Lipidome of Activated Hepatic Stellate Cells Figure 7. Incorporation of exogenous arachidonic acid into TAG is increased in activated HSCs. A. Overview of the omega-6 fatty acid synthesis pathway. B. Analysis of deuterated 18:2 and 20:4 incorporation in several TAG-PUFA species. Freshly isolated HSCs were incubated for seven days with 25 mM 18:2-d4 or 25 mM 20:4-d8. After neutral lipid extraction, HPLC-MS was performed as described. Values are expressed relative to the level of the respective undeuturated TAG species present at day 7. The results represent the means 6 SEM of three experiments. C, D. Freshly isolated HSCs were incubated at day 0, 3 or 6 with 25 mM 20:4-d8 for 24 h. Subsequently, neutral lipids were extracted and HPLC-APCI-MS analysis was performed. C. Contour plots of HPLC-APCI-MS analysis of HSC at day 0 and 7 in the m/z 880–1050 region. Positions of unlabeled species are indicated with a straight line and relative position of species with one or two d8-labeled acyl chains are indicated by dotted lines. Arrowheads indicate TAG species with three d8-labeled acyl chains. D. Quantification of several TAG species containing d8-labeled acyl chains during HSC activation. The results represent the means 6 SEM of three experiments. doi:10.1371/journal.pone.0034945.g007 unknown, but might involve specific regulation of a core motor machinery [23]. In addition to changes in LD localization and morphology, the LD lipid content alters as well. The RE concentration declines considerably and TAG species containing PUFAs rise to relatively high levels. The loss of RE, approximately 15% of total lipids in LDs, is roughly similar to the amount of newly formed PUFA- TAGs. So RE are apparently replaced by PUFA-TAGs in LDs during the first phase. In the second phase of the LD degradation process, the small LDs reduce further in size until hardly any LDs are present in the activated, myofibroblastic HSC. As demonstrated by Raman microspectroscopy no differences in lipid composition between individual LDs within one cell were observed, both in quiescent and activated HSCs. This indicates that the intracellular LD population behaves homogeneously with respect to RE degradation and other lipid metabolic pathways. The molecular identity of the enzymes involved in LD degradation in HSCs during activation is largely unexplored. Adipose triglyceride lipase (ATGL) has an important role in LD degradation in eukaryotic cells [24]. A contribution of hormone sensitive lipase (HSL), an enzyme involved in LD degradation in adipocytes, is less likely as this enzyme is relatively adipocyte specific and has not been detected in hepatic stellate cells. Also several proteins are implicated in RE hydrolysis, including hepatocyte carboxylesterase ES-3, ES-4, ES-10 and ES-22 [25,26]. Recently, autophagy was found to play a role in the degradation of lipids in activated mouse and human HSC’s [27], implicating lysosomal lipases in the degradation process of LDs. PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e34945