CIDEC/FSP27 Is Regulated by Peroxisome Proliferator-Activated Receptor Alpha and Plays a Critical Role in

Fasting- and Diet-Induced HepatosteatosisC�edric Langhi and �Angel Bald�an

The cell death-inducing DNA fragmentation factor alpha-like effector c (CIDEC; alsoknown in rodents as FSP27 or fat-specific protein 27) is a lipid droplet-associated pro-tein that promotes intracellular triglyceride (TAG) storage. CIDEC/Fsp27 is highlyexpressed in adipose tissue, but undetectable in normal liver. However, its hepatic expres-sion rises during fasting or under genetic or diet-induced hepatosteatosis in both miceand patients. Herein, we demonstrate that CIDEC/Fsp27 is a direct transcriptional targetof the nuclear receptor PPARa (peroxisome proliferator-activated receptor alpha) in bothmouse and human hepatocytes, and that preventing Fsp27 induction accelerates PPARa-stimulated fatty acid oxidation. We show that adenoviral-mediated silencing of hepaticFsp27 abolishes fasting-induced liver steatosis in the absence of changes in plasma lipids.Finally, we report that anti-Fsp27 short hairpin RNA and PPARa agonists synergize toameliorate hepatosteatosis in mice fed a high fat diet. Conclusions: Together, our datahighlight the physiological importance of CIDEC/Fsp27 in TAG homeostasis under bothphysiological and pathological liver steatosis. Our results also suggest that patients takingfibrates likely have elevated levels of hepatic CIDEC, which may limit the efficient mobi-lization and catabolism of hepatic TAGs. (HEPATOLOGY 2015;61:1227-1238)

Nonalcoholic fatty liver disease (NAFLD) is themost common form of chronic liver diseasein Western societies and its prevalence is rap-

idly increasing, paralleling the epidemic of obesity andtype 2 diabetes.1,2 Despite its rampant incidence, thetherapeutic toolbox to manage NAFLD patients isnotoriously scarce.1,2 Hepatosteatosis is classicallydefined as the nonphysiological accumulation of tri-glycerides (TAG) within lipid droplets (LDs) in thecytoplasm of hepatocytes. Whether such accumulationis pathological per se, or an adaptive response to ameli-orate lipotoxicity, is still debated.

LDs were once thought to function as mere lipidstorage depots, but more-recent studies have put LDsat the forefront of lipid metabolism, transport, and sig-naling. Several proteins at the surface of LD orchestrate

these functions and determine the metabolic fate of thelipids contained within the organelle. Among them, theCIDE (cell death-inducing DNA fragmentation factoralpha-like effector) family of LD-associated proteinshas been extensively studied. The three members of theCIDE family have distinct tissue distribution: CIDEAis abundant in brown adipose tissue; CIDEB in liver;and CIDEC/FSP27 (fat-specific protein 27 in rodents)in both white and brown adipose tissue.3 Interestingly,CIDEC/FSP27 is detected in fatty livers, but not innormal livers.3 Overexpression of Fsp27 promoted boththe accumulation and an increase in size of LDs inmultiple cell types.4-6 Conversely, knockdown of Fsp27decreased LD size and increased LD number in adipo-cytes.7 Two independent groups reported that Fsp27–/–

mice show a lean phenotype paralleled by smaller LD

Abbreviations: Acox, acyl-CoA oxidase; Act-D, actinomycin D; ApoA5, apolipoprotein A5; CIDE, cell death-inducing DNA fragmentation factor-alpha-likeeffector; CHX, cycloheximide; CoA, coenzyme A; Cpt1a, carnitine palmitoyltransferase 1a; CREB, cAMP response element-binding protein; FAO, fatty acid oxida-tion; FFA, free fatty acid; FSP27, fat-specific protein 27; HFD, high-fat diet; IR, insulin resistance; kb, kilobase; LD, lipid droplet; mRNA, messenger RNA;NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PKA, protein kinase A; Plin, perilipin; PPAR, peroxisome proliferator-activated recep-tor; PPRE, PPAR-responsive element; SCR, scrambled; sh, small hairpin; TAG, triacylglycerides; WT, wild type.

From the Edward A. Doisy Department of Biochemistry & Molecular Biology, Center for Cardiovascular Research, Saint Louis University, Saint Louis, MO.Received July 3, 2014; accepted November 10, 2014.This study was supported, in part, by the National Institutes of Health (grant no.: HL107794; to �A.B.) and the American Heart Association (grant no:

GRNT20460189; to �A.B.).Additional Supporting Information may be found at http://onlinelibrary.wiley.com/doi/10.1002/hep.27607/suppinfo.

