 µg/mL transferrin, 0.1 µM cortisol, 0.2 nM triiodotyronine (T3), 0.5 µM insulin and 0.1 µM rosiglitazone (PPAR ligand). For the first 3 days of the differentiation period, 0.25 mM methyl-isobutylxanthine (MIX) was also added. During the culture or differentiation period, some phPA were treated with 1 ng/mL FGF-1 (R&D Systems, Minneapolis, MN) and 90 µg/mL heparin (Sigma-Aldrich, Castle Hill, Victoria, Australia) as indicated.
3T3-L1 cells
3T3-L1 PA were cultured in DMEM containing 2 mM L-glutamine, 100 µg/mL streptomycin, penicillin, and 10% fetal calf serum (FCS; JRH Biosciences, Brooklyn, Victoria, Australia). For differentiation, cells were grown to confluence and induced to differentiate for 3 days in growth medium supplemented with 1.4 µM insulin, 0.22 µM dexamethasone (DEX), 0.4 nM biotin, and 500 µM methyl-isobutylxanthine (MIX). On the third day, media were changed to a medium containing 1.4 µM insulin for a further 3 days. Cells were considered to be fully differentiated on day 8 and were used for subsequent experiments from days 8–10.
SGBS preadipocytes
SGBS PA were isolated and characterized in 2001 (16)
. Cells were cultured in DMEM/F12 supplemented with 10% FCS, penicillin, streptomycin, 33 µM biotin, and 17 µM pantothenic acid. To induce differentiation, cells were grown to confluence and subsequently cultured in serum-free DMEM/F12 medium, containing penicillin/streptomycin, 33 µM biotin, 17 µM pantothenic acid, 0.01 mg/mL transferrin, 0.1 µM cortisol, 200 pM triiodotyronine (T3), 20 nM human insulin, 0.25 µM DEX, 500 µM MIX, and 2 µM rosiglitazone for 3 days. On day 3, medium was changed to differentiation medium lacking rosiglitazone and from day 7 until the completion of the differentiation regime (21 days), cells were cultured in the differentiation medium lacking rosiglitazone, MIX, and DEX. In some experiments, cells were treated with 90 µg/mL heparin and 1 ng/mL FGF-1.
Assessment of differentiation
Differentiation was assessed morphologically by observing lipid accumulation under a x 100 magnification using a light microscope. Differentiation was also determined by measuring glycerol 3-phosphate dehydrogenase (G3PDH) enzyme activity, as described previously (18)
. During the course of these studies, we observed that total protein increases during differentiation, independent of changes in cell number (data not shown). This increase in total protein appeared to correlate with the efficiency of differentiation; therefore, G3PDH activity was not normalized to protein. G3PDH activity was instead expressed as mU per square centimeter of cultured cells.
Assessment of clonal expansion
To assess mitotic clonal expansion, phPA and 3T3-L1 PA were differentiated in 12- or 24-well plates for 21 and 9 days, respectively. Cells were harvested by trypsinization and counted using a hemocytometer at days 0, 7, 14, and 21 for human cells and days 0, 3, 6, and 9 for 3T3-L1 PA. Each sample was counted twice, and for each time point, triplicate wells were counted. Alternatively, cells were fixed with 4% paraformaldehyde for 30 min, permeabilized for 5 min with 0.1% Triton-X-100 in PBS and incubated with 4'-6-diamidino-2-phenylindole (4',6'-diamidino-2-phenylidole (DAPI)) for 15 min. DAPI was visualized using an epifluorescence microscope, and five random fields were counted for each well, with triplicate wells counted for each treatment.
ERK1/2 inhibition
For inhibition of ERK1/2 activity, phPA were grown to confluence in the presence of FGF-1 in 24-well plates and subsequently differentiated in the presence of FGF-1. The MEK1 inhibitors U0126 (10 µM; Sigma-Aldrich) or PD98059 (25 µM; Sigma-Aldrich) were also added to the medium for the first four days (U0126) or 7 days of differentiation (PD98059). Lipid accumulation was observed after 21 days of differentiation, and phPA were then harvested for G3PDH assay. No cellular toxicity was observed with the described drug treatments.
