Washington University School of Medicine Digital Commons@Becker Open Access Publications 2018 Perilipin 5 and liver fatty acid binding protein function to restore quiescence in mouse hepatic stellate cells Jianguo Lin Saint Louis University Shizhong Zheng Nanjing University Alan D. Attie University of Wisconsin-Madison Mark P. Keller University of Wisconsin-Madison David A. Bernlohr University of Minnesota - Minneapolis See next page for additional authors Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Recommended Citation Lin, Jianguo; Zheng, Shizhong; Attie, Alan D.; Keller, Mark P.; Bernlohr, David A.; Blaner, William S.; Newberry, Elizabeth P.; Davidson, Nicholas O.; and Chen, Anping, ,"Perilipin 5 and liver fatty acid binding protein function to restore quiescence in mouse hepatic stellate cells." Journal of Lipid Research.59,3. 416-428. (2018). https://digitalcommons.wustl.edu/open_access_pubs/6588 This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact engeszer@wustl.edu. Authors Jianguo Lin, Shizhong Zheng, Alan D. Attie, Mark P. Keller, David A. Bernlohr, William S. Blaner, Elizabeth P. Newberry, Nicholas O. Davidson, and Anping Chen This open access publication is available at Digital Commons@Becker: https://digitalcommons.wustl.edu/open_access_pubs/6588 Supplemental Material can be found at: http://www.jlr.org/content/suppl/2018/01/09/jlr.M077487.DC1 .html Perilipin 5 and liver fatty acid binding protein function to restore quiescence in mouse hepatic stellate cells Jianguo Lin,*,† Shizhong Zheng,§ Alan D. Attie,** Mark P. Keller,** David A. Bernlohr,†† William S. Blaner,§§ Elizabeth P. Newberry,*** Nicholas O. Davidson,1,*** and Anping Chen1,* Abstract Hepatic stellate cell (HSC) activation occurs along with decreased Perilipin5 (Plin5) and liver fatty acid-binding protein (L-Fabp) expression and coincident lipid droplet (LD) depletion. Conversely, the activated phenotype is reversible in WT HSCs upon forced expression of Plin5. Here, we asked if L-Fabp expression is required for Plin5-mediated rescue of the quiescent phenotype. Lentiviral Plin5 transduction of passaged L-Fabp/ HSCs failed to reverse activation markers or restore lipogenic gene expression and LD formation. However, adenoviral L-Fabp infection of lentiviral Plin5 transduced L-Fabp/ HSCs restored both the quiescent phenotype and LD formation, an effect also mediated by adenoviral intestine-Fabp or adipocyte-Fabp. Expression of exogenous Plin5 in activated WT HSCs induced a transcriptional program of lipogenic gene expression including endogenous L-Fabp, but none of the other FABPs. We further demonstrated that selective, small molecule inhibition of endogenous L-Fabp also eliminated the ability of exogenous Plin5 to rescue LD formation and reverse activation of WT HSCs. This functional coordination of L-Fabp with Plin5 was 5′-AMP-activated protein kinase (AMPK)-dependent and was eliminated by AMPK inhibition. Taken together, our results indicate that L-Fabp is required for Plin5 to activate a transcriptional program that restores LD formation and reverses HSC activation.—Lin, J., S. Zheng, A. D. Attie, M. P. Keller,฀D.฀A.฀Bernlohr,฀W.฀S.฀Blaner,฀E.฀P.฀Newberry,฀N.฀O.฀Davidson,฀and฀A.฀Chen.฀Perilipin 5 and liver fatty acid binding protein function to restore quiescence in mouse hepatic stellate cells. J. Lipid Res. 2018. 59: 416–428. This work was supported by the Doisy Research Fund and a Research Award from the Saint Louis University Liver Center (to A. C.). N.O.D. was supported by grants from the National Institutes of Health (HL-38180, DK-112378, DK56260, DK-52574, Murine and Advanced Imaging Cores). This work was also supported by grants from the National Institutes of Health, the Wisconsin Alumni Research Foundation (WARF), and the University of Wisconsin Institute for Clinical and Translational Research (ICTR; to A.D.A. and M.P.K.) W.S.B. was supported by grants from the National Institutes of Health (RO1 DK-068437 and RO1 DK-101251). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Manuscript received 3 May 2017 and in revised form 7 December 2017. Published, JLR Papers in Press, January 9, 2018 DOI https://doi.org/10.1194/jlr.M077487 Supplementary key words฀ lipid฀droplets฀•฀perilipins฀•฀fatty฀acid-binding฀ proteins฀•฀lipid฀metabolism฀•฀stellate฀cell฀activation Nonalcoholic฀fatty฀liver฀disease฀encompasses฀a฀spectrum฀ of pathology ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis, but the mechanisms and mediators that regulate necroinflammation and disease฀progression฀are฀still฀poorly฀understood฀(1–3).฀NASHassociated hepatic fibrosis is currently the target of significant scientific and clinical interest, in particular, the mechanisms฀ regulating฀ activation฀ of฀ hepatic฀ stellate฀ cells฀ (HSCs), which are the major fibrogenic effectors (1–3). Quiescent HSCs contain abundant complex lipids that are localized within lipid droplets (LDs) (1–3). Upon HSC activation฀and฀the฀induction฀of฀fibrogenesis,฀HSCs฀undergo฀ loss฀of฀LDs฀along฀with฀enhanced฀proliferation,฀de฀novo฀expression of -smooth฀muscle฀actin฀(-SMA),฀and฀overproduction of extracellular matrix, including I(I) collagen. Among the most abundant proteins on LDs are members of the perilipin (Plin) family of lipid droplet proteins, some of which (e.g., Plin2) play important roles in the regulation฀of฀lipid฀metabolism฀in฀a฀variety฀of฀tissues฀including฀ liver฀(4–7).฀Our฀previous฀observations฀demonstrated฀that฀ Abbreviations:฀ abhd5,฀abhydrolase฀domain฀containing฀5;฀ACC,฀acetyl฀ coA฀ carboxylase;฀ AMPK,฀ AMP-activated฀ protein฀ kinase;฀ 1,฀ 8฀ ANS,฀ 8-anilinonaphthalene-1-sulfonic฀acid;฀ATGL,฀adipose฀triglyceride฀lipase;฀ Ad-L-Fabp,฀adenoviral฀liver฀Fabp;฀Ad-I-Fabp,฀adenoviral฀intestine-Fabp;฀ Ad-A-Fabp,฀adenoviral฀adipocyte-Fabp;฀-SMA,฀-smooth฀muscle฀actin;฀ HSC,฀hepatic฀stellate฀cell;฀LD,฀lipid฀droplet;฀LOX-1,฀lectin-like฀oxidized฀ LDL฀receptor-1;฀L-Fabp,฀liver฀fatty฀acid-binding฀protein;฀LRAT,฀lecithinretinol฀acyltransferase;฀LV,฀lentiviral;฀LXR,฀liver฀X฀receptor;฀NSOP313,฀ NSOP00313;฀NASH,฀nonalcoholic฀steatohepatitis;฀PKA,฀protein฀kinase฀A;฀ Plin฀ 5,฀ perilipin฀ 5;฀ PPRE,฀ peroxisome฀ proliferator฀ response฀ element;฀ TG,฀triglyceride;฀YFP,฀yellow฀fluorescent฀protein. 1 ฀To฀whom฀correspondence฀should฀be฀addressed.฀ ฀ e-mail:฀achen5@slu.edu฀(A.C.);฀nod@wustl.edu฀(N.O.D.). ฀ The฀ online฀ version฀ of฀ this฀ article฀ (available฀ at฀ http://www.jlr.org)฀ contains a supplement. Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc. 416 Journal of Lipid Research Volume 59, 2018 This article is available online at http://www.jlr.org Downloaded from www.jlr.org at Washington Univ Medical Library, on March 3, 2018 Department฀of฀Pathology,*฀School฀of฀Medicine,฀Saint฀Louis฀University,฀St.฀Louis,฀MO;฀Department฀of฀ Neurology,†฀Guangdong฀Second฀Provincial฀General฀Hospital,฀Guangzhou,฀China;฀Department฀of฀ Pharmacology,§฀School฀of฀Pharmacy,฀Nanjing฀University฀of฀Chinese฀Medicine,฀Nanjing,฀China;฀Department฀of฀ Biochemistry,฀Molecular฀Biology฀and฀Biophysics,**฀University฀of฀Wisconsin,฀Madison,฀WI,฀53706;฀Department฀ of Biochemistry, Molecular Biology and Biophysics,††฀University฀of฀Minnesota,฀Minneapolis,฀MN฀55455;฀ Department of Medicine,§§฀Columbia฀University,฀New฀York,฀NY฀10032;฀Gastroenterology฀Division,***฀ Washington฀University฀School฀of฀Medicine,฀St.