1227

in white fat, enhanced insulin sensitivity, and resistanceto diet-induced obesity.8,9 Importantly, a lipodystrophicpatient has been identified that carries a homozygousnonsense mutation in CIDEC.10

Peroxisome proliferator-activated receptors (PPARs) aremembers of the family of ligand-activated nuclear receptortranscription factors. PPARs function to maintain lipidhomeostasis and energy balance, by promoting the tran-scription of specific targets.11 Both PPARa and PPARdare expressed at high levels in the hepatocyte and play amajor role in regulating fatty acid oxidation (FAO).11 Incontrast, PPARc is not expressed in normal hepatocytes,but its abundance is markedly elevated in fatty livers,where it promotes a lipogenic phenotype in both rodentsand humans.11 Evidence shows that the expression ofFsp27 is induced in the livers of ob/ob, db/db, and ddY-Hmice, as well as in normal mice fed a high-fat diet (HFD),through a mechanism dependent on PPARc.4,12-14

Finally, Vila-Brau et al. reported on the induction ofhepatic Fsp27 expression during early fasting through thecanonical protein kinase A/cAMP response element-binding protein (PKA/CREB)-signaling pathway.15 Col-lectively, these studies suggest a role for CIDEC/Fsp27 dur-ing the metabolic adaptations of the liver to dietary insultand fasting.

In this study, we tested the functional relevance ofhepatic CIDEC/Fsp27 during fasting and in responseto an HFD. We also identified CIDEC/Fsp27 as adirect PPARa transcriptional target in the normal liver.Finally, we show that silencing Fsp27 synergized withPPARa agonists to decrease hepatosteatosis in mice,without altering plasma lipid levels. Our data highlightthe critical role of CIDEC/Fsp27 during both physio-logical and pathological hepatic TAG storage, and sug-gest that CIDEC may be a pharmacological target tomanage liver TAG contents in patients.

Materials and Methods

Mice. Male, 10-12 week-old wild-type andPPARa2/2o (on a C57BL/6 background) were pur-chased from Jackson Laboratories and maintained in a12h/12h light/dark cycle with unlimited access to foodand water. To silence Fsp27 in the liver, mice were tail-vein injected with 23109 pfu adenovirus encoding

scrambled (Ad-shSCR) or anti-Fsp27 (Ad-shFsp27)short hairpin RNA (shRNA) (sequences as described inMatsusue et al.13). Mice were sacrificed 10 days afteradenovirus infusion. Where indicated, animals werefasted 15 hours and sacrificed on the morning at thesame time as fed controls. Where indicated, mice werefed a high fat diet (Test Diets, 58Y1) for 8 weeks priorto adenoviral transduction. For Wy14643 treatment,mice were gavaged with either vehicle (1% carboxy-methyl cellulose in saline) or 10 mg/kg Wy14643(Sigma) for 7 days. For all in vivo experiments, n 5 5/group. All studies were approved by the IACUC at SLU.

Cell Lines and Primary Hepatocytes. Huh7 andHEK293 cells were maintained in DMEM supplementedwith 10% FBS. For cycloheximide (CHX) experiments,cells were pretreated with 10 mg/mL CHX for 1 hour, andthen incubated 16 hours with CHX and DMSO or 1mmol/L GW7647 (Sigma). For actinomycin D (Act-D)experiments, cells were treated 24 hours with eitherDMSO or 1 mmol/L GW7647, and then chased up to 24hours with 5 mg/mL Act-D. Mouse primary hepatocyteswere isolated from chow-fed mice, using Perfusion andDigest buffers (Invitrogen). Cells were seeded in 12- or 6-well BioCoat Collagen I plates (BD), and incubated at37�C and 5% CO2 in William’s E media 1 HepatocyteSupplements (Invitrogen). For adenovirus-mediated silenc-ing, cells were transduced with Ad-shSCR or Ad-shFsp27vectors at moi 5 3. Where indicated, cells were culturedin media supplemented with GW7647 for 24 hours. Foroleate challenge, cells were pre-treated with 1 mmol/LGW7647 for 24 hours, before addition of 300 mmol/Loleate (conjugated to BSA at a 3:1 ratio) overnight.

Statistical Analysis. Data are shown as mean 6

SEM. Differences between groups were analyzed byone-way or two-way ANOVA followed by posthocBonferroni’s test, as appropriate.

Additional experimental procedures are provided asSupporting Information.

