The obesity epidemic is associated with an increase in the prevalence of a number of chronic diseases including type 2 diabetes, cardiovascular disease, hypertension and some cancers and has been described by the World Health Organisation as one of the greatest public health challenges of the 21st century. Obesity is characterised by excessive expansion of adipose tissue mass underpinned by adipocyte hyperplasia. Central to this is the process of adipogenesis, which encompasses the proliferation and terminal differentiation of fibroblastic preadipocytes, contained within adipose tissue, to mature adipocytes. Despite the pivotal role of this process in obesity our understanding of the regulatory mechanisms governing adipocyte development, either in physiological or pathophysiological settings, is limited. Studies aimed at understanding this complex process are integral to development of more effective strategies for the prevention and/or treatment of obesity and obesity related diseases. Our laboratory recently identified a putative role for inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, in the dynamic regulation of lipid accumulation. Upon treatment of a variety of cell types with insulin or oleic acid IMPDH translocates to lipid droplets and inhibition of this translocation is correlated with reduced lipid accumulation. As lipid droplet formation and lipid accretion are defining features of adipogenesis, it was hypothesised that IMPDH may facilitate efficient lipid accumulation during adipose conversion of preadipocytes. In vitro systems have been used extensively to dissect the molecular and cellular events involved in adipogenesis. Therefore the aim of this project was to extend these investigations to examine the requirement for IMPDH activity during adipogenesis, using the well characterised murine 3T3-L1 cell line and primary human preadipocytes (phPAs). IMPDH expression and activity were transiently increased during differentiation of the 3T3-L1 cells although IMPDH did not associate with lipid droplets under these conditions. Pharmacological inhibition of IMPDH, using mycophenolic acid (MPA; 1 µM), reduced intracellular GTP by 60%, and blocked mitotic clonal expansion (MCE) and adipogenesis. Supplementation with guanosine (60 µM), a substrate in the nucleotide salvage pathway, restored both GTP levels and adipogenesis. These observations indicated that IMPDH activity is required for efficient differentiation of 3T3-L1 preadipocytes. Preliminary studies, involving differentiation of phPAs in standard serum-free medium (SFM) suggested that phPAs were resistant to MPA. To afford better comparison between the phPAs and the 3T3-L1 cells, which are differentiated in serum-containing medium (SCM), a modified 3T3-L1 like protocol facilitating efficient differentiation of the phPAs in SCM was established. Under these conditions phPAs displayed considerable variation in sensitivity to MPA which gave a trend towards decreased differentiation (reduced by 26%; p=0.07). Supplementation with guanosine significantly reduced adipogenesis (by 37%; p<0.05) in the phPAs independent of MPA. Furthermore, cells that were MPA resistant were also refractory to guanosine suggesting greater plasticity of guanine metabolism in phPAs from those subjects. A major difference between the cell types was that phPAs differentiated with high efficiency in the absence of MCE. Collectively, these data indicate that MCE is required for efficient differentiation of 3T3-L1 cells but not phPAs, even when differentiated under similar conditions, and suggest that the involvement of MCE underpins the differences in sensitivity to MPA between cell types. The differential effects of guanosine suggest there are additional differences with respect to the effects of manipulation of guanine nucleotides between cell types. In summary, the work presented in this thesis demonstrated inhibition of IMPDH blocked adipogenesis of murine 3T3-L1 cells and reduced differentiation of phPAs in some subjects. These observations provided novel insights into differences between differentiation of 3T3-L1 cells and phPAs, including their relative sensitivities to alterations in guanine nucleotides, and have implications for adipose tissue biology especially those factors involved in guanine metabolism. Ultimately this knowledge may form the basis for development of novel therapeutic strategies aimed at reduction of obesity and associated complications such as insulin resistance and type 2 diabetes.
| Identifer | oai:union.ndltd.org:ADTP/253945 |
| Creators | Ms Hua Su |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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