฀Louis,฀MO฀63110 Supplemental Material can be found at: http://www.jlr.org/content/suppl/2018/01/09/jlr.M077487.DC1 .html MATERIALS฀AND฀METHODS Animal studies The฀animal฀protocols฀for฀the฀use฀of฀mice฀in฀this฀study฀were฀approved฀by฀the฀Institutional฀Animal฀Care฀and฀Use฀Committees฀of฀ Saint฀Louis฀University฀and฀Washington฀University฀in฀St.฀Louis฀and฀ followed guidelines issued by the National Institutes of Health. Congenic L-Fabp฀knockout฀mice฀(L-Fabp/) were generated and maintained฀ as฀ described฀ previously฀ (18).฀ WT฀ male฀ and฀ female฀ C57BL/6J฀ mice฀ were฀ purchased฀ from฀ Jackson฀ Laboratory,฀ Bar฀ Harbor,฀ MA,฀ and฀ were฀ housed฀ in฀ a฀ temperature-controlled฀ animal฀facility฀(23°C)฀with฀a฀12:12฀h฀light-dark฀cycle฀and฀allowed฀free฀ access฀ to฀ regular฀ chow฀ and฀ water.฀ Male฀ and฀ female฀ WT฀ and฀ L-Fabp/ mice were maintained on a standard rodent chow diet (PicoLab฀Rodent฀Diet฀20)฀and฀mice฀of฀comparable฀ages฀(12–16฀ weeks)฀were฀used฀for฀isolation฀of฀HSCs฀for฀experiments. Isolation and culture of HSCs HSCs฀were฀isolated฀by฀pronase-collagenase฀perfusion฀in฀situ฀before฀density฀gradient฀centrifugation,฀as฀previously฀described฀(19).฀ Freshly฀ isolated฀ HSCs฀ were฀ cultured฀ in฀ DMEM฀ supplemented฀ with฀20%฀FBS.฀Cells฀were฀passaged฀in฀DMEM฀with฀10%฀FBS.฀Unless฀otherwise฀indicated,฀semi-confluent฀HSCs฀with฀four฀to฀nine฀ passages were used in all experiments. In some experiments, lentivirus฀transduced฀HSCs฀were฀treated฀with฀L-Fabp฀inhibitors฀(supplemental฀ Fig.฀ S1),฀ at฀ the฀ indicated฀ doses฀ for฀ 24฀ h.฀ All฀ FABP฀ inhibitors฀were฀synthesized฀by฀a฀commercial฀vendor฀on฀a฀contract฀ basis (Nanosyn, Santa Clara, CA). Compound purity was determined฀by฀LC-MS,฀and฀was฀>98%฀for฀all฀compounds.฀The฀design฀of฀ these฀ inhibitors฀ was฀ based฀ on฀ structural฀ similarity฀ to฀ 8-anilinonaphthalene-1-sulfonic฀acid฀(1,฀8฀ANS),฀which฀contains฀both฀a฀sulfonic acid and an amine group and competes for binding within the฀ ligand฀ binding฀ pocket฀ of฀ hydrophobic฀ ligand฀ binding฀ proteins฀as฀determined฀using฀a฀displacement฀assay฀as฀previously฀described฀ (20).฀ Purified฀ recombinant฀ FABP฀ from฀ liver,฀ intestine,฀ epithelium,฀heart,฀and฀adipose฀were฀used฀to฀evaluate฀compound฀ selectivity฀ experimentally.฀ 1,8-ANS฀ fluorescence฀ (480฀ nm฀ emission;฀397฀nm฀excitation)฀was฀determined฀in฀response฀to฀6฀µM฀purified฀FABP฀protein,฀500฀nM฀1,8-ANS,฀and฀varying฀amounts฀of฀one฀ of฀the฀test฀compounds฀in฀a฀final฀volume฀of฀50฀µl.฀A฀ten-point฀compound฀concentration฀series฀ranging฀from฀0.7฀–฀100฀µM฀was฀used฀ to determine an IC50฀ for฀ each฀ compound฀ to฀ displace฀ 1,8-ANS฀ from฀the฀ligand-binding฀pocket฀of฀each฀FABP฀isoform.฀Oleic฀acid฀ was฀ used฀ as฀ a฀ positive฀ control,฀ and฀ yielded฀ an฀ apparent฀ IC50 of 7฀µM.฀Compounds฀NSOP313฀and฀NSOP373฀were฀the฀most฀selective฀for฀liver฀FABP,฀with฀IC50฀values฀of฀3.7฀µM฀and฀7.2฀µM,฀respectively;฀for฀all฀other฀FABP฀isoforms,฀these฀compounds฀demonstrated฀ IC50฀values฀>100฀µM.฀Compounds฀NSOP318฀and฀NSOP364฀were฀ the฀most฀potent฀for฀liver฀FABP,฀with฀IC50฀values฀of฀1.5฀µM฀and฀1.8฀ µM,฀but฀substantial฀binding฀to฀the฀adipose฀and฀epithelial฀FABP฀ isoforms฀was฀observed.฀NSOP325฀was฀the฀least฀potent฀compound฀ for฀liver฀FABP฀(IC50฀9฀µM),฀and฀showed฀significant฀binding฀to฀ all฀five฀FABP฀isoforms.฀Owing฀to฀the฀combination฀of฀high฀potency฀ and฀selectivity฀toward฀liver฀FABP,฀NSOP313฀was฀used฀for฀the฀studies reported. Where indicated, the AMPK inhibitor Compound C (Sigma,฀St฀Louis,฀MO)฀was฀added฀as฀indicated฀in฀the฀Fig.฀6฀legend฀ at฀a฀dose฀of฀20฀µM฀(8). RNA extraction and real-time PCR Total฀RNA฀was฀extracted฀from฀cells฀by฀TRI-Reagent,฀following฀ the฀protocol฀recommended฀by฀the฀manufacturer฀(Sigma).฀Total฀ RNA฀ was฀ treated฀ with฀ DNase฀ I฀ before฀ the฀ synthesis฀ of฀ the฀ first฀ strand฀of฀cDNA.฀Real-time฀PCR฀was฀performed฀as฀previously฀described฀using฀SYBR฀Green฀Supermix฀(21).฀mRNA฀levels฀were฀expressed as fold change after normalization with GAPDH, as described฀ by฀ Schmittgen฀ et฀ al.฀ (22).฀ Threshold฀ cycle฀ (Ct)฀ value฀ used฀in฀this฀report฀for฀the฀housekeeping฀gene฀GAPDH฀was฀18–20.฀ Ct฀values฀for฀other฀target฀genes฀and฀corresponding฀controls฀were฀ within฀a฀range฀of฀25–30.฀Representative฀Ct฀values฀for฀Plin5฀in฀quiescent฀and฀activated฀HSCs฀are฀reported฀in฀the฀figure฀legend฀for฀ Fig.฀1.฀Fold฀change฀values฀are฀presented฀as฀2-Ct.฀Ct฀values฀higher฀ than฀35฀were฀considered฀not฀detectable.฀The฀primers฀used฀in฀realtime฀PCR฀were฀previously฀described฀(8). Recombinant expression constructs, antisera, and immunoblots Construction฀ of฀ the฀ recombinant฀ lentiviral฀ (LV)-Plin5-yellow฀ fluorescent฀protein฀(YFP)฀and฀the฀control฀LV-YFP฀were฀as฀previously฀ described฀ (8).฀ Recombinant฀ adenoviral฀ (Ad)-L-Fabp฀ with฀ expression฀of฀FLAG฀epitope-tagged฀L-Fabp,฀or฀recombinant฀adenoviral฀ Ad-LacZ฀ with฀ expression฀ of฀ LacZ฀ were฀ previously฀ described฀and฀used฀(18).฀Ad-I-Fabp฀was฀constructed฀using฀a฀mouse฀ I-Fabp฀cDNA฀containing฀three฀copies฀of฀the฀FLAG฀epitope฀tag฀at฀ the N terminus and cloned into the pVQ AdCMV shuttle plasmid for recombination, amplification, and purification (ViraQuest Labs).฀Ad-A-Fabp฀was฀generated฀as฀previously฀described฀(23).฀In฀ brief, the expression construct was recombined into pADEasy in Escherichia coli฀BJ5183฀cells฀and฀transfected฀(Lipofectamine,฀Invitrogen)฀into฀293฀cells฀(American฀Type฀Culture฀Collection,฀Manassas,฀ Plin5 regulates lipid droplet formation via L-Fabp 417 Downloaded from www.jlr.org at Washington Univ Medical Library, on March 3, 2018 activation฀of฀WT฀mouse฀HSCs฀was฀coupled฀to฀a฀dramatic฀ reduction฀in฀expression฀of฀Plin5,฀both฀in฀vitro฀and฀in฀vivo฀ (8).฀In฀addition,฀other฀work฀in฀Plin5฀knockout฀mice฀indicated฀that฀Plin5฀prevented฀hepatic฀lipotoxicity,฀suggesting฀ a broader role for this LD protein in the regulation of hepatic฀fibrogenesis฀(9).฀In฀keeping฀with฀this฀suggestion,฀we฀ recently reported that exogenous Plin5 significantly increased intracellular lipid content and restored LDs in HSCs฀ from฀ WT฀ mice฀ (8).฀ In฀ addition,฀ the฀ expression฀ of฀ exogenous฀Plin5฀attenuated฀intracellular฀oxidative฀stress฀in฀ WT฀HSCs฀(8).฀These฀actions฀collectively฀resulted฀in฀attenuated฀HSC฀activation฀(8)฀and฀support฀the฀concept฀that฀preserving฀lipid฀content฀by฀modulating฀turnover฀of฀HSC฀LDs฀ may promote a quiescent state (10, 11) and could be a functional strategy for inhibiting fibrogenesis (10, 11). Other฀players฀involved฀in฀HSC฀lipid฀metabolism฀include฀ members฀of฀the฀fatty฀acid-binding฀protein฀(FABP)฀family฀of฀ lipid-binding฀ proteins,฀ which฀ are฀ involved฀ in฀ the฀ uptake,฀ transport,฀ and฀ metabolism฀ of฀ FAs,฀ retinoids,฀ and฀ other฀ lipid฀ligands฀(12).฀Among฀these฀FABPs,฀liver฀Fabp฀(L-Fabp,฀ Fabp1)฀ is฀ abundantly฀ expressed฀ in฀ both฀ hepatocytes฀ and฀ enterocytes฀ and฀ plays฀ a฀ key฀ role฀ in฀ high-fat฀ diet-induced฀ hepatic฀steatosis฀(13–17)฀and฀also฀in฀the฀development฀and฀ progression฀of฀diet-induced฀NASH฀in฀vivo฀(18).