Results

Adenovirus-Mediated Silencing of Fsp27Expression Prevents Fasting-Induced HepaticSteatosis. Hepatic Fsp27 is highly induced duringfasting,15 paralleling the accumulation of TAG.16

Address reprint requests to: �Angel Bald�an, Ph.D., Edward A. Doisy Department of Biochemistry & Molecular Biology, Doisy Research Center, Room 427, SaintLouis University Saint Louis, MO 63104. E-mail: abaldan1@slu.edu; fax: 1-314-977-9206.

Copyright VC 2014 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.27607Potential conflict of interest: Nothing to report.

1228 LANGHI AND BALD�AN HEPATOLOGY, April 2015

To better understand the relevance of Fsp27 on themetabolic adaptation of the liver to fasting, we infusedmice with control and anti-Fsp27 adenoviral vectors.Ten days later, animals were fasted 15 hours (over-night) or allowed access to food. No changes werenoted in body weight between short hairpin scrambled(shSCR)- and shFsp27-injected mice, but, as expected,fasted animals showed �15% weight loss. SupportingFig. 1 shows that fasting induced hepatic Fsp27 expres-sion �70-fold in control mice, consistent with previ-ous reports,15 whereas mice infused with the shFsp27vector had significantly reduced Fsp27 levels. We didnot detect green fluorescent protein or shRNA effectin gonadal fat (where Fsp27 is normally expressed;data not shown), suggesting that the adenoviral trans-duction was restricted to the liver. Levels of hepaticCideb and other LD-related transcripts were notaltered by shFsp27 (Supporting Fig. 1A). As expected,fasted control mice accumulated TAG and free fattyacid (FFA) in the liver (Fig. 1A–C). In contrast,hepatic steatosis was dramatically reduced in theshFsp27-fasted group (Fig. 1A-C). No changes inhepatic cholesterol contents were noted among groups(Fig. 1C). A detailed lipidomics analysis of liversshowed that silencing Fsp27 expression did not prefer-entially change specific TAG or FFA species (Fig. 1Dand Supporting Fig. 1B). Given that hepatic lipidcatabolism during fasting triggers ketogenesis, we alsomeasured plasma b-hydroxybutyrate levels: Mice trans-duced with shFsp27 showed a �50% reduction in thismetabolite, compared to control mice (Fig. 1E). Incontrast, shFsp27 treatment did not alter other plasmametabolites (Fig. 1E and Supporting Fig. 1C).

Last, we measured the relative abundance of selectedtranscripts involved in LD metabolism, FAO, and denovo lipogenesis in those same livers. Data from con-trol mice in Supporting Fig. 1A are consistent withprevious reports on the effects of nutritional status onhepatic gene expression.17-19 Hence, fasting increasedFAO enzymes and specific perilipins (Plin) anddecreased lipogenic enzymes. Intriguingly, PPARa tar-gets (Plin2, Plin5, carnitine palmitoyltransferase 1a[Cpt1a], and acyl-coenzyme A oxidase [Acox]) wereslightly decreased in shFsp27 livers, although thefasting-mediated induction was similar compared toshSCR livers (Supporting Fig. 1A). Silencing Fsp27did not alter the expression of lipogenic genes (Sup-porting Fig. 1A). The rapid induction of Fsp27 duringearly fasting15 likely mediates hepatic accumulation oflipids, which will be used as substrates for FAO andketogenesis in late fasting. We speculate that theinability to store TAG into LDs in the shFsp27 hepa-

tocytes from the early stages of fasting results in thegradual, steady oxidation of FFA, depletion of intracel-lular lipids, and lack of ketogenic burst in late fasting.The remarkable decrease in both liver TAG andplasma ketone bodies noted in fasted mice after Fsp27silencing is consistent with this proposal.

PPARa Regulates CIDEC/Fsp27 Expression inHuman and Mouse Hepatocytes. Previous studies inobese mice suggested that PPARc mediates inductionof Fsp27 in steatotic livers.4,12-14 However, treatmentof lean mice with the PPARc agonist, rosiglitazone,failed to induce hepatic Fsp27 expression,13 likelybecause the levels of PPARc in nonsteatotic livers arevery low. In contrast, both PPARa and PPARd areabundant in healthy livers and function as major regu-lators of lipid homeostasis during the fasting and post-prandial periods.11 Previous reports using HepG2 cellsdescribed a PPAR-responsive element (PPRE) in thepromoter of CIDEC/Fsp27 that confers responsivenessto PPARc, but not to PPARa or PPARd.13 Here, weused primary hepatocytes and specific agonists to testwhether Fsp27 is responsive to the three PPARs innormal cells. Data in Fig. 2 show that Fsp27 levels,similar to Cpt1a, were increased after incubation withthe PPARa agonist, GW7647, but not with thePPARd or PPARc agonists. Other transcripts wereinduced by the other agonists (Fig. 2), thus confirmingthe biological activity of the different compounds.Contrary to in vitro approaches that require overex-pression of the PPAR isotypes, our results in primaryhepatocytes suggest that Fsp27 is an exclusive PPARatarget in normal mouse hepatocytes and confirm theremarkable subtleties of targeting of the different iso-types in vivo.20 Importantly, dose-dependent targetingof human CIDEC by the PPARa agonist was con-firmed in Huh7 hepatoma cells (Fig. 3A). The inabil-ity of GW7647 and Wy14643 to induce Fsp27expression in either hepatocytes or livers fromPPARa–/– mice, compared to wild-type (WT) mice(Fig. 3B,C), suggests that the effects of these agonistsare mediated by PPARa.