Real time reverse transcriptase-polymerase chain reaction
Subcutaneous and omental phPA were grown and differentiated in the presence or absence of 1 ng/mL FGF and 90 µg/mL heparin. Total RNA was obtained from s.c. and omental subcultured phPA and SGBS PA at confluence and following 0, 1, 4, 8 h, 4 days and 21 days of differentiation. Total RNA was obtained using the TRIzol reagent extraction procedure (Invitrogen, Mount Waverley, Victoria, Australia), and RNA was further purified using an RNAeasy column (Qiagen, Doncaster, Victoria, Australia) as per the manufacturers’ instructions. An on-column DNase digest was performed as part of the purification protocol. Total RNA (1 µg) was reverse transcribed using random hexamers and reverse transcription reagents from Applied Biosystems (Foster City, CA, USA). Primers were designed using the Primer Express 2.0 program (Applied Biosystems) and sequences are listed in Table 1
. Expression of target cDNAs was assessed by real time reverse transcriptase-polymerase chain reaction (RT-PCR) using an ABI Prism 7700 Sequence Detector system utilizing SYBR Green PCR master mix (Applied Biosystems). Amplification specificity was confirmed by visualizing PCR products on ethidium bromide stained 2.5% agarose gels and by melting curve analysis. The absence of contaminating genomic DNA was verified by including samples to which no reverse transcriptase was added. mRNA expression of the target gene was standardized to the expression of the housekeeping gene cyclophilin. Cyclophilin was chosen as the housekeeping gene as our preliminary investigations indicated expression of the cyclophilin gene (but not 18S rRNA or glyceraldehyde 3-phosphate dehydrogenase) did not change during the course of differentiation (data not shown).
|
Western blot analysis
To study the expression of adipocyte markers, human s.c. and omental PA were grown and differentiated in the presence or absence of 1 ng/mL FGF and 90 µg/mL heparin. phPA were harvested at confluence and following 0, 2, 4, 6, 8, 14, and 21 days of differentiation. In addition, 3T3-L1 PA were differentiated and harvested after 0, 1, 2, 3, 4, 6 and 8 days of differentiation. To study the activation of signaling pathways by FGF-1, s.c. phPA were grown to confluence in the presence of FGF-1. At confluence, phPA were differentiated with the standard differentiation cocktail and in the presence or absence of 1 ng/mL FGF-1 and 90 µg/mL heparin for 0, 10, 60, 480 min. Cells were harvested in buffer containing 20 mM HEPES, 1% Triton-X-100, 150 mM NaCl, 1 mM EDTA, 2 mM Na3VO4, 1 mM Na4P2O7, 10 mM NaF, and protease inhibitors (Complete Mini; Roche Diagnostics, Mannheim, Germany) and protein concentration determined by bicinchoninic acid (BCA) assay (Pierce, Rockville, IL) according to the manufacturer’s instructions. Whole cell lysates (15–30 µg) were resolved by SDS-PAGE and transferred to Immobilon-P PVDF membrane (Millipore, Bedford, MA). Membranes were blocked in a 1:1 solution of PBS and Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h and incubated in primary antibody (Ab) (diluted in PBS: Odyssey blocking buffer with 0.1% Tween-20) overnight at 4°C. Primary antibodies against the following were used for Western blot analysis: PPAR (H-100), C/EBP (14AA for murine samples and C-18 for human samples), C/EBPß (C-19) from Santa Cruz Biotechnology (Santa Cruz, CA); perilipin from Research Diagnostics (Concord, MA), ß-tubulin from Sigma-Aldrich, Akt, phospho-Akt, Ser-473, p38 MAP kinase, phospho-p38 MAP kinase, ERK1/2, and phospho-ERK1/2 from Cell Signaling Technology (Beverly, MA), and GLUT4 was obtained from Professor David James, Garvan Institute, Sydney, Australia. Membranes were then washed in PBS, 0.1% Tween-20 six times for a total of 30 min and incubated in anti-rabbit, goat, mouse or guinea pig fluorescent conjugated secondary antibodies (Invitrogen or Jackson ImmunoResearch Europe, Cambridgeshire, UK) for 1 h. Membranes were washed for a total of 30 min and then scanned on the LI-COR Odyssey Infrared Imaging System and analyzed and quantified using LI-COR Odyssey software (ver1.2).
Adiponectin secretion
Differentiated phPA were incubated in serum-free medium for 72 h. Adiponectin secretion was measured by RIA using a human adiponectin assay kit (Linco Research, Inc., MO, USA) as per the manufacturer’s instructions. The amount of adiponectin secreted was expressed as nanograms adiponectin secreted per milliliter of medium per 24 h (normalized to mg of protein whole cell lysate).
