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Abstract
Lipid droplets (LDs) long perceived to be inert organelles have now emerged to be involved in many cellular processes. Changes in cellular dynamics of LDs are associated with human metabolic diseases such as obesity and lipodystrophy. Adipocytes, the highly-specialised cells for storing lipids, are required to prevent accumulation of lipids in non-fat tissues. Adipogenesis is a tightly regulated process that leads to the transformation of preadipocytes into adipocytes capable of storing and trafficking lipids. The Berardinelli-Seip Congenital Lipodystrophy 2 gene which encodes Seipin has been found to regulate adipogenesis, as its absence leads to the most severe form of lipodystrophy. Furthermore, previous studies have shown that Seipin is also necessary for adipocyte maintenance as loss of Seipin leads to adipocyte death. This thesis has characterised the molecular regulation of Seipin and its role in both adipogenesis and lipolysis. Through proteomic screening, Seipin was found to interact with the microsomal isoforms of glycerol-3-phosphate acyltransferase (GPAT) enzymes, the rate limiting enzymes for de novo synthesis of glycerolipids. The loss of Seipin was found to cause elevated levels of phosphatidic acid (PA) due to increased GPAT enzyme activity. Overexpression of GPAT3 or 4, recapitulate the Seipin lipodystrophy phenotype, and co-overexpression of Seipin and GPAT3/4 restores adipogenesis. Moreover, the loss of Seipin in adipocytes results in ‘supersized’ LDs and decrease in hormone stimulated lipolysis in adipocytes. Down regulation of Seipin leads to a decrease in cAMP-PKA mediated phosphorylation of key proteins involved in lipolysis, perilipin1 and HSL. Protein-protein interaction screens identified hormone sensitive lipase (HSL) as a binding partner to Seipin and loss of Seipin affects its activity. Further molecular analysis showed Seipin affects the localisation of A-kinase anchoring protein, OPA1, to LD surfaces and disrupts its interaction with the PKARIIβ subunit resulting in a decrease in PKA activity. This study also identified a novel regulatory mechanism in the stability of Seipin and its disease-causing mutants. Inhibition of the autophagy pathway increased Seipin expression and a potential E3 ligase may have been identified for Seipin. Overall, this study has provided mechanistic insights into the role of Seipin in adipogenesis and lipolysis.