Lipids on the move : lipid metabolism in protein transport and hepatic stellate cell activation

Abstract

Lipids are essential in life. They are the building blocks of biomembranes, but are also stored in lipid droplets - to serve as an energy depot and maintain a balanced lipid metabolism. Both these aspects are investigated in this thesis, with the dynamic lipid droplet as a shared key organelle. In the first project, the role of phosphatidylcholine (PC), the major phospholipid of biomembranes, in eukaryotic cellular processes was investigated by using MT58 cells. These cells bear a temperature sensitive mutation in CTP:phosphocholline cytidylyltransferase (CT)?, the regulatory enzyme in the PC synthesis pathway. We showed that inhibition of PC synthesis resulted in impaired protein transport from the Golgi complex towards the plasma membrane in MT58 cells. The decreased PC levels in MT58 cells at the non-permissive temperature are accompanied by a decline of the major DAG species in isolated Golgi membranes. A direct link between PC and DAG levels is suggested by the fact that the decrease of PC species is reflected by a decrease of DAG species with a similar fatty acid composition. Furthermore, the decline in DAG in PC-depleted Golgi membranes results in a disturbed localization of the DAG-binding protein PKD. LysoPC treatment restores DAG concentration, PKD localization and protein transport at the Golgi complex. These findings demonstrate a direct interaction between PC synthesis and DAG levels in the Golgi complex. This interaction proved to be crucial for recruitment and activation of factors involved in Golgi-mediated protein transport, underlining the role of lipids in the secretory pathway. In the second project, differences in the complex neutral lipid composition of (murine) hepatic stellate cells (HSC) during activation were identified by Raman confocal microspectroscopy and a newly adapted HPLC-APCI-MS method. Hepatic stellate cells store vitamin A in lipid droplets (LDs). However, cell activation caused by liver inflammation and repair, results in LD loss. Studying the LD degradation process, we demonstrated that upon activation of the HSCs, the LDs reduced in size, but increased in number and migrated to cellular extensions. The lipid composition in activated HSCs differed strikingly from that in quiescent HSCs. After 7 days in culture HSCs had lost most of their retinyl esters, but not their triacylglycerols (TAGs) and cholesterol esters. Furthermore, we could demonstrate a large increase in the formation of TAG species containing polyunsaturated fatty acids in the activated HSCs. As these TAG-PUFAs are at least in part a result from an enhanced uptake and conversion of arachidonic acid, this suggests a role for eicosanoid metabolism in the HSC activation process. It is clear that the functions of lipids and lipid droplets are more diverse than thought. They also seem to play important roles in an increasing number of diseases. With this research project, we hope to contribute to the mapping of these lipid/lipid droplet functions.