To determine whether CIDEC/Fsp27 is a direct tran-scriptional target of PPARa, we used cycloheximide(CHX; a protein translation inhibitor) or actinomycinD (Act-D; a transcription inhibitor). Data in Fig. 3Dshow that pretreatment with CHX before addition ofGW7647 did not impair induction of either CIDECor CPT1A, suggesting that these are a direct responsethat occurs in the absence of protein synthesis. In con-trast, pretreatment with Act-D abolished the inductionof both PPARa targets (Fig. 3E), suggesting a directtranscriptional effect of the agonist on the CIDEC

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promoter. Finally, to study the potential impact of theagonist on messenger RNA (mRNA) stability, cellswere cultured in the presence or absence of the agonistto induce PPARa targets, and then Act-D was intro-duced to prevent further transcription. Data in Fig. 3Fshow that the half-life of CIDEC is long and not

altered by PPARa activation. Collectively, results fromFig. 3D-F demonstrate that induction of CIDEC/Fsp27 is mediated by PPARa-dependent transcription.

To map the promoter sequences that mediate theresponse to PPARa, we amplified a 3.3-kilobase (kb)region of the Fsp27 promoter that contained the

Fig. 1. Hepatic Fsp27 silencing abolishes fasting-induced hepatosteatosis. Mice (n 5 5) were infused with scrambled (Ad-shSCR) or anti-Fsp27 (Ad-shFsp27) adenovirus and 10 days later fasted or not. (A) Representative macroscopic appearance. (B) Representative Oil Red Ostaining of liver frozen sections. (C) Hepatic lipid contents. (D) Lipidomics heat map of hepatic TAG and FFA. (E) Plasma metabolites.*P� 0.05; **P� 0.01, fasted versus fed; ††P� 0.01, shFsp27 vs. shSCR.

1230 LANGHI AND BALD�AN HEPATOLOGY, April 2015

previously identified PPRE.13 Activity assays using thisfragment driving a luciferase reporter showed that ectopicPPARa and GW7647 increased Fsp27 promoter activity(Fig. 3G). Directed mutagenesis of the PPRE abolishedPPARa- and GW7647-mediated regulation (Fig. 3G). Areporter containing the proximal promoter of apolipo-protein A5 (ApoA5) was used as a positive control.Finally, recruitment of PPARa to the Fsp27 promoter wasverified by chromatin immunoprecipitation, usingextracts from mouse primary hepatocytes transduced witha Flag-PPARa adenovirus, and either anti-Flag or unre-lated immunoglobulin G antibodies. Data in Fig. 3Hshow that Flag-PPARa was specifically recruited to theFsp27 and Plin2 promoters, but not to intronicsequences.

Taken together, data in Figs. 2 and 3 demonstratethat CIDEC/Fsp27 is a direct transcriptional target ofPPARa in the hepatocyte.

Concomitant PPARa Activation and Fsp27 Silenc-ing Decrease Hepatic TAG Contents in Chow-FedMice. To characterize the physiological relevance ofPPARa-mediated induction of hepatic Fsp27, chow-fed mice were transduced with shSCR or shFsp27 vec-tors and then gavaged vehicle or Wy14643 for 7 days.No changes in body weight or circulating transami-nases were noted. The significant drop in circulatingTAG in mice dosed with Wy14643 (Fig. 4A) vali-dated the efficacy of the agonist, whereas no changeswere observed in plasma FFA or cholesterol (Fig. 4A).PPARa activation did not change the overall hepaticlipid contents in control mice, although a trend

toward elevated TAG was noted (Fig. 4B). In contrast,simultaneous PPARa activation and Fsp27 silencingsynergized to decrease liver TAG, but not FFA or cho-lesterol, contents (Fig. 4B). Analysis of selected tran-scripts in the same livers confirmed that shFsp27 waseffective in abrogating PPARa-mediated induction ofFsp27 (Fig. 4C). Similar to Fig. 1, the lower Fsp27expression in mice receiving both shFsp27 andWy14643 resulted in a modest decrease in PPARa tar-gets (although agonist-mediated induction was simi-lar), compared to mice treated with shSCR andWy14643 (Fig. 4C). On the other hand, overexpres-sion of Fsp27 in HuH7 cells did not change basal oragonist-induced expression of PPARa targets (Sup-porting Fig. 2). Taken together, data in Fig. 4 suggestthat PPARa activation in normal mice can onlyreduce hepatic TAG contents if induction of Fsp27 isblunted.