฀L-Fabp฀is฀ also abundantly expressed in quiescent HSCs, and our previous฀ work฀ demonstrated฀ that฀ activation฀ of฀ WT฀ HSCs฀ is฀ coupled฀to฀decreased฀L-Fabp฀expression,฀temporally฀related฀ to฀LD฀depletion,฀and฀reversed฀upon฀adenoviral-mediated฀ L-Fabp฀rescue฀(18). Here, we sought to expand understanding of the pathways฀that฀regulate฀HSC฀activation฀and฀LD฀turnover฀in฀relation฀ to฀ mechanisms฀ of฀ lipid-mediated฀ liver฀ injury.฀ Our฀ specific฀questions฀centered฀on฀the฀role฀of฀L-Fabp฀expression฀as฀a฀requisite฀component฀of฀Plin5-mediated฀rescue฀of฀ HSC฀quiescence,฀including฀LD฀formation฀and฀reversal฀of฀ the฀activated฀phenotype. Supplemental Material can be found at: http://www.jlr.org/content/suppl/2018/01/09/jlr.M077487.DC1 .html VA)฀to฀allow฀packaging฀and฀amplification.฀Large฀scale฀adenovirus฀ preparations were obtained from 10 cm plates in which the media was collected, centrifuged at 20,000 g for 10 min to pellet the cellular฀debris,฀and฀the฀supernatant฀containing฀virus฀particles฀recovered and frozen in aliquots at 70°C.฀ Antisera฀ to฀ FABPs฀ and฀ perilipins฀ were฀ previously฀ described฀ (16).฀ Western฀ blotting฀ analyses฀ were฀ conducted฀ as฀ we฀ previously฀ described฀ (8).฀ Thirty฀ micrograms of proteins per well were used in all immunoblots, except฀where฀indicated฀as฀in฀Fig.฀1. Knockdown of perilipin 2 by shRNA Plasmids, transient transfection, and luciferase activity assays The฀ luciferase฀ reporter฀ plasmid฀ pPPRE-Luc฀ contains฀ three฀ copies฀ of฀ peroxisome฀ proliferator฀ response฀ elements฀ (PPREs)฀ from฀the฀acyl-CoA฀oxidase฀gene,฀as฀we฀previously฀described฀(25).฀ The฀luciferase฀reporter฀plasmid฀LXR-Luc฀has฀three฀copies฀of฀liver฀ X฀receptor฀(LXR)฀binding฀elements.฀It฀was฀a฀gift฀from฀Dr.฀Knut฀ Steffensen฀(26).฀The฀plasmid฀TOPFlash฀is฀a฀Wnt/-catenin฀signaling฀luciferase฀activity฀reporter.฀It฀was฀kindly฀provided฀by฀Dr.฀Randall฀ Moon฀(27).฀Plasmid฀transfection฀and฀luciferase฀activity฀assays฀were฀ undertaken฀as฀previously฀described฀(28). In situ quantification of LDs and lipid assays Oil฀Red฀O฀staining฀was฀undertaken฀on฀HSCs฀seeded฀on฀autoclaved฀ cover฀ slips฀ in฀ a฀ 6-well฀ plate฀ and฀ cultured฀ in฀ DMEM฀ with฀ 10%฀ FBS฀ with฀ or฀ without฀ transduction฀ and/or฀ treatment,฀ followed฀by฀fixation฀with฀4%฀paraformaldehyde฀(30฀min).฀LDs฀were฀ stained฀as฀previously฀described฀(28).฀Numbers฀of฀LDs฀in฀Oil฀Red฀ O-stained฀HSCs฀were฀quantified฀using฀the฀Nuance฀multispectral฀ imaging฀ system฀ (Perkin-Elmer,฀ MA),฀ as฀ we฀ previously฀ described฀ (18).฀ Cells฀ of฀ interest฀ were฀ digitized฀ and฀ circled฀ individually฀ within the image and the numbers of LDs per cell automatically calculated.฀ Positive฀ staining฀ was฀ adjusted฀ by฀ subtracting฀ background฀control฀signals.฀The฀results฀were฀collected฀and฀expressed฀ as a mean number of LDs per cell based on at least 10 cells. Intracellular฀FFAs฀and฀triglycerides฀(TG)฀were฀colorimetrically฀determined฀ using฀ kits฀ from฀ BioVision,฀ Inc.฀ (Mountain฀ View,฀ CA),฀ following the manufacturer’s protocol (29). Analysis of cellular retinol and retinyl esters WT-passaged฀HSCs฀were฀transduced฀with฀or฀without฀LV-Plin5YFP,฀or฀LV-YFP,฀or฀LV-Plin5-YFP฀plus฀Ad-L-Fabp.฀HSC฀concentrations of retinol and retinyl esters were determined by HPLC protocols฀ described฀ previously฀ (30).฀ Briefly,฀ frozen฀ HSC฀ pellets฀ were฀resuspended฀and฀homogenized฀in฀1.0฀ml฀of฀ice-cold฀PBS฀(10฀ mM฀sodium฀phosphate,฀pH฀7.2,฀150฀mM฀sodium฀chloride)฀using฀ a฀Polytron฀homogenizer฀(Brinkmann฀Instruments,฀Westbury,฀NY)฀ set฀at฀half-maximal฀speed฀for฀10฀s.฀The฀HSC฀homogenate฀was฀then฀ treated฀ with฀ an฀ equal฀ volume฀ of฀ absolute฀ ethanol฀ containing฀ a฀ known฀amount฀of฀retinyl฀acetate฀as฀an฀internal฀standard.฀The฀retinoids present in the homogenates were extracted into hexane. After฀one฀backwash฀against฀doubly฀distilled฀water,฀the฀hexane฀extract฀was฀evaporated฀to฀dryness฀under฀a฀gentle฀stream฀of฀nitrogen.฀ Immediately฀upon฀reaching฀dryness,฀the฀retinoid-containing฀film฀ was฀redissolved฀in฀40฀µL฀of฀benzene฀for฀injection฀onto฀the฀HPLC฀ column.฀ The฀ extracted฀ retinoids฀ were฀ separated฀ on฀ a฀ 4.6฀ ×฀ 250฀ mm฀ Ultrasphere฀ C18฀ column฀ (Beckmann,฀ Fullerton,฀ CA)฀ preceded by a C18 guard column (Supelco, Bellefonte, PA) using 70%฀ acetonitrile-15%฀ methanol-15%฀ methylene฀ chloride฀ as฀ the฀ running฀ solvent฀ flowing฀ at฀ 1.8฀ ml/min.฀ Retinol฀ and฀ individual฀ retinyl esters (retinyl palmitate, oleate, linoleate, and stearate) were detected at 325 nm and identified by comparing the retention times and spectral data of experimental compounds with those of authentic standards. Concentrations of retinol and retinyl esters in the HSCs were quantitated by comparing integrated peak฀ areas฀ of฀ each฀ retinoid฀ against฀ those฀ of฀ known฀ amounts฀ of฀ purified standards. Loss during extraction was accounted for by adjusting฀for฀the฀recovery฀of฀internal฀standard฀added฀immediately฀ after homogenization of the samples. Statistical analyses Differences฀between฀means฀were฀evaluated฀using฀an฀unpaired฀ two-sided฀ Student’s฀ t-test฀ (P < 0.05 considered as significant). Where appropriate, comparisons of multiple treatment conditions฀with฀controls฀were฀analyzed฀by฀ANOVA฀with฀the฀Dunnett’s฀ test for post hoc analysis. Fatty acid uptake and lipolysis assays WT-passaged฀HSCs฀were฀transduced฀with฀or฀without฀LV-Plin5YFP,฀or฀LV-YFP,฀or฀LV-Plin5-YFP฀plus฀Ad-L-Fabp.฀Cells฀were฀incubated฀ in฀ media฀ containing฀ 2µCi฀ [3H]oleic฀ acid฀ (ART฀ 0198,฀ American฀ Radiolabeled฀ Chemicals,฀ St.฀ Louis,฀ MO)฀ and฀ 250฀ µM฀ oleic฀ acid฀ albumin฀ (O-3008,฀ Sigma-Aldrich)฀ for฀ 4฀ h฀ at฀ 37°C,฀ washed,฀ and฀ extracted฀ with฀ chloroform-methanol฀ as฀ described฀ (15).฀Radiolabeled฀lipids฀were฀separated฀by฀thin฀layer฀chromatography,฀and฀the฀position฀of฀3H-lipids฀was฀determined฀by฀migration฀ of unlabeled standards. Assays were performed in triplicate, and normalized to cellular protein content. For฀ lipolysis฀ assays,฀ cells฀ (groups฀ as฀ described฀ above)฀ were฀ collected in PBS and frozen. Cell pellets were homogenized in 418 Journal of Lipid Research Volume 59, 2018 RESULTS L-Fabp is critical for Plin5-mediated rescue of LD formation in HSCs To฀determine฀the฀role฀of฀L-Fabp฀in฀Plin5-mediated฀rescue of LD formation, we examined cultured HSCs from / WT฀and฀L-Fabp ฀mice,฀transduced฀with฀recombinant฀LVPlin5-YFP฀or฀an฀empty฀lentiviral฀LV-YFP฀control.฀As฀shown฀ in Fig. 1A,฀LV-Plin5-YFP฀(but฀not฀the฀empty฀vector฀control)฀ restored฀the฀formation฀of฀LDs฀in฀activated฀WT฀HSCs,฀demonstrated฀by฀staining฀with฀Oil฀Red฀O.฀In฀contrast,฀LV-Plin5-YFP฀ Downloaded from www.jlr.org at Washington Univ Medical Library, on March 3, 2018 The฀RNAi฀sequences฀targeting฀mouse฀Plin2฀mRNA฀was฀selected฀ using฀ online฀ RNAi฀ design฀ program฀ from฀ Thermo฀ Fisher฀ Scientific,฀ Dharmacon฀ RNAi฀ Technologies฀ (Lafayette,฀ CO).฀ Three฀ shRNA฀constructs฀were฀generated฀and฀tested.฀The฀best฀inhibitory฀ results฀were฀from฀the฀following฀Plin2฀shRNA฀sequence:฀5′-GGA฀ CCA฀AGT฀CTG฀TGG฀TCA฀A-3′.฀The฀anti-sense฀sequence฀of฀the฀ Plin2฀ shRNA฀ was฀ used฀ as฀ a฀ negative฀ control.฀ Construction฀ of฀ shRNA฀expression฀cassettes฀and฀subsequent฀cloning฀in฀the฀lentiviral฀ vector฀ pFLRu฀ were฀ conducted฀ as฀ we฀ previously฀ described฀ (24). 