Towards a Subcellular Description of Cholesterol Homeostasis

Abstract

The role of autophagy in the maintenance of skeletal muscle cholesterol homeostasis has not been investigated prior to this thesis. Cholesterol abundance has two known inputs: (i) de novo synthesis via the mevalonate pathway, and (ii) adsorption of dietary cholesterol via lipoprotein receptor mediated uptake. Breakdown of cellular cholesterol depots by autophagy, known as macrolipophagy, may contribute as a third source of cellular cholesterol. Furthermore, the selectivity of macrolipophagy and the subcellular distribution of cholesterol may play integral roles in cholesterol homeostasis that have not been investigated. The driving hypothesis in the field is that autophagy contributes to cholesterol abundance through macrolipophagy and regulation of cholesterol synthesis. In this thesis, new methods were developed for macrolipophagy organelle analysis in tissues and to determine lipid droplet size. The first method is the initial step in measuring macrolipophagy at the individual organelle level. The second method, which involves the characterization of lipid droplet size, is critical because of the impact of dysfunctional macrolipophagy on cholesterol homeostasis. Equally important is the characterization of the inter-regulation of autophagy and cholesterol homeostasis. Inhibition of autophagy using ATG7 siRNA suggests that autophagy plays a key role in mediating cholesterol abundance and the mevalonate pathway (the parent pathway of cholesterol synthesis and protein prenylation). Together, these two advances could contribute to the future refinement of how autophagy and cholesterol homeostasis are inter-regulated at the subcellular level.