More extended proteomic screening during HSC activation may reveal enzymes associated with the degradation/conversion of cholesterol esters, TAGs and RE and unravel their roles in HSC activation. We demonstrated that the levels of TAGs and RE were not decreased simultaneously upon HSC activation, suggesting a specific lipolytic regulation for these two classes of lipids. Alternatively, the lipid content (both TAGs and RE) of existing LDs may be degraded simultaneously, but the resulting retinol and fatty acids may be re-esterified at different rates. In support of this latter possibility it was shown that in activated HSCs the enzyme involved in esterification of retinol, i.e. LRAT, was more down regulated than DGAT2, an enzyme involved in incorporation of fatty acids into TAG [28] (Fig. S2). The observation that RE are degraded in the initial phase of HSC activation suggests a role for the released retinol or its metabolites like retinoic acid, during liver injury and/or repair. Recently, HSCs devoid of retinoid containing LDs, were shown to have a similar effect on liver fibrosis as normal RE containing HSCs, but caused a decrease in hepatic carcinogenesis [29]. Together with the loss of RE in the initial phase of HSC activation, we demonstrated an enhanced incorporation of exogenous arachidonic acid (AA) in TAG and formation of TAG-PUFA species. The selective incorporation of PUFAs in TAG implies the involvement of either i) specific transporters for PUFAs, ii) enzymes which selectively couple PUFAs to CoAs or iii) enzymes which selectively incorporate PUFA-CoAs into TAG. Among the various enzymes known to be involved in these pathways only the long-chain acyl-CoA synthetase 4 (ACSL4) was described to be selective for AA and other C20 fatty acids [30]. However, no large increase in mRNA levels of this enzyme was observed during HSC activation (unpublished observation; [31]). Elevation of TAG-PUFA species during HSC activation could be physiologically relevant in at least two different cellular processes. First, long chain PUFAs are known to be abundantly present in membrane phospholipids of sperm and neuronal cells, affecting membrane fluidity and regulating the function of several membrane associated proteins, including those involved in fusion of transport organelles with the plasma membrane [32–33]. Neurons and spermatocytes have in common that they have long cellular extensions, similar to activated HSC. As during transdif- ferentiation from quiescent HSC to myofibroblast long extensions are formed, phospholipids have to be synthesized to increase both the membrane surface area and the fluidity of the extensions. Although phospholipid analysis of total cell homogenates did not show an enhancement of PUFA containing phospholipid species in activated HSCs, specific enrichment of PL-PUFAs in the plasma membranes of activated HSCs cannot be excluded. A second process in which the increase in TAG-PUFAs may play a role is the AA/eicosanoid metabolism. AA plays a central role in inflammation and is involved in many pathological conditions [34]. Cubero and colleagues report that in alcoholic liver disease AA can accelerate ECM synthesis by HSCs, resulting in liver cirrhosis [35]. AA is also a precursor for eicosanoids, which are signaling lipids that play a role in a broad range of processes, such as modulation of the immune system. In several types of immune cells esterified AA has been found in isolated LDs [36]. It has been suggested that in leukocytes such LDs serve as an AA reservoir for local activation of essential cellular functions [16]. Also proteins involved in AA metabolism and transport were found on the surface of LDs supporting the hypothesis that LDs might have a central role in eicosanoid synthesis and secretion [36]. The HSC has also been recognized as antigen presenting cell involved in a range of immunological functions [37]. TAG-PUFAs Lipidome of Activated Hepatic Stellate Cells found in our lipidomic screen could therefore be storage pools, waiting to be incorporated in phospholipids or used for local eicosanoid synthesis, as described in immune cells. In conclusion, we demonstrated that drastic lipidomic changes take place during early HSC activation, including preferential RE depletion and an increase in the formation of TAG-PUFA species. These lipid species proved to be very characteristic markers for HSCs in their initial activation state and underline the importance of the lipidome during (patho)physiological alterations. Materials and Methods Ethics statement Rats were treated according to the strict governmental and international guidelines on animal experimentation, and were approved by the Animal Experimentation Committee (Dierexper- imentencommissie; DEC) of Utrecht University (DEC-numbers: 2009.III.08.076 and 2010.III.09.110). Chemicals Collagenase (Clostridium histolyticum Type I) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagles’s medium (DMEM) and fetal bovine serum (FBS) were from Gibco (Grand Island, NY, USA). Bodipy 558/568-C12 (D- 3835) and Bodipy 493/503 (D-3922) were from Molecular Probes (Invitrogen, Carlsbad, CA, USA). Antibody against glial fibrillary acidic protein (GFAP) was from BD Pharmingen (San Jose, CA, USA), against a-smooth muscle actin (a-SMA) from Thermo Scientific (Waltham, MA, USA), against diacylglycerol acyltrans- ferase 2 (DGAT2) and b-actin from Abcam (Cambridge, MA, USA), against lecithin:retinol acyltransferase (LRAT) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and against CTP:phosphocholine cytidylyltransferase alpha (CCTa) from Cell Signaling Technology (Danvers, MA, USA), All HPLC-MS solvents were from Biosolve (Valkenswaard, the Netherlands) with exception of chloroform (Carl Roth, Karlsruhe, Germany) and were of HPLC grade. Silica-G (0,063–0,200 mm) was purchased from Merck (Darmstadt, Germany). Diacylglycerol (16:0,16:0), TAG (18:1, 18:1, 18:1), cholesterol, CE (18:1), and retinyl palmitate standards were from Sigma-Aldrich (St. Louis, USA). Linoleic acid-d4 (18:2-d4) and arachidonic acid-d8 (20:4-d8/AA- d8) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Isolation and culture of HSC Adult male Wistar rats (350–400 g) were used in all experi- ments. Stellate cells were isolated from rat liver by collagenase digestion followed by differential centrifugation [38]. Cell isolations were performed under reduced light conditions to prevent retinoid oxidation. After isolation, HSC were plated in 25 cm2 culture flasks or on glass coverslips. Purity of the plated cell population was checked routinely by immuno-staining of glial fibrillary acidic protein (GFAP). Cells were maintained in Dulbecco’s modified Eagles’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100-units/ml penicillin and 100 mg/ml streptomycin and 4 ml/ml Fungizone. Medium was changed every 4 days. Cells were protected from light exposure by wrapping culture flasks and dishes in aluminium foil. Fluorescence and live cell microscopy Freshly isolated HSCs were grown on glass coverslips. Cells were fixed at day 0, 4, 7 and 14 with 4% paraformaldehyde in phosphate-buffered saline (PBS). For staining of the LDs, cells were incubated with Bodipy 493/503 (final concentration PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e34945