Investigating The Role Of BAMBI In Human Adipogenesis

Obesity epidemic is the most challenging health issue facing the world in the 21st century. Adipose tissue dysfunction underpins the association of obesity with the development of cardiovascular disease, type 2 diabetes, atherosclerosis, hepatic steatosis and some cancers. Adipogenesis is required for maintenance of adipose tissue function. It consists of the proliferation, commitment and subsequent differentiation of preadipocytes and involves the coordination of a tightly regulated transcriptional program via numerous paracrine and endocrine factors.In our laboratory, we previously reported that Fibroblast Growth Factor-1 (FGF-1) acts as a paracrine factor, to prime primary human preadipocytes (phPAs) and SGBS PAs (a human preadipocyte strain derived from an individual with Simpson-Golabi-Behmel syndrome) and increases adipogenesis through a FGFR1/FRS2/Ras/Erk1/2 cascade. This provides a useful platform for investigations aimed at obtaining more comprehensive understanding of adipogenesis at the molecular level. This also provides the opportunity for identification of novel regulatory factors downstream of FGF/FGFR signalling as potential candidates for future obesity therapies. To address these aims, DNA microarray analysis of +/- FGF-1-treated primary phPAs was performed. According to change fold by FGF-1 and novelty regarding adipogenesis, BMP and activin membrane-bound inhibitor (BAMBI) was chosen for further investigation in this thesis. Therefore, the aims of this thesis were to identify the function of BAMBI and investigate the mechanisms of BAMBI action in human adipogenesis.BAMBI is a transmembrane protein and modulator of paracrine factors that regulate adipogenesis including TGF superfamily members (TGF-βand BMP) and Wnts. Consistent with 13-fold decrease in BAMBI expression in response to FGF-1 from microarray data, qRT-PCR data demonstrated that BAMBI expression was decreased upon FGF-1 treatment in phPAs and SGBS PAs. Moreover, using pharmacological approaches to inhibit activation of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3 kinase (PI3K) and p38 mitogen activated protein kinase (p38MAPK) signalling, it was identified that FGF-1-induced decrease in BAMBI expression is dependent on activation of PI3K pathway. The effects of BAMBI knockdown on adipogenesis were subsequently examined. Using an optimised siRNA knockdown system, the expression levels of BAMBI mRNA and protein were significantly reduced at all stages of differentiation. BAMBI knockdown resulted in induction of PPARγand a change in cell morphology prior to induction of differentiation and these effects mirrored those of FGF-1. In this thesis, it was also demonstrated that BAMBI knockdown, like FGF-1, decreased expression of FGF-2, which was identified recently by our group as a negative regulator of preadipocyte commitment. In addition, although more preliminary, the BAMBI overexpression data presented in this thesis confirm the negative role BAMBI plays in preadipocyte commitment. The ability of FGF-1 to induce expression of PPARγmRNA was significantly reduced in the BAMBI overexpressing cells.The following data presented in this thesis confirms the adipogenic effect of BAMBI knockdown. Consistent with increased lipid accumulation, BAMBI siRNA treated phPAs and SGBS PAs expressed higher levels of PPARγ, G3PDH, adiponectin, GLUT4 and perilipin than the SCR siRNA treated cells. Furthermore, adipocytes derived from BAMBI siRNA treated SGBS PAs were also shown to be functionally more active, secreting higher levels of the insulin-sensitising hormone adiponectin and being more insulin responsive in terms of glucose uptake.Functional investigations indicated that BAMBI is likely to play a central role in the modulation and integration of signalling from multiple autocrine / paracrine adipogenic factors (Wnt3a, TGF-β1 and BMP-4) that represent therapeutic anti-obesity targets. BAMBI knockdown attenuated anti-adipogenic effects of Wnt3a and TGF-β1 as well as pro-adipogenic effects of BMP-4 through suppressing their canonical signalling pathways respectively. Further studies showed that BAMBI expression was significantly reduced in adipose tissue from high fat diet (HFD)-induced obese and genetically obese (ob/ob) mice.Therefore, the work presented in this thesis establishes BAMBI as a potent negative regulator of adipogenesis that affects commitment and subsequent differentiation of human PAs. Furthermore, BAMBI acts as a signalling nexus to modulate the affects of several key autocrine / paracrine adipogenic regulators (Wnt, TGF-βand BMP). In addition, BAMBI expression is reduced in adipose tissue from mice model of diet-induced obesity. These findings suggest that alterations in BAMBI may play a role in the (patho)physiology of obesity and manipulation of BAMBI may present a novel therapeutic approach to improve adipose tissue function.