A putative role for endogenous FGF-2 in FGF-1 mediated differentiation of human preadipocytes
Highlights
► FGF-1 reduces expression and secretion of FGF-2 in human preadipocytes. ► FGF-2 siRNA reduces proliferation/increases early expression of adipogenic markers. ► FGF-1 induced decrease in FGF-2 contributes to FGF-1's adipogenic effects.
Introduction
The current obesity pandemic is having significant adverse consequences on the health and functionality of both individuals and healthcare systems worldwide. Obesity, characterized by excessive body fat (adipose tissue), occurs in a setting of positive energy balance and is strongly associated with many chronic diseases including type 2 diabetes, cardiovascular disease and many cancers. Given the public health significance of disorders of adipose tissue mass, increased knowledge of the molecular and cellular mechanisms governing adipose tissue expansion is critical to development of effective strategies in prevention and treatment of obesity and its metabolic complications.
Both size and number of adipocytes underpins adipose tissue mass. Initially hypertrophy of existing adipocytes is sufficient to store excess energy in the form of triglycerides but in the face of continuing positive energy balance the system requires generation of new adipocytes. This occurs via the complex process of adipogenesis whereby multi-potent mesenchymal stromal cells within adipose tissue undergo commitment to the adipocyte lineage and proliferate to form a pool of preadipocytes which, upon further stimulation, differentiate into new adipocytes.
Healthy adipose tissue maintains a balance between preadipocyte differentiation and lipid storage within mature adipocytes and impairment of this balance is an important factor linking obesity to its metabolic sequelae (Medina-Gomez et al., 2007, Sethi and Vidal-Puig, 2007, Spalding et al., 2008). Excessive adipocyte hyperplasia and, conversely, excessive adipocyte hypertrophy in the absence of new adipocyte generation have each been implicated in the aetiology of obesity and its complications (Heilbronn et al., 2004, Kim et al., 2007, Spalding et al., 2008, Lebeche et al., 1999, Li et al., 2000, Sherman et al., 1993). It is possible that each of these processes may be important in obesity under different settings, such as individual genetic differences or differences in developmental stages of obesity. Together, this highlights the importance of adipogenesis as a therapeutic target – for either inhibition of excessive adipocyte number or for promotion of the acquisition of new and metabolically healthy adipocytes. Underpinning both approaches is the absolute requirement for a comprehensive understanding of the molecular regulation of adipogenesis.
For the last few years we have investigated human adipogenesis and have identified FGF-1 as a novel and potent adipogenic factor secreted from adipose-derived microvascular endothelial cells (Hutley et al., 2004). FGF-1 stimulates all stages of adipogenesis including preadipocyte proliferation, commitment and differentiation (Hutley et al., 2004, Newell et al., 2006). Further we identified FGF receptor 1 (FGFR1) and signalling via the MAPK/ERK pathway as essential components in the adipogenic actions of FGF-1 in human preadipocytes (Widberg et al., 2009). These effects of FGF-1 are specific for this family, in contrast to a range of growth factors, including PDGF-AA, PDGF-BB and IGF-1, which also signal via tyrosine kinase receptors but are not adipogenic (Widberg et al., 2009). Importantly, we also demonstrated that human adipogenesis can be modulated in vitro by targeting FGF/FGFR signalling (Hutley et al., 2004, Newell et al., 2006, Widberg et al., 2009). Preadipocytes treated with recombinant FGF-1 during proliferation have a greatly increased capacity for differentiation and adipocytes generated from this model express all examined genetic and metabolic markers of the mature phenotype including insulin sensitivity (Hutley et al., 2004, Newell et al., 2006, Widberg et al., 2009). A paracrine role for the FGF system in the regulation of adipose tissue in vivo is strongly suggested by expression of FGF ligands and their receptors in this tissue and by reports demonstrating increased expression of FGF-1 in adipose tissue from obese individuals (Mejhert et al., 2010, Teichert-Kuliszewska et al., 1992). Together this data highlights the FGF-1 model of human adipogenesis as an important platform for further studies of this process particularly of the earliest commitment stage about which little is currently understood.
One potential mechanism by which FGF-1 primes cells for differentiation is by inducing alterations in gene expression. We demonstrated that FGF-1 treatment of proliferating human preadipocytes (hPA) mediates altered expression of genes regulating adipogenesis, including the key adipogenic transcription factor, PPARγ (Newell et al., 2006, Widberg et al., 2009). In embryonic development FGF ligands are known to act in a paracrine fashion to modulate the expression of other FGF family members and this co-regulation is an important mechanism in cell fate decisions and developmental processes (Lebeche et al., 1999, Li et al., 2000, Sherman et al., 1993). Currently little is known concerning the role of endogenous FGFs in adult physiology and, more specifically, in human adipogenesis. The present work aims to identify effects of FGF-1 on preadipocyte expression of endogenous FGFs, specifically FGF-2 and FGF-10 which have each been implicated in development of white adipose tissue (Hutley et al., 2004, Krieger-Brauer and Kather, 1995, Neubauer et al., 2004, Prusty et al., 2002, Yamasaki et al., 1999). Further, we aim to determine if modulation of expression of these growth factors is a component of the molecular regulation governing the complex process of adipogenesis which involves proliferation, commitment and differentiation of preadipocytes.
Section snippets
Primary human preadipocytes (phPA)
Subcutaneous adipose tissue biopsies were obtained from 5 male (average age 50 years [range 21–76], average BMI 25.8 kg/m2 [range 19–33]) and 12 female (average age 43 years [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
Effects of FGF-1 on expression of FGF-10 and FGF-2 at confluence
In confluent phPA in response to exogenous FGF-1 treatment, FGF-10 mRNA was increased approximately 20-fold compared to ‘no FGF-1’ control (p < 0.05) (Fig. 1A) and, conversely, FGF-2 mRNA showed a 5-fold decrease (p < 0.05) (Fig. 1B). We were unable to establish whether differences in FGF-10 mRNA were recapitulated at the protein level with FGF-10 protein levels being undetectable by western blot, although recombinant human FGF-10 protein was readily detected (Fig. 1C). In contrast, the
Discussion
We previously identified a novel role of FGF-1 as a potent regulator of human adipogenesis (Hutley et al., 2004). This factor is produced in adipose tissue and acts in a paracrine manner to enhance all aspects of the adipogenic program including proliferation and adipose commitment of multi-potent mesenchymal stromal cells (preadipocytes) (Hutley et al., 2004, Hutley et al., 2001a, Newell et al., 2006). Proliferation and commitment to the adipocyte lineage of these cells is integral to the
Acknowledgements
This work was funded by The National Health and Medical Research Council of Australia and The Diabetes Australia Research Trust and The National Heart Foundation. We acknowledge the expertise and technical assistance of Dr. Anthony Bachmann in developing methodologies used in this study.
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Permanent address: Laboratory of Animal Fat Deposition and Muscle Development, College of Animal Science and Technology, Northwest A&F University, People's Republic of China.