Lipid Dynamics in Hepatic Stellate Cells. Uncovering Molecular Mechanisms: from Lipids to Lipidomes

Hepatic Stellate Cells (HSCs) are key players in liver physiology. Nevertheless, much of the molecular mechanisms behind vitamin A (retinol) storage and release—the main function of HSCs—is still poorly understood. In this thesis, I aimed to address several questions related to HSCs, vitamin A storage, and LD biology. The thesis consists of two main parts.

In Chapters 2, 3, and 4, I studied the role of lecithin acyltransferase (LRAT) in the storage of retinyl ester (RE) and lipid droplet (LD) formation. First, I investigated the LD formation capacity of LRAT-null HSCs in Chapter 2. I show that, although the in vitro RE synthesis capacity is low in freshly isolated LRAT-null HSCs, these cells are still able to form small triacylglycerol (TAG)-filled LDs. In Chapter 3, I investigated whether vitamin A-filled LDs can be formed without requiring the machinery that is used to form “normal” TAG-filled LDs. By making use of molecular dynamics and the non-mammalian model system Saccharomyces cerevisiae, I show that vitamin A-filled lipid droplets can be formed without the requirement of pre-existing TAG droplets and without seipin, a protein dictating the site of biogenesis and size of TAG-filled LDs. In Chapter 4, I studied the in Chapter 2 observed differences in size between LDs from wild-type and LRAT-null HSCs in more detail. In this chapter, I describe that the hydrophobic N-terminus of LRAT has affinity for neutral lipids in general and an enhanced affinity for RE. Moreover, truncation of the N-terminus abolishes the ability of LRAT to form large LDs, indicating that this domain plays an important role in determining the size of the large vitamin-A-filled LDs.

In Chapters 5 and 6, the second main part of this thesis, I follow a data-driven approach using lipidomics to study HSC activation. In Chapter 5, I describe Lipid Ontology (LION), a novel ontology that formalizes scientific knowledge about lipids. In addition, I report LION/web, an online bioinformatic webtool to perform LION-enrichment analysis of user-provided lipidomic datasets. In Chapter 6, I use LION-enrichment, together with a pathway analysis tool making use of LION as well as a new LION heatmap tool, to investigate the dynamics of lipids of activating rat HSCs over the course of three weeks. Our lipidomics data reveal two distinct stages during HSC activation. In the first stage, polyunsaturated bis(monoacylglycero)phosphates (BMPs), a lipid class typically localized at endo- and lysosomes, increase at the expense of saturated PCs, SMs and PAs. In the second activation stage, BMPs, hexosylceramides and ether-linked PCs are increased, in line with a lysosomal lipid storage disease profile. These findings suggest that lysosomes play a critical role during HSC activation.

In the last chapter, Chapter 7, the combined findings of the thesis chapters are summarized and discussed.