Replacement of Retinyl Esters by Polyunsaturated Triacylglycerol Species in Lipid Droplets of Hepatic Stellate Cells during Activation Nicole Testerink 1 , Mokrish Ajat1, Martin Houweling1, Jos F. Brouwers1, Vishnu V. Pully2¤a, Henk-Jan van Manen2¤b, Cees Otto2, J. Bernd Helms 1 , Arie B. Vaandrager1* 1 Department of Biochemistry and Cell Biology, Faculty of Veterinary Sciences, Utrecht University, Utrecht, The Netherlands, 2 Medical Cell BioPhysics, MIRA Institute for Biomedical Technology and Technical Medicine, Department of Science and Technology, University of Twente, Enschede, The Netherlands Abstract Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation. Citation: Testerink N, Ajat M, Houweling M, Brouwers JF, Pully VV, et al. (2012) Replacement of Retinyl Esters by Polyunsaturated Triacylglycerol Species in Lipid Droplets of Hepatic Stellate Cells during Activation. PLoS ONE 7(4): e34945. doi:10.1371/journal.pone.0034945 Editor: Antonio Moschetta, University of Bari & Consorzio Mario Negri Sud, Italy Received December 9, 2011; Accepted March 11, 2012; Published April 20, 2012 Copyright: 2012 Testerink et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the seventh framework program of the European Union-funded ‘‘LipidomicNet’’ project (proposal number 202272). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: H-JvM and VVP joined a commercial company (AkzoNobel Research and Philips Research, respectively) after the completion of the experimental work. The other authors have declared that no competing interests exist. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: A.B.Vaandrager@uu.nl ¤a Current address: Philips Research, Eindhoven, The Netherlands ¤b Current address: Department of Development and Innovation Expert Capability Group-Measurement & Analytical Science, AkzoNobel Research, Deventer, The Netherlands Introduction Hepatic stellate cells (HSCs) are non-parenchymal cells located perisinusoidally in the space of Disse and comprise about 5–10% of the total liver cell population [1]. HSCs play an important role in the turnover of hepatic extracellular matrix (ECM). They synthesize extracellular matrix proteins and secrete metalloprotei- nases to maintain the 3D structure of the liver in a dynamic way [2,3]. During the process of liver injury and repair, HSCs become activated, and the quiescent HSC undergoes a gradual transfor- mation from a non-dividing phenotype into a proliferative myofibroblastic phenotype [4,5]. HSC activation and subsequent production of excessive ECM are therefore recognized as initial steps in the process of liver cirrhosis [6]. It is therefore important to understand the molecular mechanism that underlies the activation process of HSCs. Quiescent HSCs have a lipid storing phenotype as indicated by the presence of large lipid droplets (LDs). During the activation process the HSCs lose their LDs [7]. LDs exist of a core of neutral lipids, surrounded by a phospholipid monolayer [8]. In most cells the neutral lipid stored in the LDs consists of triacylglycerols (TAG) and cholesterol esters. In HSCs, the LDs contain in addition to these neutral lipids also retinyl esters (RE). In fact, the surplus of retinol/vitamin A is mainly stored as RE in HSCs [9,10]. The existence of two types of LDs is described in HSCs [10], although it is unknown whether one of these pools exclusively contains RE. Of the stored RE, retinyl palmitate is the most abundant species in rat HSC, followed by retinyl stearate and retinyl oleate [11]. The preferred esterification of retinol with saturated fatty acid species is mediated by the enzyme lecithin:re- tinol acyltransferase (LRAT) [12]. One of the unresolved issues in the field of HCS research is, whether the decrease in LDs is causally related to the activation PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34945