0.25M฀sucrose,฀1฀mM฀EDTA,฀1฀mM฀DTT,฀with฀protease฀inhibitors and centrifuged at 1,000 g฀ to฀ remove฀ insoluble฀ material.฀ Lipase฀activity฀was฀measured฀using฀0.5฀µCi฀[3H]Triolein฀(ART฀ 0199,฀American฀Radiolabeled฀Chemicals)฀in฀the฀presence฀of฀80฀ µM฀ glyceryl฀ trioleate฀ (T-7140,฀ Sigma-Aldrich)฀ and฀ 1%฀ FA-free฀ BSA฀ for฀ 1฀ h฀ at฀ 37°C.฀ Assays฀ were฀ stopped฀ by฀ the฀ addition฀ of฀ chloroform-methanol,฀ extracted,฀ and฀ analyzed฀ by฀ thin฀ layer฀ chromatography฀ as฀ described฀ above,฀ and฀ regions฀ comigrating฀ with unlabeled oleate were scraped and counted. Data were corrected for nonspecific hydrolysis and normalized to cellular protein. Supplemental Material can be found at: http://www.jlr.org/content/suppl/2018/01/09/jlr.M077487.DC1 .html failed to restore LD formation in HSCs from L-Fabp/mice (Fig.฀1A). We found that Plin5 was expressed in both freshly isolated฀ WT฀ and฀ L-Fabp/฀ HSCs฀ (Fig.฀ 1B,฀ C),฀ although฀ the฀ abundance฀of฀Plin5฀mRNA฀was฀lower฀in฀L-Fabp/฀(Ct฀value฀ ≈28.5)฀compared฀with฀WT฀HSCs฀(Ct฀value฀≈28.1) (Day 1) (Fig.฀ 1B).฀ These฀ findings฀ led฀ us฀ to฀ ask฀ whether฀ germline฀ L-Fabp deletion impaired endogenous Plin5 expression in freshly฀isolated฀HSCs.฀However,฀Western฀blotting฀analyses฀ of฀HSCs฀revealed฀comparable฀abundance฀of฀Plin5฀at฀Day฀0฀ in฀both฀genotypes฀(Fig.฀1C).฀We฀further฀showed฀that฀Plin5฀ was reduced in HSCs of both genotypes after culture for 7฀days฀(Day฀7)฀(Fig.฀1B฀and฀C),฀a฀time฀in฀culture฀that฀other฀ studies฀have฀shown฀quiescent฀HSCs฀spontaneously฀become฀ fully฀activated฀(31).฀Transduction฀of฀LV-Plin5-YFP฀resulted฀ in a comparable increase in (exogenous) Plin5 expression in฀both฀passaged-WT฀and฀L-Fabp/฀HSCs฀(Fig.฀1D).฀Considered฀together,฀these฀findings฀reveal฀expression฀of฀Plin5฀ protein in freshly isolated, quiescent HSCs in both genotypes,฀albeit฀at฀lower฀abundance฀than฀observed฀in฀passaged฀ HSCs฀transduced฀with฀LV-Plin5฀(compare฀Fig.฀1C฀with฀Fig.฀ 1D),฀particularly฀when฀taking฀into฀consideration฀that฀40฀µg฀ total฀ protein฀ was฀ loaded฀ per฀ well฀ in฀ Fig.฀ 1C฀ versus฀ 20฀ µg฀ in฀Fig.฀1D.฀Nevertheless,฀the฀data฀established฀that฀Plin5฀ Downloaded from www.jlr.org at Washington Univ Medical Library, on March 3, 2018 Fig. 1.฀ L-Fabp฀is฀critical฀for฀Plin5-mediated฀rescue฀of฀LD฀formation฀in฀HSCs.฀Passaged฀HSCs฀from฀WT฀mice฀(wt฀HSC)฀and฀from฀L-Fabp฀ germline฀knockout฀mice฀(L-Fabp/HSC)฀were฀transduced฀with฀the฀recombinant฀lentiviral฀LV-Plin5-YFP฀or฀the฀empty฀lentiviral฀LV-YFP,฀respectively,฀as฀a฀vehicle฀mock฀control.฀Positive฀transductants฀were฀selected฀with฀puromycin฀at฀5฀g/ml฀for฀48฀h.฀A:฀Oil฀Red฀O฀staining฀of฀cellular฀LDs.฀Representative฀views฀from฀three฀independent฀experiments฀are฀presented.฀B:฀Real-time฀PCR฀assays฀of฀Plin5฀in฀freshly฀isolated฀WT฀ HSCs or L-Fabp/HSCs฀after฀culture฀for฀1฀day฀or฀7฀days฀(n฀=฀3),฀*P฀<฀0.05฀vs.฀corresponding฀HSCs฀at฀day฀1,฀and฀‡P฀<฀0.05฀vs฀wtHSCs฀at฀day฀1฀ (Ct฀Plin5฀in฀WT฀HSC฀≈28.1;฀Ct฀Plin5฀in฀L-Fabp/HSC≈28.5;฀Ct฀GAPDH≈18).฀C:฀Western฀blotting฀analyses฀of฀Plin5฀in฀freshly฀isolated฀WT฀ HSCs or L-Fabp/HSCs฀(D0)฀or฀after฀culture฀for฀7฀days฀(D7)฀(40฀g฀protein/well฀was฀loaded).฀-actin฀was฀used฀as฀an฀invariant฀control฀for฀ equal฀loading.฀D:฀Western฀blotting฀analyses฀of฀Plin5฀in฀WT฀HSCs฀or฀L-Fabp/HSCs,฀respectively,฀transduced฀with฀LV-YFP฀or฀LV-Plin5-YFP฀ (20 g฀protein/well฀was฀loaded).฀E:฀Real-time฀PCR฀analyses฀of฀expression฀of฀LD฀protein฀genes฀in฀wt฀HSCs฀and฀in฀L-Fabp/HSCs฀(n฀=฀3).฀ *P฀<฀0.05฀vs฀corresponding฀control฀(Ctr).฀F:฀Analyses฀of฀cellular฀FFA฀(n฀=฀3),฀and฀cellular฀TG฀(n฀=฀3)฀content฀following฀lentiviral฀transduction;฀ *P฀<฀0.05฀vs.฀corresponding฀nontransduced฀Ctr฀HSCs.฀G:฀Uptake฀of฀FA฀and฀incorporation฀into฀TG฀in฀wt฀HSCs฀following฀transduction฀with฀or฀ without฀LV-YFP,฀LV-Plin5,฀or฀LV-Plin5฀plus฀Ad-L-Fabp฀(n฀=฀3);฀*P฀<฀0.01฀vs.฀the฀CtrHSCs;฀‡P฀<฀0.04฀vs.฀HSCs฀with฀LV-YFP. Plin5 regulates lipid droplet formation via L-Fabp 419 Supplemental Material can be found at: http://www.jlr.org/content/suppl/2018/01/09/jlr.M077487.DC1 .html L-Fabp is required for Plin5-mediated reversal of HSC activation and transcriptional induction of prolipogenic signaling pathways To฀explore฀the฀underlying฀mechanisms฀by฀which฀Plin5฀ and฀ L-Fabp฀ function฀ in฀ promoting฀ cellular฀ lipid฀ content,฀ we฀asked฀whether฀exogenous฀Plin5฀might฀stimulate฀prolipogenic฀and/or฀attenuate฀antilipogenic฀transduction฀pathways,฀in฀a฀manner฀dependent฀on฀L-Fabp.฀For฀this฀purpose,฀ we฀examined฀LXR฀and฀PPAR induction as models of prolipogenic฀ induction฀ (32–34)฀ and฀ also฀ Wnt/-catenin฀ signaling฀which,฀via฀cross-talk฀with฀PPAR, interferes with its functions฀and฀is฀considered฀antilipogenic฀(35,฀36).฀We฀ transduced฀ WT฀ or฀ L-Fabp/฀ HSCs฀ with฀ LV-Plin5-YFP฀ or฀ LV-YFP฀and฀after฀selection,฀transfected฀those฀cells฀with฀luciferase฀ reporter฀ plasmids฀ LXR-Luc,฀ PPRE-Luc,฀ or฀ TOPFlash฀(see฀details฀in฀Methods).฀As฀shown฀in฀Fig. 2A,฀elevated฀ luciferase฀ activity฀ was฀ found฀ in฀ LXR-Luc฀ and฀ PPRE-Luctransfected฀ WT฀ but฀ not฀ L-Fabp/ HSCs transduced with Plin5.฀Conversely,฀we฀observed฀reduced฀luciferase฀activity฀ in฀WT฀but฀not฀L-Fabp/฀HSCs฀transfected฀with฀TOPFlashLuc and transduced with Plin5. We฀ further฀ examined฀ mRNA฀ expression฀ of฀ acetyl฀ coA฀ carboxylase฀(ACC),฀FAS,฀and฀lectin-like฀oxidized฀LDL฀receptor-1฀(LOX-1),฀which฀are฀surrogate฀downstream฀target฀ genes฀of฀LXR฀(37),฀PPAR฀(38)฀and฀Wnt/-catenin฀signaling฀(39),฀respectively.฀We฀found฀that฀expression฀of฀exogenous฀ Plin5฀ significantly฀ increased฀ mRNA฀ abundance฀ of฀ ACC฀and฀FAS฀and฀reduced฀LOX-1฀mRNA฀in฀WT฀but฀not฀ L-Fabp/฀HSCs฀(Fig.฀2B).฀Using฀the฀same฀experimental฀design,฀forced฀expression฀of฀Plin5฀in฀WT,฀but฀not฀L-Fabp/ HSCs,฀significantly฀inhibited฀mRNA฀abundance฀of฀-SMA฀ and I(I)฀procollagen,฀two฀markers฀of฀fibrogenic฀activation฀ (Fig.฀2C).฀We฀also฀found฀that฀exogenous฀Plin5฀significantly฀ reduced฀mRNA฀abundance฀of฀pro-lipolytic฀genes฀patatinlike฀ phospholipase-3,฀ adipose฀ triglyceride฀ lipase฀ (ATGL),฀ and฀abhydrolase฀domain฀containing฀5฀(abhd5),฀in฀WT฀but฀ 420 Journal of Lipid Research Volume 59, 2018 not L-Fabp/฀HSCs฀(Fig.฀2D).฀These฀changes฀were฀accompanied฀by฀induction฀of฀SREBP-1฀and฀PPAR฀mRNAs฀by฀3-฀to฀ 5-fold฀(Fig.฀2D).฀As฀noted฀above,฀these฀effects฀of฀exogenous฀ Plin5฀on฀lipogenic฀gene฀expression฀were฀not฀observed฀in฀ L-Fabp/฀HSCs฀(Fig.฀2D).฀Taken฀together,฀these฀findings฀ reinforce the conclusion that L-Fabp deletion significantly attenuates฀the฀ability฀of฀Plin5฀to฀inhibit฀HSC฀activation฀and฀ elevate฀cellular฀lipid฀content,฀likely฀through฀a฀network฀of฀ transcriptional signaling pathways. Introduction of L-Fabp into L-Fabp/ HSCs rescues Plin5-mediated reversal of activation and prolipogenic activity We฀introduced฀Ad฀L-Fabp฀(or฀LacZ฀control฀virus)฀into฀ / HSCs as deLV-Plin5-YFP฀or฀LV-YFP฀transduced฀L-Fabp tailed฀in฀Methods.฀Western฀blotting฀revealed฀(as฀expected)฀ that฀endogenous฀L-Fabp฀in฀both฀passaged฀WT฀HSCs฀and฀ L-Fabp/฀HSCs฀was฀not฀detectable,฀while฀Ad-L-Fabp฀transduction฀led฀to฀a฀comparable฀increase฀in฀L-Fabp฀expression฀ in both genotypes (Fig. 3A).฀Real-time฀PCR฀analyses฀indicated฀that฀forced฀expression฀of฀L-Fabp฀induced฀expression฀ of฀ Plin5฀ in฀ both฀ passaged฀ WT฀ HSCs฀ and฀ L-Fabp/ HSCs (Fig.