2-deoxyglucose uptake assay
Cells were differentiated in 12-well plates for 21 days for phPA or 9 days for 3T3-L1 PA. Some phPA were additionally differentiated in 0.25 µM dexamethasone for the first 4 days of differentiation, which had no effect on the subsequent insulin responsiveness of the cells. Twenty hours before assay, medium was changed to low Glc (1000 mg/l) DMEM plus 0.1% BSA. Cells were subsequently washed three times and incubated in warm KRH buffer (136 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 1.25 mM MgSO4, 10 mM HEPES, pH 7.4) for 2 h at 37°C. Quadruplicate wells were stimulated with 100 nM insulin for 20 min at 37°C. As a negative control, 25 µM cytochalasin B was added to appropriate wells 1 min before the addition of [3H]2-deoxyglucose (Amersham, Little Chalfont, UK). Glc transport was carried out for 20 min at 37°C with the addition of 50 µM 2-deoxyglucose and 1 µCi/mL [3H]deoxyglucose. The reaction was stopped with cold PBS, and cells were harvested into 1% Triton X-100 (in PBS). An aliquot was saved for protein assay, and the remaining sample solubilized in Optiphase "supermix" liquid scintillant (Perkin Elmer, Wellesley, MA) and counted in a Microbeta Jet 1450 LSC beta counter (Perkin Elmer). An aliquot of each sample was used for protein concentration determination by BCA assay. Results were expressed as counts per minute per milligram of protein.
Lipolysis assay
3T3-L1 PA and phPA were differentiated in 24-well plates. For phPA, media were changed to DMEM/F12 containing 10% FCS 24 h prior to assay. To induce lipolysis, cells were washed twice in DMEM/F12 medium and then triplicate wells were incubated in either DMEM/F12 supplemented with 2% BSA alone, or with 10 nM isoproterenol, 10 nM isoproterenol and 100 nM insulin, or 100 nM insulin alone. Lipolysis was allowed to proceed for 4 h at 37°C. Media were then collected for assay of free glycerol. Cells were harvested in 1% Triton-X-100 in PBS, and protein concentration was determined using the BCA assay. Free glycerol was assayed by comparison against a glycerol standard curve. Twenty-five microliters of media was added to 200 µl of free glycerol assay reagent (0.75 mM ATP, 3.75 mM magnesium salt, 0.188 mM 4-aminoantipyrine, 2.11 mM sodium-N-ethyl-N(3-sulfopropyl) m-anisidine, 1.25 U/mL microbial glycerol kinase, 2.5 U/mL microbial glycerol phosphate oxidase, 2.5 U/mL horseradish peroxidase buffer pH 7.0, 0.05% sodium azide) from Sigma-Aldrich and incubated at room temperature for 15 min. Samples were then read in a spectrophotometric plate reader at 550 nM.
Statistics
Data were analyzed using ANOVA for differences across experimental groups. Student’s t test was used to evaluate the significance of the difference in mean values between different treatments. Data are expressed as the means ± SEM.
RESULTS
FGF-1 up-regulates expression of PPAR in confluent preadipocytes
Treatment of phPA with FGF-1 during the proliferation phase alone is sufficient to increase the subsequent differentiation of these cells suggesting a priming effect of FGF-1 during this period (13)
. To highlight potential mechanisms by which FGF-1 may prime the cells for differentiation, the expression of the adipogenic transcription factors in phPA grown to confluence was determined. phPA were grown in serum-containing medium in the presence or absence of FGF-1 for up to 8 wk and were harvested at confluence, before induction of differentiation. Following RNA extraction, real-time RT-PCR was performed to determine the relative levels of the adipogenic transcription factors PPAR, C/EBPß, C/EBP (Fig. 1
). Relative levels of the target mRNAs were normalized to the expression of the housekeeping gene cyclophilin. The expression of C/EBP at confluence in these cells was detectable but not quantifiable and was therefore not examined further. The expression of C/EBPß and C/EBP was not significantly different between treatments. However, the expression of PPAR was significantly increased (2.5-fold higher) in phPA exposed to FGF-1 during proliferation.
|
FGF-1 treatment increases expression of adipocyte markers in human preadipocytes
To confirm that FGF-1-treated cells express higher levels of adipogenesis-related genes as a result of increased differentiation, phPA grown and differentiated in the presence or absence of FGF-1 were harvested at various time points up to day 21 of differentiation. Cells were derived from both s.c. and omental depots of human subjects, as there are depot-specific differences in adipocyte gene expression and differentiation potential in phPA (19
, 20)
. Gene and protein expression of the adipogenic transcription factors C/EBP, C/EBPß, and PPAR was determined by quantitative real-time RT-PCR and Western blot analysis (Fig. 2
). There was considerable interindividual variation in expression levels, but similar trends in expression were observed for all individuals examined. The expression of PPAR was increased by FGF-1 treatment throughout the 21-day time period and was higher in the s.c. depot than the omental depot (Fig. 2A
). The expression of C/EBP in FGF-1-treated cells followed a similar pattern (Fig. 2B
).
