Lipidome of Activated Hepatic Stellate Cells Figure 5. HSC activation results in a preferential decrease in retinyl esters. A. Neutral lipid composition of quiescent HSCs (day 0) analyzed by HPLC-APCI-MS. The results represent the means 6 SEM of three experiments. B. Quantification of RE, cholesterol esters (CE), total TAG (TAG), and TAG(18:2,18-2,18:2) content in HSC at day 0, 4 and 7. Values are expressed relative to the level of lipid present at day 0. The results represent the means 6 SEM of three experiments. doi:10.1371/journal.pone.0034945.g005 shown). Hence, the increase in PUFA species is specific for TAG and not observed in phospholipids. Incorporation of exogenous arachidonic acid is increased during HSC activation PUFAs can be imported into cells or synthesized from various precursors by elongation and desaturation, starting from linoleic acid (18:2) or linolenic acid (18:3; for an overview of the PUFA (22:5) synthesis pathway see Fig. 7A). To investigate the origin of the observed TAG-PUFAs species, freshly isolated HSCs were incubated with deuterated linoleic acid (18:2-d4) or arachidonic acid (20:4-d8) for 7 days. Analysis of TAG species by HPLC-MS showed a higher incorporation of 20:4-d8 in comparison with 18:2-d4, (Fig. 7B). Not only 20:4-d8 containing TAG species were increased, but deuterated 22:4 and 22:5 species were also present, indicating elongation and desaturation of the imported fatty acids (Fig. 7B). As arachidonic acid was preferentially incorporated, we studied its incorporation and subsequent conversion in closer detail during the HSC activation process. HSCs in different states of activation were incubated for 24 h with 20:4-d8 (Fig. 7C). Whereas quiescent cells incorporate only minor quantities 20:4-d8 in 24 h, the incorporation in activated cells was drastically increased (Fig. 7C and D). At day 7, activated HSCs incorporated 15–20 times more deuterated lipid than HSCs at day 1, in particular in the 20:4, 22:4 and 22:5 containing TAG species (Fig. 7D). Discussion Raman microspectroscopy and HPLC-MS showed that the relative retinyl ester content in quiescent HSCs amounted to 10% and 15–20%, respectively, of the neutral lipid fraction. The somewhat lower percentage RE obtained by Raman measure- ments might be explained by laser-induced photo-oxidation effects of retinoids, as described by other groups [17]. Our findings indicate a lower percentage RE than the approximately 40–45% described in other studies, but a similar species profile (i.e. retinyl palmitate..retinyl stearate.retinyl oleate<retinyl linoleate) [11,18]. It has been reported that the HSC LD composition is significantly influenced by the dietary retinol intake and age [19]. The rats in our experiments were fed a comparable amount of retinol (5 mg retinol/g dry food) and had similar weights (around 400 g) as described in the most frequently cited papers. However, the detection method for retinoids and other lipids differed between this study and the previous ones [11,18]. Upon activation HSC were thought to start loosing both their RE and their LDs simultaneously. Based on our observations we suggest that LD turnover during HSC activation occurs in two phases. Initially, LDs redistribute from perinuclear regions towards newly formed cellular extensions. During this first phase the total LD volume does not decrease, but the LDs reduce in size and increase in number. This might indicate fission of LDs during the redistribution process. Fission of LDs has been observed recently in fission yeast during cell cycle [20], although the mechanism of LD fission remained obscure. Interestingly, rat HSCs are reported to upregulate cell cycle proteins and DNA synthesis upon activation [21]. The increase in the number of LDs also suggests an increase in the amount of phospholipids, mainly PC, surrounding the LDs. CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme involved in PC synthesis, was recently shown to be required for the increase in LD number upon oleate addition to mammalian cells [22]. However, we could not observe an PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e34945