฀3B)฀as฀previously฀reported฀(18).฀As฀shown฀in฀Fig.฀3C,฀ upon฀rescue฀of฀L-Fabp฀expression฀in฀L-Fabp/ HSCs, Plin5 transduction decreased expression of -SMA฀ and฀ I(I) procollagen฀mRNAs฀and฀also฀increased฀mRNA฀abundance฀ of฀ SREBP-1฀ and฀ PPAR฀ (Fig.฀ 3C).฀ Forced฀ L-Fabp฀ expression฀ also฀ restored฀ cellular฀ FFA฀ and฀ TG฀ content฀ in฀ Plin5฀ transduced L-Fabp/฀HSCs฀(Fig.฀3D). As฀ noted฀ above฀ (Fig.฀ 1A),฀ Plin5฀ transduction฀ into฀ WT฀ HSCs฀led฀to฀increased฀mRNA฀expression฀of฀Plin2.฀In฀view฀ of prior studies demonstrating a role of Plin2 in LD formation฀in฀HSCs฀(40),฀we฀sought฀to฀clarify฀the฀role฀of฀Plin2฀in฀ the฀coordinated฀interactions฀of฀L-Fabp฀and฀Plin5฀in฀LD฀formation.฀For฀this฀purpose,฀we฀cultured฀L-Fabp/ HSCs with LV-YFP฀or฀LV-Plin5,฀with฀or฀without฀Ad-L-Fabp,฀in฀the฀presence฀of฀Plin2฀shRNA.฀This฀knockdown฀strategy฀resulted฀ in฀more฀than฀80%฀Plin2฀knockdown฀efficiency฀(Fig.฀3E).฀ We confirmed that exogenous Plin5 increased endogenous Plin2 protein expression in L-Fabp/฀HSCs,฀but฀Ad-L-Fabp฀ rescue caused no further upregulation of Plin2 expression (Fig.฀3E).฀Furthermore,฀Oil฀Red฀O฀staining฀revealed฀that฀ shRNA฀inhibition฀of฀Plin2฀had฀no฀effect฀on฀LD฀formation฀ in L-Fabp/฀ HSCs฀ transduced฀ with฀ Plin5฀ and฀ Ad฀ L-Fabp฀ (Fig.฀3E).฀Finally,฀to฀examine฀the฀role฀of฀L-Fabp฀and฀Plin5฀ in฀ coordinating฀ transcriptional฀ activation฀ of฀ lipogenic฀ pathways,฀Ad-L-Fabp฀infected,฀Plin5฀transduced฀L-Fabp/ HSCs฀ were฀ transiently฀ transfected฀ with฀ LXR-Luc,฀ PPRELuc,฀or฀TOPFlash฀plasmids฀as฀above.฀Luciferase฀activity฀assays฀ revealed฀ that฀ Ad-L-Fabp฀ rescued฀ Plin5-mediated฀ transcriptional induction in L-Fabp/ HSCs transfected with LXR-Luc฀or฀PPRE-Luc,฀and,฀conversely,฀reduced฀luciferase฀ activities฀ in฀ L-Fabp/฀ HSCs฀ transfected฀ with฀ TOPFlash฀ (Fig.฀3F).฀These฀results฀collectively฀reinforce฀the฀conclusions฀ that฀ expression฀ of฀ exogenous฀ L-Fabp฀ in฀ L-Fabp/ HSCs฀rescues฀the฀ability฀of฀Plin5฀to฀reverse฀HSC฀activation,฀ elevate฀cellular฀lipid฀content,฀and฀regulate฀prolipogenic฀ pathways.฀ Furthermore,฀ these฀ effects฀ appear฀ to฀ be฀ independent of Plin2. Downloaded from www.jlr.org at Washington Univ Medical Library, on March 3, 2018 expression฀ from฀ the฀ lentiviral฀ transduction฀ experiments฀ reflect฀a฀reasonable฀range฀of฀forced฀overexpression฀in฀relation฀to฀endogenous฀levels฀in฀freshly฀isolated฀HSCs. Having฀ established฀ these฀ baseline฀ parameters,฀ we฀ next฀ evaluated฀the฀role฀of฀Plin5฀in฀regulating฀cellular฀lipid฀content฀ in HSCs. We found that forced expression of exogenous Plin5฀in฀both฀WT฀and฀L-Fabp/ HSCs induced expression of Plin2฀(Fig.฀1E),฀but฀that฀the฀expression฀of฀other฀lipid฀droplet฀ protein฀ genes,฀ including฀ Plin3฀ and฀ hypoxia-induced฀ gene 2 (HIG2), were unaffected. We further examined the coordinated฀ effects฀ of฀ Plin5฀ and฀ L-Fabp฀ in฀ modulating฀ cellular฀lipid฀content฀(Fig.฀1F).฀As฀expected,฀LV-Plin5-YFP฀ transduction฀ increased฀ intracellular฀ FFA฀ and฀ TG฀ content฀ in฀ WT฀ but฀ not฀ L-Fabp/฀ HSCs฀ (Fig.฀ 1F).฀ In฀ addition,฀ we฀ found฀that฀Plin5฀and฀L-Fabp฀together฀promote฀uptake฀of฀ radiolabed฀FA฀and฀TG฀formation฀in฀WT฀HSCs฀(Fig.฀1G).฀ These฀ results฀ collectively฀ suggest฀ that฀ L-Fabp฀ plays฀ a฀ permissive฀ role,฀ along฀ with฀ Plin5,฀ in฀ increasing฀ cellular฀ lipid฀ content฀ and฀ restoring฀ LD฀ formation฀ in฀ WT฀ HSCs.฀ However,฀for฀reasons฀explored฀below,฀and฀despite฀comparable฀ expression฀ of฀ exogenous฀ Plin5฀ following฀ lentiviral฀ transduction, this response fails to occur in L-Fabp/ HSCs. Supplemental Material can be found at: http://www.jlr.org/content/suppl/2018/01/09/jlr.M077487.DC1 .html Expression of other FABPs in L-Fabp/ HSCs rescues the ability of Plin5 to restore LD formation Consistent฀ with฀ the฀ findings฀ above,฀ lentiviral฀ Plin5฀ restored฀ LD฀ formation฀ in฀ WT฀ HSCs฀ with฀ Ad-LacZ฀ or฀ with฀ Ad-L-Fabp,฀ suggesting฀ that฀ endogenous฀ expression฀ of฀ L-Fabp฀is฀sufficient฀for฀these฀effects฀in฀WT฀HSCs.฀However,฀ LDs฀ were฀ only฀ restored฀ in฀ lentivirally฀ transduced฀ Plin5฀ L-Fabp/฀HSCs฀transfected฀with฀Ad-L-Fabp,฀but฀not฀with฀ Ad-LacZ฀ (the฀ lower฀ right฀ panel฀ with฀ arrow฀ of฀ the฀ lower฀ four panels in Fig. 4A).฀We฀previously฀observed฀that฀other฀ FABPs฀were฀expressed฀in฀quiescent฀HSCs,฀albeit฀at฀very฀ low฀levels฀relative฀to฀L-Fabp฀(18).฀To฀address฀the฀question฀ whether฀other฀FABPs฀could฀replace฀L-Fabp฀in฀Plin5-mediated฀ rescue฀ of฀ LD฀ formation,฀ WT฀ and฀ L-Fabp/ HSCs were฀sequentially฀transduced฀with฀LV-Plin5-YFP฀or฀LV-YFP฀ followed฀by฀recombinant฀adenoviral฀intestinal฀Fabp฀(Ad-IFabp),฀adipocyte฀Fabp฀(Ad-A-Fabp),฀or฀Ad-LacZ฀transduction฀(Fig.฀4B).฀We฀found฀that฀either฀Ad-I-Fabp฀or฀Ad-A-Fabp฀ together with Plin5 restored LDs in L-Fabp/ HSCs (panels฀with฀arrows฀in฀Fig.฀4C).฀In฀addition,฀and฀as฀found฀with฀ the฀Ad-L-Fabp฀transduction฀experiments฀reported฀above,฀ neither฀ Ad-I-Fabp฀ or฀ Ad-A-Fabp฀ transduction฀ alone฀ in฀ L-Fabp/฀HSCs฀restored฀LD฀formation฀(Fig.฀4฀C).฀These฀ findings฀ again฀ point฀ to฀ a฀ role฀ for฀ endogenous฀ L-Fabp฀ in฀ LD฀ formation฀ in฀ response฀ to฀ Plin5฀ transduction฀ in฀ WT฀ HSCs. Although we did not systematically examine the entire฀range฀of฀FABPs,฀our฀results฀suggest฀that฀the฀expression฀of฀exogenous฀L-Fabp฀as฀well฀as฀other฀FABPs฀effectively฀ rescues the ability of Plin5 to restore LD formation in L-Fabp/ HSCs. Small molecule inhibitors of L-Fabp impair the ability of Plin5 to restore LD formation in HSCs To฀address฀how฀Plin5฀induces฀LD฀formation฀through฀an฀ L-Fabp-dependent฀mechanism,฀we฀were฀guided฀by฀the฀finding฀that฀exogenous฀Plin5฀significantly฀increased฀mRNA฀ Plin5 regulates lipid droplet formation via L-Fabp 421 Downloaded from www.jlr.org at Washington Univ Medical Library, on March 3, 2018 Fig. 2.฀ L-Fabp฀is฀required฀for฀Plin5-mediated฀reversal฀of฀HSC฀activation฀and฀transcriptional฀induction฀of฀prolipogenic฀signaling฀pathways.฀ Passaged฀WT฀or฀L-Fabp/HSCs฀were฀transduced฀with฀LV-Plin5-YFP฀or฀LV-YFP.฀After฀selection฀with฀puromycin,฀cells฀were฀prepared฀for฀experiments;฀*P฀<฀0.05฀vs.฀HSCs฀transduced฀with฀LV-YFP฀(corresponding฀solid฀columns).฀A:฀Luciferase฀activity฀assays฀of฀WT฀(wt)฀or฀L-Fabp/ HSCs transduced฀with฀LV-Plin5-YFP฀or฀LV-YFP฀and฀followed฀by฀transfection฀with฀the฀luciferase฀reporter฀plasmids฀LXR-Luc,฀PPRE-Luc,฀or฀TOPFlash฀ (n฀=฀6).฀B:฀Real-time฀PCR฀analyses฀of฀downstream฀target฀genes฀of฀the฀signaling฀of฀SREBP-1฀(ACC),฀PPAR฀(FAS),฀and฀Wnt-1฀(LOX-1).฀C:฀ Real-time฀PCR฀analyses฀of฀genes฀relevant฀to฀HSC฀activation฀(n฀=฀3).฀D:฀Real-time฀PCR฀analyses฀of฀genes฀relevant฀to฀lipolysis฀(left฀panel)฀and฀ lipogenesis฀(right฀panel)฀(n฀=฀3). Supplemental Material can be found at: http://www.jlr.org/content/suppl/2018/01/09/jlr.M077487.DC1 .html abundance฀of฀endogenous฀L-Fabp฀in฀WT฀HSCs฀(Fig. 5A), and฀the฀transcriptional฀reporter฀GFP฀in฀our฀GFP-knockin฀ L-Fabp/฀ HSCs฀ (17)฀ (Fig.฀ 5A).฀ Further฀ studies฀ indicated฀ that the expression of exogenous Plin5 had no apparent impact฀on฀mRNA฀expression฀of฀other฀FABPs฀in฀activated฀ WT฀HSCs฀(Fig.฀5B).฀These฀findings฀further฀reinforce฀the฀ concept฀that฀endogenous฀L-Fabp฀induction฀may฀be฀a฀key฀ step฀in฀the฀Plin5-mediated฀restoration฀of฀LDs฀in฀WT฀HSCs.