|
The expression of C/EBPß was more variable between individuals, particularly in the omental depot (Fig. 2)
. Analysis of mRNA expression demonstrated that C/EBPß expression remained relatively constant at the time points examined in cells, which had not been exposed to FGF-1, except for a trend toward a small increase in C/EBPß expression in the early hours of differentiation. In cells that had been exposed to FGF-1, there was also a trend toward increased expression of C/EBPß, with a twofold increase in mRNA expression 1 h after the initiation of differentiation. Western blot analysis for C/EBPß revealed the presence of a number of bands, which increased in the first hours and days of the differentiation process.
Additional features of differentiating phPA were observed by Western blot analysis for other differentiation markers, examining lipid accumulation and measuring the activity of the enzyme G3PDH (Fig. 3
). As observed with the adipogenic transcription factors, there was higher expression of perilipin and GLUT4 in cells that had been treated with FGF-1 (Fig. 3A
). There was also increased lipid accumulation (Fig. 3B
) and G3PDH activity (Fig. 3C
). There were depot-specific differences, with increased expression of differentiation markers and lipid accumulation in cells isolated from the s.c. depot compared with those isolated from the omental depot. In summary, FGF-1 treated phPA differentiated to a greater extent than untreated PA, expressing higher levels of PPAR, C/EBP, C/EBPß, perilipin, and GLUT4, and they also exhibited greater lipid accumulation and G3PDH activity.
|
FGF-1-treated preadipocytes have similar expression patterns of adipocyte genes as 3T3-L1 preadipocytes
The ontogeny of gene expression during adipogenesis in murine models of differentiation such as 3T3-L1 PA is well characterized. However, there is a paucity of similar studies using human models of differentiation. In the past, such studies have been hampered by the small sample size attainable from human adipose biopsies and by the poor differentiation capacity of the isolated cells. However, with the addition of FGF-1, phPA can be passaged and expanded and still retain their ability to differentiate to a high level. Hence, FGF-1-treated phPA were used to characterize the pattern of protein expression of differentiation markers in s.c. and omental phPA in comparison with 3T3-L1 PA.
In 3T3-L1 PA, C/EBPß expression was transiently increased with the highest expression on the first day of differentiation (Fig. 4
A). The expression of PPAR and C/EBP increased throughout differentiation. The expression of perilipin appeared midway through differentiation, with GLUT4 appearing late in the differentiation period. These observations agree with previously published reports (10
, 11)
. In phPA, a similar expression pattern was observed, although this took place over a period of 21 days compared to the 8 days required for the differentiation of 3T3-L1 PA (Fig. 4A, B
). In omental samples, expression of C/EBP was detected at low levels only in those cells, which had the highest levels of differentiation. There was considerable variation between individuals with respect to the differentiation capacity of the cells and the expression levels of adipocyte markers in s.c. and omental depots. For example, differences in GLUT4 expression ranged from 1.5 to 5-fold higher in s.c. compared to omental phPA. However s.c. phPA consistently showed increased differentiation, compared to omental phPAs, as evidenced by higher expression of adipocyte-specific proteins and/or the appearance of such proteins at an earlier time point.
|
Human adipocytes differentiated in the presence of FGF-1 secrete increased adiponectin and are more insulin responsive
We next characterized the features of mature adipocytes by determining the insulin responsiveness of these cells and their ability to secrete adipokines. Subcutaneous phPA were grown and differentiated for 21 days in the presence or absence of FGF-1. Secretion of adiponectin into the medium was measured by RIA (Fig. 5
A). Cells differentiated in the presence of FGF-1 secreted significantly greater (>10-fold) levels of adiponectin than control cells.
