ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6

Thomas O Eichmann,Lukas Grumet,Ulrike Taschler,Jürgen Hartler,Christoph Heier,Aaron Woblistin,Laura Pajed,Manfred Kollroser,Gerald Rechberger,Gerhard G Thallinger,Rudolf Zechner,Günter Haemmerle,Robert Zimmermann,Achim Lass

doi:10.1194/jlr.m062372

Abstract

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.

Highlights

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs
Despite the small amounts of vitamin A required for biological activities (e.g.,
We found that incubation of HSC-T6 cells with increasing FA18:1 concentration led to a dose-dependent increase in TG and cholesteryl ester (CE)(+RE) content, while for the latter, this increase was much less pronounced (Fig. 1B)

Summary

Introduction

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. For instance, this hepatic vitamin A pool is sufficient to ensure and maintain vitamin A supply for Abbreviations: ADRP, adipose differentiation-related protein; Ai, Atglistatin; AIF2, apoptosis inducing factor 2; ATGL, adipose TG lipase; CE, cholesteryl ester; CGI-58, comparative gene identification-58; Clps, procolipase; DGAT, diacylglycerol O-acyl transferase; HSC, hepatic stellate cell; HSL, hormone-sensitive lipase; IRE1␣, inositol requiring protein 1␣; ISTD, internal standard; LacZ, ␤-galactosidase; LD, lipid droplet; LRAT, lecithin:retinol O-acyltransferase; PC, phosphatidylcholine; PL, phospholipid; PLIN2, perilipin 2; PLRP2, pancreatic lipaserelated protein 2; PNPLA, patatin-like phospholipase domain containing protein; qTOF, quadrupole time of flight; rATGL, rat adipose TG lipase; RBP4, retinol-binding protein 4; rCGI-58, rat comparative gene identification-68; RE, retinyl ester; ROH, retinol; SDHA, succinate dehydrogenase complex subunit A; UBXD8, UBX domain containing protein 8; UPLC, ultra-performance LC; 14-3-3/YWHA, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein

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