฀ To฀examine฀this฀possibility฀directly,฀we฀turned฀to฀a฀series฀of฀ small฀ molecule฀ inhibitors฀ of฀ L-Fabp฀ function฀ engineered฀ with฀ varying฀ degrees฀ of฀ specificity฀ and฀ potency฀ (supplemental฀Fig.฀S1). We฀ focused฀ on฀ the฀ most฀ selective฀ L-Fabp฀ inhibitor฀ (NSOP00313,฀abbreviated฀as฀NSOP313)฀at฀increasing฀doses฀ in฀ WT฀ HSCs฀ for฀ 24฀ h฀ before฀ and฀ after฀ transduction฀ with฀ LV-Plin5-YFP.฀ We฀ found฀ that฀ treatment฀ with฀ NSOP313฀ 422 Journal of Lipid Research Volume 59, 2018 caused฀a฀dose-dependent฀decrease฀in฀the฀formation฀of฀LDs฀ (Fig.฀ 5C),฀ along฀ with฀ decreased฀ cellular฀ FFA฀ and฀ TG฀ content฀ (Fig.฀ 5E).฀ Moreover,฀ higher฀ doses฀ of฀ NSOP313฀ triggered฀ upregulation฀ of฀ mRNAs฀ associated฀ with฀ HSC฀ activation฀and฀decreased฀expression฀of฀PPAR฀mRNA฀(Fig.฀ 5D). We฀ also฀ asked฀ whether฀ the฀ effects฀ of฀ Ad-L-Fabp฀ in฀ increasing฀endogenous฀Plin5฀expression฀(see฀Fig.฀3B)฀might฀ be฀reversed฀following฀L-Fabp฀inhibitor฀treatment.฀Accordingly,฀ NSOP313฀ at฀ increasing฀ doses฀ was฀ used฀ to฀ treat฀ AdL-Fabp฀ transduced฀ WT฀ HSCs,฀ the฀ data฀ showing฀ a฀ dosedependent฀reduction฀in฀Plin5฀mRNA฀expression฀(Fig.฀5F).฀ These฀results฀together฀confirm฀the฀critical฀role฀of฀L-Fabp฀ in฀regulating฀Plin5฀in฀inhibiting฀HSC฀activation,฀increasing cellular lipid content, and restoring LD formation in HSCs. Downloaded from www.jlr.org at Washington Univ Medical Library, on March 3, 2018 Fig. 3.฀ Introduction฀of฀L-Fabp฀into฀L-Fabp/HSCs฀rescues฀Plin5-mediated฀reversal฀of฀activation฀and฀prolipogenic฀activity.฀L-Fabp/HSCs were฀transduced฀with฀LV-Plin5-YFP฀or฀LV-YFP,฀and฀in฀some฀experiments฀with฀Plin2฀shRNA,฀then฀transduced฀with฀recombinant฀adenoviral฀ Ad-L-Fabp฀or฀the฀control฀recombinant฀adenoviral฀Ad-LacZ.฀Twenty-four฀hours฀later,฀cells฀were฀prepared฀for฀experiments;฀*P฀<฀0.05฀vs.฀HSCs฀ transduced฀with฀Ad-LacZ฀(corresponding฀solid฀columns).฀A:฀Western฀blotting฀analyses฀of฀L-Fabp฀in฀WT฀(wt)฀HSC฀or฀L-Fabp/HSCs transducted฀with฀Ad-L-Fabp฀or฀Ad-LacZ,฀respectively.฀-actin฀was฀an฀internal฀control฀for฀equal฀loading.฀Representative฀views฀of฀three฀independent฀ tests฀are฀presented.฀B:฀Real-time฀PCR฀analyses฀of฀Plin5฀in฀WT฀HSC฀and฀L-Fabp/HSCs฀transducted฀with฀Ad-L-Fabp฀or฀Ad-LacZ฀(n฀=฀3).฀C:฀ Real-time฀PCR฀analyses฀of฀genes฀(n฀=฀3).฀D:฀Analyses฀of฀cellular฀FFA฀and฀TG฀(n฀=฀3).฀E:฀Western฀blotting฀analyses฀of฀the฀inhibitory฀efficiency฀ of฀shRNAPlin2฀and฀Oil฀Red฀O฀staining.฀Representative฀views฀from฀three฀independent฀experiments฀are฀presented.฀F:฀Luciferase฀activity฀assays฀ of฀the฀transduced฀HSCs฀transfected฀with฀the฀luciferase฀reporter฀plasmids฀LXR-Luc,฀PPRE-Luc,฀or฀TOPFlash(n฀=฀6). Supplemental Material can be found at: http://www.jlr.org/content/suppl/2018/01/09/jlr.M077487.DC1 .html Fig. 4.฀ Expression฀of฀other฀FABPs฀in฀L-Fabp/HSCs rescues the ability of Plin5 to restore LD formation. Passaged฀WT฀(wt)฀or฀L-Fabp/HSCs were transduced with฀LV-Plin5-YFP฀or฀LV-YFP,฀respectively.฀After฀selection฀with฀puromycin,฀cells฀were฀transduced฀with฀Ad-LFabp฀or฀Ad-LacZ฀(A฀and฀B)฀or฀were฀transduced฀with฀a฀ recombinant฀adenovirus฀expressing฀I-Fabp฀(Ad-I-Fabp)฀ or฀A-Fabp฀(Ad-A-Fabp)฀(C).฀Representative฀views฀from฀ three฀independent฀experiments฀are฀presented.฀A:฀Oil฀ Red฀O฀staining฀of฀LDs฀in฀HSCs฀transduced฀with฀AdL-Fabp฀ or฀ Ad-LacZ.฀ B:฀ Western฀ blotting฀ analyses฀ of฀ I-Fabp฀or฀A-Fabp.฀C:฀Oil฀Red฀O฀staining฀of฀LDs฀in฀HSCs฀ transduced฀with฀Ad-I-Fabp,฀Ad-A-Fabp,฀or฀Ad-LacZ. DISCUSSION We฀ recently฀ reported฀ that฀ the฀ activation฀ of฀ HSCs฀ was฀ temporally coincident with the loss of LDs, the depletion of Plin5฀(8),฀and฀the฀suppression฀of฀L-Fabp฀expression฀(18)฀ both฀in฀vitro฀and฀in฀vivo.฀The฀current฀findings฀extend฀those฀ observations฀ by฀ demonstrating฀ that฀ exogenous฀ Plin5฀ induces฀endogenous฀L-Fabp฀expression฀in฀WT฀HSCs,฀suggesting฀a฀requirement฀for฀L-Fabp฀in฀the฀rescue฀of฀quiescence฀ mediated by Plin5. We further show that either lifelong absence฀of฀L-Fabp฀(in฀L-Fabp/ HSCs) or pharmacologic inhibition฀ of฀ endogenous฀ L-Fabp฀ in฀ WT฀ HSCs฀ abrogated฀the฀ability฀of฀Plin5฀to฀reverse฀HSC฀activation฀and฀also฀ eliminated LD formation and lipid accumulation. Conversely,฀ adenoviral-mediated฀ expression฀ of฀ exogenous฀ L-Fabp฀ in฀ L-Fabp/ HSCs rescued the ability of Plin5 to restore quiescence. Accordingly, a central conclusion of this฀work฀is฀that฀L-Fabp฀is฀required฀for฀Plin5-mediated฀rescue of cellular quiescence in HSCs, through pathways that intersect with the induction of lipogenic gene programs. That฀being฀said,฀we฀recognize฀that฀the฀range฀of฀functions฀of฀ Plin5 implied from studies in animal models is still controversial฀(9,฀41). Accumulating฀ evidence฀ has฀ shown฀ the฀ importance฀ of฀ cellular lipids in maintaining HSCs in a state of quiescence, although฀the฀precise฀roles฀of฀key฀LD฀proteins฀remain฀elusive.฀ At฀ the฀ molecular฀ level,฀ independent฀ work฀ strongly฀ supports the idea that Plin5 functions as a scaffold for three major฀ key฀ lipolytic฀ players,฀ abhd5,฀ ATGL,฀ and฀ hormonesensitive฀lipase,฀in฀hydrolysis฀of฀TG฀in฀LDs฀(42–45).฀Plin5฀is฀ highly฀expressed฀in฀oxidative฀tissues฀such฀as฀skeletal฀muscle,฀liver,฀and฀heart฀and฀is฀central฀to฀lipid฀homeostasis฀in฀ these฀tissues.฀Studies฀in฀cell฀systems฀have฀ascribed฀several฀ metabolic roles to Plin5 and demonstrated interactions with other proteins that are requisite for these functions (41).฀ Our฀ results฀ support฀ an฀ important฀ role฀ for฀ Plin5฀ in฀ elevating฀cellular฀lipid฀content฀in฀HSCs฀(8);฀namely,฀induction฀of฀lipogenic฀pathways฀through฀transcriptional฀activation฀ of฀ SREBP฀ and฀ PPAR dependent pathways. We recognize that further studies will be required to explore the฀precise฀mechanisms฀of฀increased฀de฀novo฀lipogenesis฀ observed. Studies฀ suggest฀ that฀ lecithin-retinol฀ acyltransferase฀ (LRAT)฀acts฀as฀the฀sole฀retinol฀acyltransferase฀in฀the฀liver฀ and is responsible for the formation of retinyl esters in HSCs฀ (46).฀ LRAT฀ is฀ highly฀ expressed฀ in฀ quiescent฀ HSCs฀ and฀downregulated฀during฀HSC฀activation฀(47).฀Lrat฀ / Plin5 regulates lipid droplet formation via L-Fabp 423 Downloaded from www.jlr.org at Washington Univ Medical Library, on March 3, 2018 AMPK activation is required for L-Fabp-mediated Plin5-dependent restoration of lipogenesis and / HSCs quiescence in L-Fabp We฀previously฀reported฀that฀exogenous฀Plin5฀stimulated฀ AMPK฀activation฀in฀WT฀HSCs,฀which฀in฀turn฀induced฀the฀ expression of endogenous Plin5, a pathway required for restoring฀LD฀formation฀and฀inhibiting฀HSC฀activation฀(8).฀ To฀address฀the฀role฀of฀AMPK฀activation฀in฀the฀coordination฀ of฀L-Fabp฀with฀Plin5,฀we฀first฀explored฀the฀requirement฀of฀ L-Fabp฀ in฀ the฀ activation฀ of฀ AMPK฀ by฀ Plin5฀ in฀ L-Fabp/ HSCs. As shown in Fig. 6A, forced expression of exogenous Plin5 increased the abundance of phosphorylated AMPK in฀ both฀ WT฀ and฀ L-Fabp/ HSCs. By contrast, forced expression฀ of฀ L-Fabp฀ alone฀ could฀ not฀ activate฀ AMPK฀ in L-Fabp/฀HSCs฀(Fig.฀6A).฀These฀findings฀suggest฀that฀ L-Fabp฀is฀not฀required฀for฀Plin5฀to฀activate฀AMPK฀in฀HSCs.฀ They฀also฀suggest฀that฀the฀introduction฀of฀Ad-L-Fabp฀alone฀ into L-Fabp/฀HSCs฀is฀not฀sufficient฀for฀AMPK฀activation.