|
The insulin responsiveness of the adipocytes was determined by 2-deoxyglucose uptake assay and lipolysis assay. For 2-deoxyglucose uptake assay, s.c. phPA differentiated in the absence of FGF-1 had low levels of differentiation and little, if any, insulin-stimulated Glc uptake (Fig. 5B
). However, cells differentiated in the presence of FGF-1 were insulin responsive, with insulin stimulating an average three-fold increase in Glc uptake. There was considerable interindividual variation in the insulin response, which ranged from two- to eight-fold (data not shown). In addition, there was a significant decrease (P<0.05) in basal Glc uptake in phPA differentiated in the presence of FGF-1 in comparison with non-FGF-1-treated phPA. As a comparison, 2-deoxyglucose uptake in 3T3-L1 adipocytes was also determined (Fig. 5B
, inset). 3T3-L1 PA exhibited higher net basal and insulin-stimulated Glc uptake than phPA. They also displayed greater insulin-responsiveness with a mean 10-fold increase in Glc uptake in response to insulin. The insulin responsiveness of phPA grown and differentiated in the presence of FGF-1 was further assessed by measuring the antilipolytic effect of insulin. Lipolysis was stimulated in FGF-1-treated differentiated PA by exposure to 10 nM isoproterenol for 4 h. Treatment of cells with 100 nM insulin protected the adipocytes from lipolysis, reducing glycerol release by 45% (±6.4%, P<0.05).
In summary, FGF-1 treatment of phPA during growth and differentiation results in functional adipocytes that secrete high levels of adiponectin. In addition, these cells are insulin responsive in terms of both Glc and lipid metabolism.
FGF-1 treatment increases the adipogenic potential of SGBS PA
To determine whether FGF-1 would have additional effects on differentiation in human PA, which already have high adipogenic potential, SGBS PA were treated with 90 µg/ml heparin and 1 ng/ml of FGF-1 for either 7 days prior to differentiation, or during 21 days of differentiation, or both. In contrast to phPA (13)
, FGF-1 treatment during differentiation alone significantly increased the differentiation of SGBS PA in comparison with untreated cells. There was higher differentiation in cells, which were treated with FGF-1 during proliferation alone and, as with phPA, the greatest differentiation was observed in cells, which had been proliferated and differentiated in the presence of FGF-1 (Fig. 6
A).
|
Western blot analysis was carried out on differentiated SGBS samples derived from both non-FGF-1 treated cells and cells exposed to FGF-1 during both proliferation and differentiation (Fig. 6B
). As with phPA, a similar pattern of gene expression for all adipocyte markers was observed in SGBS PA in comparison with 3T3-L1 PA. Furthermore, as previously observed in phPA, FGF-1 treatment of SGBS PA resulted in increased expression of adipogenic transcription factors PPAR and C/EBP and differentiation markers GLUT4 and perilipin.
The overall effect of FGF-1 treatment in both SGBS PA and phPA was to increase the differentiation potential of both cell types. In the absence of FGF-1, SGBS PA displayed a much greater differentiation potential than similarly treated phPA. However, with FGF-1 treatment, the differentiation capacity of phPA dramatically increased to levels higher than those seen in non-FGF-1-treated SGBS PA. The human in vitro model, which consistently displayed the greatest levels of differentiation was FGF-1 treated SGBS PA. Generally, 100% of FGF-1-treated SGBS cells accumulated lipid and expressed high levels of all adipocyte markers examined (C/EBP, PPAR, perilipin, GLUT4, G3PDH).
Insulin responsiveness of SGBS adipocytes was determined by 2-deoxyglucose uptake assay. SGBS adipocytes not treated with FGF-1 exhibited a two-fold insulin-stimulated 2-deoxyglucose uptake (Fig. 6C
), whereas cells proliferated and differentiated in the presence of FGF-1 exhibited a five-fold insulin stimulated Glc uptake. These data demonstrate that the differentiation potential of a human cell model, SGBS PA (which exhibit an intrinsic capacity for adipogenesis) is further augmented by treatment with FGF-1.
ERK1/2 activation is required for human adipogenesis in the absence of clonal expansion.
FGF-1 is known to stimulate a number of downstream signaling molecules, including ERK1/2, p38 MAP kinase, and Akt, which have postulated roles in PA proliferation and differentiation (21
, 22)
. Various reports suggest a role for ERK1/2, PI 3-kinase, and p38 MAP kinase in murine PA differentiation (10
, 23
, 24)
. Studies in human PA have not been extensive, although treatments with chemical inhibitors suggest roles for PI 3-kinase and p38 MAP kinase (25
, 26)
.
