Characterisation of Lipid Droplet Metabolism

Samantha Murphy (2011). Characterisation of Lipid Droplet Metabolism PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Samantha Murphy
Thesis Title Characterisation of Lipid Droplet Metabolism
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2011-04
Thesis type PhD Thesis
Supervisor Prof Robert Parton
Dr Sally Martin
Total pages 139
Total colour pages 26
Total black and white pages 113
Subjects 03 Chemical Sciences
Abstract/Summary As obesity reaches epidemic proportions globally, the need for a more thorough understanding of cellular, tissue and whole body lipid regulation is becoming apparent. Within the cell, lipid droplets (LDs) are the primary sites for the storage of excess fatty acids. LDs have only recently been ascribed many of the functional characteristics of bona fide organelles and despite their importance in maintaining lipid homeostasis; many fundamental aspects of LDs remain largely unknown. One of the characteristic features of LDs is their size and distribution, which can vary depending on cell type and metabolic state. Recent studies have suggested that LDs can undergo constitutive homotypic fusion under basal conditions. However, in lipid-loaded cells and in model cells lines such as 3T3-L1 adipocytes, LDs frequently form closely packed clusters, implying that LD fusion does not readily occur. In Chapter Three of this thesis, we developed refined light microscopic methods and strict quantitative criteria to investigate LD fusion. We have now shown that under normal growth conditions, LDs in diverse cell types exhibit very low fusogenic activity. However, by screening a number of pharmacological agents we found that homotypic LD fusion could be triggered in a variety of cell types. This provided a novel cell system in which we could study the regulated fusion of phospholipid monolayers. Using this system we have shown that LD fusion involves a two-step process to produce a single spherical LD. Analysis of LD fusion events revealed that fused LDs maintained their initial volume, with a corresponding decrease in surface area, which suggests rapid removal of membrane from the fused LD. These results highlighted the lack of LD fusion observed under control conditions as well as the extent of LD restructuring that occurred when homotypic LD fusion was triggered. After demonstrating that LDs could undergo regulated homotypic fusion, and in view of the number of studies suggesting a link between LD size and lipid metabolism, we went on to investigate the regulation of LD size and fusion during fatty acid release and lipid storage in adipocytes. Despite being fundamental to adipocyte biology, very little is known about the morphological changes that occur during these processes. The remodelling of lipid droplets to form microlipid droplets (mLDs) is a striking feature of lipolysis in adipocytes. However, once lipolysis ceases, the cell must regain basal morphology. In Chapter Four of this thesis, we have characterised the mLDs that appear throughout the cell during lipolysis. We have shown for the first time that mLDs are bone fide LDs that form in both cultured and primary adipocytes. Using high-resolution electron microscopy and tomography techniques we have demonstrated that mLDs have essentially the same ultrastructure as large LDs and identified novel tethering structures between mLDs and other organelles. We also show that contrary to popular assumption, mLD biogenesis does not involve fission from large LDs or fatty acid esterification, suggesting a role for triglyceride transfer to peripheral sites of mLD formation. Analysis of mLD formation in live cells revealed that an average of 6 mLDs were formed per minute, increasing the total LD surface area by 36.5% after 30 min. We propose that mLDs are formed specifically in response to activation of the lipolysis pathway to increase the surface area available to lipases to augment lipid hydrolysis. We then went on to examine the changes in LD morphology that occurred when lipolysis ceased and lipid storage was promoted. In Chapter Five we have shown that insulin inhibited the lipolytic signalling pathway, rapidly reversing the phosphorylation of perilipin and returning the cell to basal morphology. Further analysis showed that the return to basal morphology was achieved through the formation of macroLDs. For the first time we have demonstrated insulin- and microtubule-dependent restructuring of LDs following lipolysis and shown that this occurred via two mechanisms; mLD growth and homotypic mLD fusion. mLD growth was defined as the gradual increase in mLD size without any apparent interaction with other LDs, suggesting the addition of lipid directly into the mLDs. Two types of mLD fusion were also observed, complete fusion analogous to that triggered by the addition of chemical fusogens where two LDs rapidly fuse to create one spherical LD, and controlled fusion where lipid appeared to be transferred from a donor LD to an acceptor LD. These results demonstrate that in adipocytes LD morphology is altered in accordance with changes to the metabolic state of the cell. Altogether this study highlights the inherent stability of LD structure, providing evidence against LD fission and fusion in cells under normal growth conditions, whilst demonstrating the dynamic restructuring that occurs during the cycle of lipolysis and lipid storage. This work provides novel insights into fundamental characteristics of LD biology.
Keyword lipid droplet, adipocyte, fusion, lipolysis, microlipid droplet, macrolipid droplet, insulin, Rab18.
Additional Notes 22,26,28,31,35,53,56,58,61,71,72,76,79,82,84,86,89,92,94,103,105,107,110,112,122,124

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Created: Fri, 16 Sep 2011, 09:49:48 EST by Miss Samantha Murphy on behalf of Library - Information Access Service