฀ We further studied L-Fabp/฀ HSCs฀ transduced฀ with฀ LVPlin5-YFP฀or฀LV-YFP฀and฀subsequent฀Ad-L-Fabp฀rescue,฀followed฀ by฀ treatment฀ with฀ the฀ selective฀ AMPK฀ inhibitor฀ Compound C at 20 M฀for฀24฀h.฀AMPK฀inhibition฀significantly฀attenuated฀the฀effects฀of฀Plin5฀and฀L-Fabp฀on฀regulating฀ expression฀ of฀ genes฀ relevant฀ to฀ HSC฀ activation,฀ lipogenesis, and lipolysis in L-Fabp/฀HSCs฀(Fig.฀6B)฀and฀ also฀ attenuated฀ LD฀ formation฀ (Fig.฀ 6C).฀ Additionally,฀ AMPK฀inhibition฀dose-dependently฀reversed฀Plin5-dependent฀ increases฀in฀endogenous฀L-Fabp฀mRNA฀in฀WT฀HSCs฀(Fig.฀ 6D).฀These฀findings฀suggest฀that฀Plin5-induced฀expression฀ of฀endogenous฀L-Fabp฀in฀HSCs฀is฀likely฀AMPK-dependent.฀ AMPK฀ inhibition฀ also฀ reversed฀ the฀ transcriptional฀ programs฀ of฀ lipogenic฀ induction฀ of฀ LXR฀ and฀ PPAR and of Wnt suppression as indicated by the luciferase assays using reporter฀ constructs฀ outlined฀ above฀ (Fig.฀ 6E).฀ Taken฀ together,฀these฀results฀strongly฀suggest฀that฀AMPK฀activation฀ is฀ required฀ for฀ the฀ coordination฀ of฀ L-Fabp฀ with฀ Plin5฀ in฀ mediating rescue of quiescence in L-Fabp/ HSCs. Supplemental Material can be found at: http://www.jlr.org/content/suppl/2018/01/09/jlr.M077487.DC1 .html mice฀showed฀a฀striking฀total฀absence฀of฀large฀lipid-containing฀ droplets that normally store hepatic retinoid within HSCs (48).฀ However,฀ the฀ absence฀ of฀ retinyl฀ ester-containing฀ LDs฀does฀not฀promote฀either฀spontaneous฀HSC฀activation,฀or฀worsen฀bile฀duct฀ligation-induced฀or฀carbon฀tetrachloride-induced฀ liver฀ fibrosis฀ (47).฀ Our฀ earlier฀ studies฀ showed that exogenous Plin5 had no impact on the expression฀of฀endogenous฀LRAT฀in฀WT฀HSCs฀(8).฀We฀considered฀the฀possibility฀that฀Plin5฀and฀L-Fabp฀transduction฀ might modify retinol content of HSCs but our findings demonstrated฀only฀very฀low฀levels฀of฀retinol฀with฀no฀differences by genotype and retinyl esters were undetectable฀(supplemental฀Table฀S1).฀Because฀these฀studies฀were฀ 424 Journal of Lipid Research Volume 59, 2018 undertaken฀ in฀ passaged฀ HSCs,฀ those฀ findings฀ would฀ be฀ predicted. Our฀findings฀also฀highlight฀a฀potential฀functional฀redundancy฀ in฀ the฀ FABP฀ requirement฀ for฀ Plin5-dependent฀ rescue฀of฀quiescence฀and฀LD฀formation.฀We฀have฀previously฀ shown฀ that฀ among฀ all฀ FABPs,฀ L-Fabp฀ is฀ by฀ far฀ the฀ most฀ abundantly expressed member in quiescent HSCs (18). Other฀ FABP฀ members฀ in฀ quiescent฀ HSCs฀ are฀ either฀ expressed฀at฀a฀very฀low฀level฀or฀their฀expression฀is฀unchanged฀ during฀HSC฀activation฀(18).฀We฀observed฀that฀exogenous฀ Plin5฀in฀WT฀HSCs฀induced฀endogenous฀L-Fabp฀expression,฀ but฀not฀other฀FABP฀members฀(Fig.฀5B),฀suggesting฀specificity฀in฀Plin5-dependent฀effects฀via฀L-Fabp฀in฀promoting฀LD฀ Downloaded from www.jlr.org at Washington Univ Medical Library, on March 3, 2018 Fig. 5.฀ Small฀molecule฀inhibitors฀of฀L-Fabp฀impede฀the฀ability฀of฀Plin5฀to฀restore฀LD฀formation฀in฀HSCs.฀A:฀Passaged฀WT฀(wt)฀HSCs฀or฀ L-Fabp/HSCs฀were฀transduced฀with฀LV-Plin5-YFP฀or฀LV-YFP.฀Total฀RNA฀was฀prepared฀for฀real-time฀PCR฀analyses;฀*P฀<฀0.05฀vs.฀HSCs฀transduced฀with฀LV-YFP฀(corresponding฀solid฀columns)฀(n฀=฀3).฀B:฀Real-time฀PCR฀analyses฀of฀WT฀HSCs฀transduced฀with฀LV-Plin5-YFP฀or฀LV-YFP.฀ All฀data฀were฀normalized฀to฀the฀expression฀of฀L-Fabp฀in฀HSCs฀with฀LV-YFP;฀*P฀<฀0.05฀vs.฀HSCs฀transduced฀with฀LV-YFP฀(corresponding฀solid฀ columns)฀(n฀=฀3).฀C–E:฀WT฀HSCs฀were฀transduced฀with฀LV-Plin5-YFP฀followed฀by฀treatment฀with฀the฀selective฀L-Fabp฀inhibitor฀NSOP00313฀ (referred฀to฀as฀NSOP313฀in฀text)฀at฀the฀indicated฀doses฀for฀24฀h.฀C:฀Oil฀Red฀O฀staining฀of฀cellular฀LDs฀(Representative฀views฀from฀three฀independent฀experiments฀are฀presented)฀and฀the฀numbers฀of฀LDs฀per฀cell฀(mean฀from฀10฀cells).฀D:฀Real-time฀PCR฀analyses;฀*P฀<฀0.05฀vs.฀HSCs฀ without฀NSOP313฀treatment฀(n฀=฀3).฀E:฀Analyses฀of฀cellular฀FFA฀and฀TG฀content.฀*P฀<฀0.05฀vs.฀HSCs฀without฀NSOP313฀treatment฀(n฀=฀3).฀ F:฀Real-time฀PCR฀analyses฀of฀endogenous฀Plin5฀mRNA฀in฀Ad-L-Fabp฀transduced฀WT฀HSCs฀with฀treatment฀of฀NSOP313฀at฀the฀indicated฀doses.฀ *P฀<฀0.05฀vs.฀HSCs฀without฀NSOP313฀treatment. Supplemental Material can be found at: http://www.jlr.org/content/suppl/2018/01/09/jlr.M077487.DC1 .html formation฀ and฀ restoration฀ of฀ quiescence.฀ On฀ the฀ other฀ hand,฀our฀findings฀also฀suggest฀that฀L-Fabp฀is฀not฀functionally฀unique฀and฀its฀role฀could฀be฀replicated฀by฀other฀FABPs,฀ including฀ I-Fabp฀ or฀ A-Fabp.฀ Although฀ we฀ did฀ not฀ undertake฀a฀comprehensive฀evaluation฀of฀the฀entire฀FABP฀family,฀ it is tempting to speculate that under circumstances where other฀FABPs฀might฀be฀induced฀(not฀examined฀here),฀there฀ may฀yet฀be฀L-Fabp-independent฀pathways฀of฀restoring฀quiescence. Along these lines, whereas we found that the most฀specific฀and฀potent฀small฀molecule฀L-Fabp฀inhibitor฀ (NSOP313)฀effectively฀replicated฀the฀phenotypes฀observed฀ with L-Fabp฀ deletion฀ (Fig.฀ 5),฀ we฀ also฀ found฀ that฀ other฀ L-Fabp฀inhibitors฀(supplemental฀Fig.฀S1)฀showed฀similar฀effects฀but฀were฀either฀less฀selective฀or฀more฀toxic. Our฀ earlier฀ work฀ suggested฀ that฀ germline฀ L-Fabp deletion฀ functions฀ to฀ attenuate฀ high-fat,฀ fibrogenic,฀ dietinduced hepatic steatosis and fibrosis, raising the possibility that฀ inhibition฀ of฀ L-Fabp฀ might฀ be฀ a฀ feasible฀ strategy฀ to฀ mitigate the effects of hepatic steatosis and necroinflammatory฀ disease฀ progression.฀ However,฀ it฀ bears฀ emphasis฀ that฀L-Fabp฀is฀abundantly฀expressed฀in฀hepatocytes฀as฀well฀ as in HSCs and it is possible that its roles and functions are exerted฀in฀a฀distinct฀cell-฀and฀metabolic-state-specific฀context.฀ The฀ current฀ findings฀ support฀ the฀ concept฀ of฀ this฀ cell-type-specific฀ function฀ by฀ demonstrating฀ that฀ genetic฀ deletion฀or฀pharmacologic฀inhibition฀of฀L-Fabp฀essentially฀ eliminates the ability of Plin5 to restore LD formation in either฀WT฀or฀L-Fabp/฀HSCs.฀The฀findings฀from฀these฀in฀ vitro฀studies฀additionally฀suggest฀that฀pharmacologic฀inhibition฀ of฀ L-Fabp,฀ by฀ mitigating฀ LD฀ formation฀ and฀ induction of a lipogenic program, might simultaneously induce HSC฀activation. An฀ overarching฀ question฀ in฀ our฀ approach฀ relates฀ to฀ whether฀ the฀ expression฀ levels฀ of฀ exogenous฀ Plin5฀ are฀ reflective฀of฀those฀encountered฀physiologically.฀We฀demonstrated that Plin5 protein is indeed detectable in freshly isolated฀HSCs,฀albeit฀at฀levels฀somewhat฀lower฀than฀those฀ seen฀ with฀ forced฀ lentiviral฀ expression฀ in฀ passaged฀ HSCs฀ (Fig.฀1C,฀D).฀In฀addition,฀we฀previously฀reported฀that฀transduction฀ of฀ WT฀ HSCs฀ with฀ LV-Plin5-YFP฀ or฀ the฀ empty฀ Plin5 regulates lipid droplet formation via L-Fabp 425 Downloaded from www.jlr.org at Washington Univ Medical Library, on March 3, 2018 Fig. 6.฀ AMPK฀activation฀is฀required฀for฀L-Fabp-dependent฀Plin5-mediated฀restoration฀of฀lipogenesis฀and฀quiescence฀in฀L-Fabp/HSCs. A: Western฀blotting฀analyses฀of฀phosphorylated฀AMPK฀(p-AMPK)฀in฀WT฀HSCs฀or฀in฀L-Fabp/HSCs฀transduced฀with฀recombinant฀virus฀as฀indicated.฀Total฀AMPK฀abundance฀was฀used฀as฀an฀internal฀control฀for฀equal฀loading.฀Representative฀views฀from฀3฀independent฀experiments฀are฀ presented;฀(B,฀C,฀and฀E).