To elucidate how FGF-1 may act to increase the differentiation of phPA independent of its priming effect in proliferation, the phosphorylation of Akt, p38 MAP kinase, and ERK1/2 signaling molecules was examined. phPA were proliferated in the presence of FGF-1 and at confluence, treatment with FGF-1 for 10 min resulted in strong phosphorylation of Akt, p38 MAP kinase, and ERK1/2 as observed by Western blot analysis with phospho-specific antibodies. phPA were also treated with differentiation cocktail ± FGF-1, harvested at time points early in differentiation and subjected to Western blot analysis with phospho-specific antibodies (Fig. 7
A). In the early hours of differentiation, the standard chemically defined differentiation cocktail induced sustained phosphorylation of Akt and p38 MAP kinase, and the levels of phosphorylation were increased when FGF-1 was included in the differentiation cocktail. In the absence of FGF-1, levels of ERK1/2 phosphorylation were low or undetectable. In the presence of FGF-1, however, there was a robust, sustained ERK1/2 phosphorylation throughout the first 8 h of differentiation.
|
To determine whether ERK1/2 activity was required for differentiation, phPA were differentiated in the presence of FGF-1 for 21 days, and MEK1 inhibitors were differentiated for the first 4–7 days. phPA treated with 10 µM U0126 for the first four days of differentiation had reduced levels of lipid accumulation (Fig. 7B
) and G3PDH activity (Fig. 7C
) at day 21. Similar treatment with the inactive analog, U0124 was without effect (data not shown). As PD98059 is a less potent MEK1 inhibitor than U0126, phPA were treated with 25 µM PD98059 for the first 7 days of differentiation. As before, lipid accumulation (Fig. 7B
) and G3PDH activity (Fig. 7C
) were reduced following 21 days of differentiation. Similar results were also obtained when SGBS PA were treated with PD98059 or U0126 (data not shown). U0126 treatment of phPA during differentiation in the absence of FGF-1 also resulted in decreased differentiation as assessed by morphology and G3PDH assay (data not shown). Therefore ERK1/2 activity early in adipogenesis is required for human PA differentiation.
As mentioned previously, mitotic clonal expansion occurs in 3T3-L1 and 3T3-F442A murine PA cell models. Although controversial, the process is thought to be necessary for efficient differentiation and to involve the activation of ERK1/2. In contrast, evidence suggests that phPA do not undergo mitotic clonal expansion when differentiated in vitro shortly after isolation (27
, 28)
. We have demonstrated that FGF-1 treatment of phPA increases adipogenic capacity and ERK1/2 phosphorylation and that ERK1/2 activity is required for adipogenesis of phPA. Given that FGF-1 is also a potent mitogen, we next investigated whether FGF-1 promoted adipogenesis by inducing mitotic clonal expansion (MCE) in the early stages of differentiation. 3T3-L1 PA and s.c. phPA were differentiated, and MCE was assessed by cell counting throughout the differentiation period. 3T3-L1 cells were differentiated for 9 days, and there was a two-fold increase in cell number by day 3, increasing to almost three-fold by day 6 (Fig. 7D
). phPA were differentiated for 21 days in the presence or absence of FGF-1. There was no increase in cell number, either in the presence or absence of FGF-1. In fact, there was a loss in cell number with only 60% of cells remaining by day 21 (Fig. 7D
). To confirm that a loss in cell number was not merely due to an effect of trypsinization, cells were also counted by fixing cells directly in wells and staining nuclei with DAPI. The cells in five representative fields were counted for each condition, and similar results were obtained as those reported in Fig. 7D
(data not shown). Additionally, to confirm that clonal expansion is not influenced by the presence of serum, phPA were differentiated in serum-containing medium in a protocol similar to that used for the differentiation of 3T3-L1 PA. No MCE was observed under these conditions (data not shown). Taken together, these data indicate that ERK1/2 activity is required for phPA differentiation in the absence of clonal expansion.
DISCUSSION
In this report, we have characterized the features of important new in vitro models of human preadipocyte differentiation, which have a high capacity for differentiation. The work in this study also highlights potential mechanisms of FGF-1 action in the regulation of human adipogenesis. We determined that FGF-1 treatment results in up-regulated PPAR expression prior to the induction of differentiation. FGF-1 treatment also results in increased expression of adipocyte markers, and the adipocytes derived from this process are functionally active. In addition, there are many similarities with respect to gene expression and functional characteristics of adipocytes between the phPA, SGBS PA, and 3T3-L1 PA. Finally, we demonstrated that FGF-1 treatment results in activation of the Ras-MAP kinase pathway. ERK1/2 activity is required for efficient differentiation of phPA and, unlike murine preadipocytes, this occurs in the absence of clonal expansion.
