Molecular Analysis of Lipid Storage and Trafficking

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Embargoed until 2015-06-30
Copyright: Zhang, Yuxi
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Abstract
Lipid storage and trafficking are essential cellular activities to deliver nutrients and reserve energy for cell growth. The malfunction of either one of these processes could lead to the accumulation of harmful lipids in non-fat tissues and cells. Adipocytes, the master storage depot of fats provides reservoir for energy storage as neutral lipids in the core of lipid droplets. The process of adipogenesis is elegantly regulated under specific physiological condition to favour the development of adipocyte. Seipin has been found as an important protein during adipogenesis and its absence results in lipodystrophy, hypertriglyceridemia, insulin resistance, fatty liver and diabetes. Previous study has shown that PPARγ, the master regulator of adipogenesis, may bind to the promoter region of mouse Seipin gene. The cloning of the mouse Seipin promoter region in a luciferase reporter plasmid showed PPARγ promoted the expression of the reporter gene and the co-expression with RXRα further increased the expression. Gel shift assay and ChIP qRT-PCR further proved the binding of PPARγ to the mouse Seipin promoter region. Immunofluorescence study showed PPARγ was absent in the nucleus of Seipin knockout MEFs during adipogenesis. The addition of PPARγ agonist rosiglitazone rescued the differentiation defect and restored the localisation of PPARγ to the nucleus. The absence of PPARγ might be due to high level of phosphatidic acid (PA) that was detected with PA sensor plasmid. Interesting, the knockdown of Seipin in newborn mouse hippocampus neuronal cells greatly influenced the growth of neurons and promoted the formation of supersized lipid droplets under fatty acid loading. The second part of this thesis discovered a novel degradation mechanism of the PI3K/Akt/mTORC1 pathway. The activation of the mTOR complex 1 (mTORC1) pathway under high phospho-Akt level accelerated the degradation of the cholesterol trafficking protein Niemann-Pick C1 (NPC1). This mechanism was observed in several cell lines and the knockdown of the essential component of mTORC1 raptor using siRNA greatly inhibited the degradation of NPC1. Glucose infusion study in rats showed the activation of the PI3K/Akt pathway in the liver cells greatly decreased NPC1 protein level. The degradation of NPC1 by the mTORC1 pathway was hypothesised as a feedback mechanism to shut down the insulin-signalling pathway. Overall, this thesis provided important insights into lipid trafficking and storage.
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Author(s)
Zhang, Yuxi
Supervisor(s)
Yang, Robert
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Publication Year
2014
Resource Type
Thesis
Degree Type
PhD Doctorate
UNSW Faculty
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