฀L-Fabp/HSCs฀were฀sequentially฀transduced฀with฀LV-Plin5-YFP฀or฀LV-YFP,฀and฀then฀with฀Ad-L-Fabp,฀followed฀by฀ treatment฀with฀the฀AMPK฀inhibitor฀Compound฀C฀(CompdC),฀at฀the฀indicated฀concentrations.฀B:฀Real-time฀PCR฀analyses฀(n฀=฀3);฀*P < 0.05 vs.฀HSCs฀transduced฀with฀LV-YFP฀(corresponding฀solid฀columns).฀C:฀Oil฀Red฀O฀staining฀of฀cellular฀LDs.฀Representative฀views฀from฀three฀independent฀experiments฀are฀presented.฀D:฀Real-time฀PCR฀analyses฀of฀endogenous฀L-Fabp฀in฀WT฀HSC฀transduced฀with฀LV-Plin5฀(n฀=฀3);฀*P < 0.05฀vs.฀HSCs฀with฀no฀CompdC.฀E:฀Luciferase฀activity฀assays฀of฀the฀transduced฀HSCs฀transfected฀with฀the฀luciferase฀reporter฀plasmids฀LXRLuc,฀PPRE-Luc,฀or฀TOPFlash฀(n฀=฀6). Supplemental Material can be found at: http://www.jlr.org/content/suppl/2018/01/09/jlr.M077487.DC1 .html 426 Journal of Lipid Research Volume 59, 2018 Fig. 7.฀ Summary฀overview฀of฀the฀proposed฀pathways฀explored฀in฀ this฀ report.฀ Stellate฀ cell฀ (HSC)฀ activation฀ is฀ temporally฀ accompanied by loss of lipid droplets (LDs) along with decreased expression of฀both฀Plin5฀and฀L-Fabp.฀Lentiviral฀(LV)฀transduction฀of฀Plin5฀into฀ WT฀HSCs฀restores฀exogenous฀(exo)฀Plin5,฀leading฀to฀AMPK฀activation฀and฀increased฀endogenous฀L-Fabp฀expression฀which฀in฀combination฀further฀increases฀endogenous฀(endo)฀Plin5฀expression.฀The฀ coordination฀of฀Plin5฀and฀L-Fabp฀promotes฀lipid฀(fatty฀acid)฀uptake฀ and฀increases฀lipogenesis,฀leading฀to฀the฀elevation฀of฀cellular฀lipid฀ content.฀Upregulation฀of฀endogenous฀L-Fabp฀is฀a฀requisite฀step฀in฀ this pathway, the net effects leading to accumulation of LDs and a return฀to฀quiescence฀with฀HSC฀deactivation. L-Fabp฀→฀AMPK฀activation).฀This฀suggestion฀is฀supported฀ by experiments showing that, although forced expression of฀Plin5฀induced฀expression฀of฀endogenous฀L-Fabp฀in฀WT฀ HSCs฀(Fig.฀5A),฀inhibition฀of฀Plin5฀signaling฀by฀AMPK฀inhibition฀dose-dependently฀reduced฀L-Fabp฀expression.฀These฀ findings฀ beg฀ the฀ question฀ of฀ whether฀ AMPK฀ activation฀ modulates฀L-Fabp฀expression,฀which฀is฀a฀subject฀of฀future฀ investigation. Forced฀expression฀of฀L-Fabp฀elevated฀the฀mRNA฀level฀of฀ endogenous฀Plin5฀by฀2-฀to฀3-fold฀in฀both฀WT฀HSCs฀and฀in฀ L-Fabp/฀HSCs฀(Fig.฀3B),฀consistent฀with฀our฀prior฀observations฀ (18).฀ However,฀ forced฀ expression฀ of฀ exogenous฀ L-Fabp฀itself฀did฀not฀inhibit฀expression฀of฀HSC฀activationrelevant฀ genes฀ (Fig.฀ 3C),฀ augment฀ cellular฀ lipid฀ content฀ (Fig.฀3D),฀nor฀rescue฀LD฀formation฀in฀L-Fabp/฀HSCs฀(Fig.฀ 4A).฀ These฀ observations฀ imply฀ that฀ despite฀ the฀ ability฀ of฀ forced฀ L-Fabp฀ expression฀ to฀ increase฀ endogenous฀ Plin5฀ mRNA฀ by฀ 2-฀ to฀ 3-fold,฀ the฀ levels฀ of฀ Plin5฀ protein฀ remain฀ Downloaded from www.jlr.org at Washington Univ Medical Library, on March 3, 2018 control฀virus฀LV-YFP฀shows฀significant฀differences฀in฀inhibiting฀HSC฀activation฀(8).฀Introduction฀of฀LV-Plin5-YFP,฀but฀ not฀ LV-YFP,฀ significantly฀ reduces฀ cell฀ growth฀ and฀ expression฀of฀activation-relevant฀genes฀and฀restores฀LD฀formation฀ in HSCs. Additionally, we showed that the major functional domain of Plin5 for the LD formation in HSCs resided in a domain spanning amino acids 1–188 (aa1–188) of Plin5 (8)฀ and฀ that฀ overexpression฀ of฀ a฀ subdomain฀ of฀ Plin5฀ (aa189–463)฀had฀no฀function฀in฀LD฀formation.฀These฀prior฀ results,฀coupled฀with฀the฀new฀findings฀shown฀in฀Fig.฀1C฀and฀ D, greatly diminish the possibility of a neomorphic phenotype฀ of฀ Plin5฀ transduction฀ or฀ of฀ an฀ artifact฀ derived฀ from฀ nonspecific฀recombinant฀lentiviral฀transduction. It was reported that introduction of Plin2 promotes downregulation฀ of฀ HSC฀ activation฀ and฀ is฀ functionally฀ linked฀to฀the฀expression฀of฀fibrogenic฀genes฀(40).฀We฀observed฀that฀forced฀expression฀of฀Plin5฀was฀coupled฀with฀the฀ expression฀of฀endogenous฀Plin2฀in฀HSCs฀(Fig.฀1E).฀Inhibition฀ of฀ the฀ Plin2฀ expression฀ by฀ Plin2฀ shRNA฀ showed฀ no฀ impact฀on฀the฀Plin5฀function฀in฀restoring฀LD฀in฀HSCs฀(Fig.฀ 3E).฀ Our฀ results฀ and฀ others฀ suggest฀ that฀ Plin5,฀ Plin2,฀ or฀ even฀ other฀ lipid฀ droplet฀ proteins,฀ could฀ independently฀ function in HSCs. Plin5฀was฀reported฀to฀be฀phosphorylated฀by฀protein฀kinase฀A฀(PKA)฀(49–51),฀and฀the฀serine฀at฀155฀in฀Plin5฀has฀ been proposed as the PKA phosphorylation site (50). Phosphorylation of Plin5 by PKA stimulated the interaction of Plin5฀and฀ATGL฀(51),฀which฀might฀play฀a฀critical฀role฀in฀ Plin5-regulated฀lipolysis฀(50).฀Plin5฀has฀been฀proposed฀to฀ be฀ an฀ important฀ molecular฀ link฀ that฀ couples฀ the฀ coordinated฀catecholamine฀activation฀of฀the฀PKA฀pathway฀and฀of฀ lipid droplet lipolysis with transcriptional regulation to promote฀efficient฀fatty฀acid฀catabolism฀(49).฀The฀possibility฀ that Plin5 modulates lipid hydrolysis in HSCs and the role, if฀any,฀for฀L-Fabp฀will฀require฀further฀study. AMPK฀ is฀ a฀ well-conserved฀ serine/threonine-protein฀ kinase, which acts as a cellular energy and nutrient sensor and plays a crucial role in regulation of metabolic pathways and cellular energy homeostasis (52). Phosphorylation of AMPK฀at฀Thr-172฀stimulates฀its฀kinase฀activity฀(53).฀AMPK฀ acts฀as฀an฀anti-lipolytic฀factor฀by฀phosphorylating฀and฀inactivating฀ hormone-sensitive฀ lipase฀ (54)฀ and฀ blocking฀ its฀ translocation฀to฀LDs฀(55).฀We฀previously฀showed฀that฀the฀ activation฀of฀AMPK฀increased฀cellular฀TG฀in฀HSCs฀and฀inhibited฀ activation฀ (29).฀ We฀ also฀ showed฀ that฀ PKA฀ and฀ AMPK฀played฀opposite฀roles฀in฀regulating฀HSC฀activation฀ (56).฀In฀the฀current฀study,฀we฀observed฀that฀activation฀of฀ AMPK฀played฀a฀key฀role฀in฀the฀coordination฀of฀L-Fabp฀with฀ Plin5฀in฀inhibiting฀HSC฀activation฀and฀restoring฀LD฀formation.฀ Our฀ findings฀ lead฀ us฀ to฀ hypothesize฀ that฀ a฀ positivefeedback฀loop฀exists฀between฀Plin5฀and฀AMPK฀activation฀in฀ HSCs฀in฀which฀expression฀of฀Plin5฀results฀in฀activation฀of฀ AMPK฀and฀induction฀of฀pro-lipogenic฀genes฀(Fig. 7). Our฀results฀also฀suggest฀that฀forced฀expression฀of฀Plin5฀ stimulated฀ AMPK฀ activation฀ in฀ WT฀ and฀ L-Fabp/ HSCs. However,฀overexpression฀of฀exogenous฀L-Fabp฀alone฀could฀ not฀activate฀AMPK.฀These฀results฀suggest฀that฀AMPK฀might฀ be฀a฀downstream฀target฀of฀Plin5,฀and฀upstream฀of฀L-Fabp฀ (i.e., Plin5 →฀ AMPK฀ activation฀ →฀ L-Fabp;฀ not Plin5 → Supplemental Material can be found at: http://www.jlr.org/content/suppl/2018/01/09/jlr.M077487.DC1 .html below the threshold necessary for complete rescue of quiescence฀as฀previously฀observed฀in฀WT฀HSCs฀(18).฀In฀addition,฀as฀noted฀above,฀the฀introduction฀of฀Ad-L-Fabp฀alone฀ into L-Fabp/฀HSCs฀failed฀to฀activate฀AMPK.฀Based฀on฀the฀ collective฀observations฀presented฀above,฀a฀simplified฀model฀ is฀ proposed฀ to฀ address฀ the฀ role฀ of฀ AMPK฀ and฀ L-Fabp฀ in฀ mediating/coordinating฀ the฀ effects฀ of฀ Plin5฀ in฀ restoring฀ HSC฀quiescence฀(Fig.฀7).฀It฀bears฀emphasis฀that฀this฀model฀ does฀ not฀ exclude฀ other฀ pathways฀ by฀ which฀ L-Fabp฀ functions in coordinating with Plin5 the inhibition of HSC activation.฀ In฀ summary,฀ our฀ results฀ demonstrate฀ that฀ L-Fabp฀ plays a critical role in coordinating with Plin5 in modulating expression of lipogenic genes, restoring LD formation, elevating฀lipid฀content,฀and฀inhibiting฀HSC฀activation. 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