Our data demonstrate that the adipogenic transcriptional process is similar between 3T3-L1 PA and human PA, albeit over a different timescale. FGF-1 treatment of s.c. and omental phPA and SGBS PA results in similar expression patterns of the differentiation markers C/EBP, C/EBPß, PPAR, GLUT4, and perilipin in comparison with murine 3T3-L1 cells. Adipocytes derived from phPA and SGBS PA are also functionally similar to 3T3-L1 adipocytes, secreting adiponectin and being insulin responsive in terms of Glc uptake and lipolysis. A decrease in basal Glc uptake is also observed in adipocytes differentiated from FGF-1-treated phPA in comparison with non-FGF-1-treated cells. This is consistent with data from both 3T3-L1 fibroblasts (29)
and human preadipocytes (30)
, demonstrating decreased basal Glc uptake during differentiation. In these studies, this differentiation-dependent decrease in basal Glc uptake correlated with decreased GLUT1 expression (29
, 30)
.
Our data demonstrate that FGF-1-treated phPA and SGBS PA are excellent models for the study of the human adipogenic process. Furthermore, the cells derived from this process are biochemically differentiated and functionally active adipocytes, which are suitable for subsequent metabolic studies. FGF-1-treated SGBS PA is the human in vitro model, which consistently attains the highest levels of differentiation. There is high expression of a range of adipocyte markers; nearly 100% of cells accumulate lipids, and the differentiated adipocytes are functionally active with respect to insulin responsiveness. However, major advantages of the FGF-1-treated phPA model system over SGBS PA and murine 3T3-L1 cell lines include the ability to compare adipogenic and metabolic processes in human cells at different stages of postnatal development and, even more importantly, in cells from different anatomical depots. Epidemiological evidence strongly correlates intra-abdominal, or visceral, adiposity with the metabolic and cardiovascular complications that accompany obesity (31)
. Depot-specific differences in the differentiation capacity of preadipocytes has previously been well established (19
, 20)
. FGF-1 increases the differentiation capacity of cells derived from both s.c. and omental preadipocytes; however, depot-specific differences persist. The underlying reasons for these depot-specific differences are not yet understood; therefore, the use of phPA derived from specific anatomical sites provides an excellent model for identification of regulatory factors governing site-specific adipose tissue development.
The work described in this study identifies potential mechanisms of FGF-1 action in human PA. FGF-1 has a role both in the proliferation and differentiation stages of human PA development, as described previously (13)
and demonstrated in Fig. 6A
. FGF-1 treatment results in increased expression of PPAR even prior to differentiation, which suggests a role for FGF-1 in the commitment of precursor cells to the adipocyte lineage. Increased expression of the PPAR2 isoform has previously been observed in rat mesenchymal stem cells following treatment with FGF-2 (32)
. The mechanism for increased PPAR expression at confluence is unknown but could reflect changes in the activity of a number of factors. The C/EBP transcription factors play an important role in the induction of PPAR expression during the process of differentiation. These events occur after the induction of differentiation however, and the same may not be true at confluence. There is no difference in the expression of C/EBPß and between ± FGF-1 treated phPA at confluence. In addition, only low levels of C/EBP mRNA are detected in confluent phPA, as has previously been observed (33)
. Alternatively, these levels of C/EBP expression at confluence may be sufficient to interact with other required factors to induce increases in PPAR expression. PPAR also is influenced by a wide range of other factors, and one possible mechanism for direct effects of FGF-1 on PPAR expression is by decreasing the expression of the GATA transcription factors. GATAs are known to interact with FGFs in other cell types and have also been recently shown to be negative regulators of adipogenesis, binding to the promoters of PPAR and the C/EBPs (34
35
36
37)
. FGFs also interact with many other factors, which may be involved in PA commitment and differentiation, including bone morphogenic proteins (BMPs) and members of the Wnt signaling pathway (21)
.
Although FGF-1 exerts its greatest effects during proliferation, the growth factor also has additional effects during differentiation. One potential mechanism for such effects is through coordinated activation of the signaling pathways necessary for differentiation, which include the PI-3-kinase and Ras-MAP kinase pathways. To elucidate how FGF-1 may act to increase the differentiation of PA independent of its priming effect in proliferation, the phosphorylation of Akt, p38 MAP kinase and ERK1/2 signaling molecules was examined. We observed that all three pathways are activated by the standard differentiation cocktail in the early hours of differentiation and that FGF-1 treatment increases the phosphorylation of Akt, p38 MAPK, and ERK1/2. Therefore, FGF-1 appears to increase differentiation by amplifying the signals of pathways that are already activated during adipogenesis.
