|dc.description.abstract||Adipose tissue homeostasis is regulated by a combination of extracellular and intracellular signalling pathways. Activation of surface G protein-coupled receptors (GPCR) and insulin receptors, influence the rate of lipolysis within adipocytes, the pathway responsible for triacylglycerol (TAG) breakdown into non-esterified fatty acids (NEFA) and glycerol. A GPCR for short chain fatty acids (SCFA) has been identified on the surface of mature adipocytes and activation of this receptor by SCFA including acetate, butyrate and propionate has resulted in a decrease in lipolysis, measured as a reduction in NEFA and glycerol concentration in the media. However, evidence for a mechanism of action of SCFA within adipocytes has remained unclear. Therefore, this thesis has aimed to better understand the mechanism(s) by which the SCFA, acetate, regulates adipose tissue metabolism and function, and in particular the pathway of lipolysis.
Through the development of experimental methods in vitro, it was determined that the dose of isoproterenol, a β-adrenergic receptor activator, required to stimulate lipolysis by 50 % in murine 3T3-L1 adipocytes, was 5 μM, and that no interference with metabolic assays was observed in the presence of any potential treatment condition, either in the basal or stimulated state. In 3T3-L1 cells, in the basal state, treatment of cells with the short chain fatty acid acetate (4 mM), significantly reduced lipolysis, as a measure of NEFA and glycerol (P = 0.004 and P = 0.020, respectively) after a 180 min incubation. Similarly, in the stimulated state, acetate also reduced NEFA significantly (P = 0.020), however, glycerol was not reduced (P = 0.529) compared with controls. To evaluate whether the metabolic changes in NEFA and glycerol concentration reflected an intracellular change to the pathway of lipolysis, phosphorylation of the key enzyme, hormone sensitive lipase (HSL), was also analysed. It was identified that in the stimulated state, phosphorylation of HSL, at serine residue 563, was reduced by 15 % in the presence of 4 mM acetate, compared with control, complimentary to metabolic data. However, treatment of isolated primary mouse mature adipocytes with acetate did not produce results comparable to those found in the literature. Instead, accumulation of NEFA and glycerol in the media were found to be negligible. Cell viability may have been a limiting factor regarding the outcome of these studies, and therefore development of a protocol to improve cell viability without compromising cell yield
would prove useful. Furthermore, future work should consider the lack of reduction in glycerol in the media in the stimulated state from 3T3-L1 mature adipocytes. This may be accounted for by fatty acid re-esterification, as the NEFA:glycerol ratios were 1.5:1, compared with the expected 3:1 ratio. Similarly, to re-enforce the effects of acetate on stimulated lipolysis, other phosphorylation sites within HSL may be considered, including SER565.
Overall, the results obtained in this thesis demonstrate that in the mature adipocyte cell line 3T3-L1, an increase in the availability of the SCFA, acetate, resulted in a change in the pathway of lipolysis. This was evidenced by a reduction in the phosphorylation of HSL(SER563) under sub-maximal stimulation with isoproterenol, similar to levels observed in the presence of insulin. Complimentary to these data, under the same treatment conditions, incubation with acetate also resulted in a small but significant reduction in NEFA concentration in the media.||en_US