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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
SPECIFIC AIMS
We recently established that FGF-1 promotes both proliferation and differentiation of primary human preadipocytes (phPA), thus providing a powerful model for in-depth study of human adipogenic processes. In the current report, we aimed to characterize the mechanisms by which FGF-1 exerts its adipogenic actions and to identify key events in the differentiation process in phPA and in a human preadipocyte strain (SGBS PA), which exhibits a high intrinsic differentiation capacity. We also sought to compare and contrast findings in the human preadipocytes with those obtained using the extensively studied murine 3T3-L1 model of adipogenesis. The specific aims were to 1) determine FGF-1 effects on expression of key adipogenic transcription factors, including PPAR
and C/EBP family members, 2) identify the signal transduction pathways downstream of FGF-1 mediating the observed adipogenic effects, and 3) determine whether FGF-1 affected mitotic clonal expansion (MCE) of phPA because MCE appears to be tightly associated with adipogenesis in 3T3-L1 cells.
PRINCIPAL FINDINGS
1. FGF-1 up-regulates the adipogenic program in human preadipocytes
We previously demonstrated that FGF-1 treatment of phPA during proliferation primed the cells for subsequent differentiation. To further explore this priming effect, we examined the expression of key adipogenic transcription factors at confluence (prior to the induction of differentiation). Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that expression of PPAR, a key regulator of adipose conversion, was increased three-fold in response to FGF-1, whereas there was no detectable difference in C/EBP
or ßbeta; expression.
Subsequent work examined expression of these adipogenic transcription factors throughout the differentiation process. Results demonstrated increased expression of PPAR, C/EBPßbeta;, and C/EBP in response to FGF-1, which correlated with the increased differentiation potential of FGF-1-treated phPA. Similarly, there was increased expression of the adipocyte genes GLUT4 and perilipin, as well as increased activity of the differentiation marker glycerol 3-phosphate dehydrogenase (G3PDH). FGF-1-treated phPA were also more functionally active, secreting higher levels of the adipokine adiponectin and demonstrating increased insulin responsiveness with an average three-fold insulin-stimulated 2-deoxy-D-glucose uptake.
SGBS PA comprise a preadipocyte cell strain derived from an infant with Simpson-Golabi-Behmel syndrome. Unlike normal phPA, these cells retain the ability to differentiate to a high degree even after 30 population doublings. FGF-1 treatment of SGBS PA had similar effects to those observed in phPA, resulting in increased differentiation as measured by G3PDH activity and increased expression of adipogenic markers.
We also compared the human preadipocyte models with 3T3-L1 murine fibroblasts, the most widely studied model of preadipocyte differentiation. Results demonstrated both similarities and also important differences between the human and murine models. With respect to temporal order of expression of PPAR, C/EBPßbeta;, C/EBP, perilipin, and GLUT4, the human and murine models were broadly similar, however, differentiation in the human models occurred over 14 to 21 days, while 3T3-L1 PA were fully differentiated by day 8 (Fig. 1
).
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2. ERK1/2 is required for human preadipocyte differentiation, in the absence of clonal expansion
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. 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 further increased when FGF-1 was included in the differentiation cocktail (data not shown). Figure 2A
demonstrates that in the absence of FGF-1, levels of ERK1/2 phosphorylation were low or undetectable. In the presence of FGF-1, however, there was robust, sustained ERK1/2 phosphorylation throughout the first 8 h of differentiation (Fig. 2A
).
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To determine whether ERK1/2 activity was required for differentiation, phPA were treated with the MEK1 inhibitors U0126 (10 µM) or PD98059 (25 µM) in the early days of differentiation. Treatment of phPA with these inhibitors for the first 4–7 days of differentiation resulted in reduced lipid accumulation (Fig 2B
) and G3PDH activity (data not shown) at 21 days. These data demonstrate a requirement for ERK1/2 activity early in adipogenesis of phPA.
Mitotic clonal expansion (MCE) is observed in murine models of differentiation such as 3T3-L1 PA. The process is thought to be necessary for efficient differentiation and requires the activation of ERK1/2. Given that FGF-1 is a potent mitogen and causes robust phosphorylation of ERK1/2, we wished to determine whether FGF-1 affected clonal expansion in phPA. Human and murine preadipocytes were differentiated on reaching confluence and cell number assessed (by cell count, Syto60 and MTS) at various time points throughout differentiation. Whereas 3T3-L1 preadipocytes underwent at least one round of cell division in the early days of differentiation, we did not observe any evidence of this process during differentiation of human preadipocytes (Fig 2C
). Thus, ERK1/2 activity is required for adipogenesis in both murine preadipocyte systems and phPA, but a key difference between these systems is that human preadipocytes do not undergo associated MCE.
CONCLUSIONS AND SIGNIFICANCE
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 (see summary in Fig. 3
).
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We confirmed that FGF-1-treated phPA share many features with the murine 3T3-L1 model, with similar expression patterns of the differentiation markers C/EBP, C/EBPßbeta;, PPAR, GLUT4, and perilipin. phPA and SGBS adipocytes were also functionally similar to 3T3-L1 adipocytes. These results demonstrate that FGF-1-treated phPA and SGBS PA are excellent models for the study of the human adipogenic process. Further, cells derived from this process are biochemically differentiated and functionally active adipocytes suitable for subsequent metabolic studies. A prominent advantage of the FGF-1-treated phPA model system is the ability to compare adipogenic and metabolic processes in human cells from different anatomical depots. Epidemiological evidence strongly correlates intra-abdominal, or visceral, adiposity with the metabolic and cardiovascular complications that accompany obesity. The use of phPA derived from specific anatomical sites therefore provides an excellent model for identification of regulatory factors governing site-specific adipose tissue development.
FGF-1 has effects during both proliferation and differentiation of human preadipocytes. PPAR is central to the adipose conversion process and increased expression of this factor prior to exposure of the cells to a prodifferentiation environment suggests a role for FGF-1 in commitment of phPA to the adipocyte lineage.
Previously, we determined that although FGF-1 exerts its greatest effects on differentiation potential of phPA during proliferation, the growth factor also has additional effects during differentiation. Specifically, FGF-1 appears to increase differentiation by amplifying the signals of pathways that are already activated during adipogenesis. One such pathway is the Ras-MAP kinase pathway, and chemical inhibition of ERK1/2 activity significantly reduced phPA differentiation. Recent data in murine cells suggest that the intensity and duration of ERK signaling is important in the control of differentiation. Therefore FGF-1 treatment of phPA appears to stimulate ERK1/2 activity to a level that is optimal for differentiation.
The role of ERK1/2 in human adipogenesis is of particular interest as its role in murine models has been linked to clonal expansion. ERK1/2 phosphorylates C/EBPßbeta;, and this phosphorylation is required for MCE, DNA-binding activity, and terminal differentiation. We show that human PA do not undergo MCE, but that ERK1/2 is strongly phosphorylated by FGF-1 and is necessary for subsequent differentiation. This indicates roles for ERK1/2 activity independent of an effect on MCE. This key difference between murine and human models in the requirement for clonal expansion highlights the necessity for readily available human preadipocyte models with a high capacity for differentiation in order to further investigate the molecular differences with respect to this process. FGF-1-treated phPA and SGBS PA will therefore 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 paper, we highlight new models for the study of human preadipocyte differentiation. FGF-1-treated human preadipocytes offer an excellent in vitro model to study many features of human adipogenesis and provide further insights into the processes that ultimately lead to obesity.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5710fje
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