Intriguingly, ERK activity in the 3T3-L1 model has been intimately associated with MCE early in the differentiation process (10)
; however, no requirement for differentiation associated MCE has previously been demonstrated in human preadipocytes (27
, 28)
. As FGF-1 is a known mitogen, with ERK activity central to this effect, we were interested to determine whether the greatly increased differentiation of phPA in response to this growth factor may be associated with postconfluent MCE following induction of differentiation. For the purposes of the present study, therefore, we went on to investigate the role of ERK activity in phPA differentiation in more depth. We determined that ERK1/2 is strongly phosphorylated by FGF-1 and is necessary for subsequent differentiation. Inhibition of ERK activity by both U0126 and PD98059 reduced adipogenesis, by up to 72% and 45%, respectively. U0126 treatment in the absence of FGF-1 also resulted in decreases in the inherently low levels of differentiation observed in non-growth factor-treated PA. This is consistent with inhibition of the low levels of ERK activity demonstrated in the absence of FGF-1 and provides further evidence of the importance of ERK signaling in human PA differentiation. The role of ERK1/2 in human adipogenesis is of particular interest, as its role in murine models has been somewhat controversial. It is now generally accepted that ERK1/2 activation is important for differentiation as ERK1 knockout mice have reduced adiposity and ERK1 (–/–) murine embryonic fibroblasts have a reduced capacity for adipogenesis (38)
. Activation of ERK1/2 is required to phosphorylate C/EBPß, a process thought to be necessary for MCE, DNA-binding activity, and terminal differentiation in murine PA (3)
. Moreover, elegant studies involving careful titration of expression of the molecular scaffold protein kinase suppressor of Ras 1 (KSR), which facilitates efficient activation of the MEK/ERK cascade, demonstrate that the strength and duration of ERK activation must be tightly coordinated for optimal differentiation (39)
. Therefore FGF-1 treatment of phPA appears to stimulate ERK1/2 activity to a level that is optimal for differentiation.
This work also provides further insights into ERK1/2 activity and its role in MCE. A striking difference between the 3T3-L1 and human PA is the lack of MCE during differentiation of human PA. In the 3T3-L1 murine model, we demonstrated that the cells undergo at least one round of replication in the early stages of differentiation, as has previously been established (40)
. In contrast, we observed no clonal expansion in phPA, even in the presence of FGF-1, a known mitogenic agent. As has been previously postulated, human PA may already have undergone any requisite clonal expansion in vivo (27)
. Our data, which demonstrates differentiation in the absence of MCE after multiple rounds of replication in vitro, is consistent with this and suggests that phPA may have the ability to retain such programming. Alternatively, phPA may not require the process of clonal expansion at any stage of development in order to undergo adipogenesis. In either case, human preadipocytes appear to be intrinsically different from 3T3-L1 PA in their requirements for clonal expansion when cultured in vitro. A recent report suggests that it may be progress through S-phase rather than clonal expansion per se that is the event required for adipogenesis and as such, further investigations of human adipogenesis in the presence of cell cycle inhibitors may also yield valuable data (41)
. Therefore, ERK1/2 activity is important in human adipogenesis, independent of clonal expansion and FGF-1-treated phPA, and SGBS PA will be useful models to investigate the functional roles of ERK1/2 that are not related to MCE.
In conclusion, in the work described in this study, we have started to elucidate the mechanisms of FGF-1 action in human PA. FGF-1-treated PA have the potential to offer insights into the mechanisms of commitment to the PA lineage and into the signaling pathways involved in both proliferation and differentiation of human PA. FGF-1-treated phPA and SGBS PA display a high differentiation capacity and are, in many ways, similar to murine 3T3-L1 PA. They therefore provide reproducible and efficient in vitro human cell models to study insulin signaling, adipokine secretion, and the mechanisms of PA differentiation. The difference in the requirement for MCE between murine and human systems highlights the necessity for further studies in human models in parallel with studies in murine models. Furthermore, these cells offer a unique opportunity to investigate the role of ERK1/2 in human adipogenesis, particularly in relation to C/EBPß phosphorylation, in an environment where clonal expansion does not take place. Therefore, FGF-1-treated human PA offer an attractive in vitro model to study many features of adipogenesis in a human model and provide further insights into the processes that ultimately lead to obesity.
ACKNOWLEDGMENTS
This work was supported by grants from Adipogen, Princess Alexandra Hospital Foundation and Diabetes Australia Research Trust. FSN holds an NHMRC Biomedical Postgraduate Scholarship and a Queensland Government Smart State Ph.D. funding grant. H.S. is the recipient of a University of Queensland Joint Research Scholarship. L.J.H. holds a Smart State Fellowship. J.P.W. is the recipient of a Lions Senior Medical Research Fellowship. The authors give special thanks to the volunteers and surgeons at the Princess Alexandra Hospital for providing the tissue for study.
Received for publication March 20, 2006. Accepted for publication August 7, 2006.
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