Fsp27 Silencing and PPARa ActivationSynergize to Ameliorate Hepatosteatosis in HFD-FedMice. The data above suggest that the combinationof PPARa activation with Fsp27 silencing may curbdiet-induced hepatosteatosis. Hence, we fed mice anHFD for 8 weeks and then performed adenoviraltransduction followed by a 7-day agonist gavage. Nochanges in body weight or circulating transaminaseswere noted, although both parameters were signifi-cantly elevated, compared to chow-fed mice. Asexpected, HFD raised both plasma TAG and choles-terol, as well as hepatic TAG contents, compared tochow-fed animals (Fig. 5A,B vs. 4A,B). PPARa

Fig. 2. Fsp27 is a PPARa-specific target. Mouse primary hepatocytes were cultured overnight in the presence of 1 mmol/L of agonists forPPARa (GW4767), PPARd (GW0742), or PPARc (rosiglitazone; Rosi). Expression of selected transcripts was measured by quantitative reverse-transcriptase polymerase chain reaction. Data from three independent experiments (n 5 4); *P� 0.05; **P� 0.01, compared to vehicle. Abbre-viation: DMSO, dimethyl sulfoxide.

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Fig. 3. Transcriptional control of CIDEC/Fsp27 by PPARa. (A) Dose-dependent induction of CIDEC by GW7647 in Huh7 cells. (B) GW7647-mediated induction of Fsp27 is lost in primary hepatocytes from PPARa–/– mice. (C) Wy14643-mediated induction of Fsp27 is lost in the liversof PPARa–/– mice. (D) Relative expression of CIDEC and CPT1A in Huh7 cells cultured 16 hours in media supplemented with GW7647, with orwithout 10 mg/mL of CHX. (E) Relative expression of CIDEC and CPT1A in Huh7 cells cultured 1 hour in media supplemented with 5 mg/mL ofActi-D, then with or without GW7647 for an extra 16 hours. (F) Relative expression of CIDEC and CPT1A in Huh7 cells cultured 16 hours inmedia supplemented with GW7647, then with or without 5 mg/mL of Act-D for an extra 6-24 hours. (G) Normalized activities of luciferase report-ers under control of a 3.3-kb fragment containing the Fsp27 proximal promoter and first intron, which includes the putative PPRE, or the samefragment carrying a mutated PPRE (PPRE*), or the proximal promoter of ApoA5 (positive control), in the presence or absence of an expressionvector for human PPARa and/or 1 mmol/L GW7647. (H) Relative chromatin enrichment shows specific recruitment of PPARa to the proximal pro-moters of Fsp27 and Plin2 (positive control), but not to intronic regions (negative controls). See Experimental Procedures in the Supporting Infor-mation. Data from three independent experiments (n 5 4); *P� 0.05; **P� 0.01, agonist versus DMSO; †P� 0.05; ††P� 0.01 PPARa–/– (orCHX, or Act-D, or PPRE*) versus WT (or DMSO, or PPRE). Abbreviations: DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline.

Fig. 4. Hepatic Fsp27 silencing and PPARa activation reduce TAG contents in chow-fed mice. Mice (n 5 5) were infused with scrambled (Ad-shSCR) or anti-Fsp27 (Ad-shFsp27) adenovirus and 3 days later gavaged with vehicle or 10 mg/kg/day of Wy14643 for 7 days and sacrificedwithout earlier fasting. (A) Hepatic lipid contents. (B) Plasma metabolites. (C) Relative expression of selected transcripts. *P� 0.05; **P� 0.01Wy14643 versus vehicle; †P� 0.05; ††P� 0.01, Ad-shFsp27 versus Ad-shSCR. Abbreviations: Acc, acetyl-CoA-carboxylase; Fasn, fatty acidsynthase.

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Fig. 5. Hepatic Fsp27 silencing and PPARa activation synergize to reverse diet-induced hepatosteatosis. Mice (n 5 5) were fed an HFD for 8weeks, then infused with scrambled (Ad-shSCR, closed bars) or anti-Fsp27 (Ad-shFsp27, open bars) adenovirus, 3 days later gavaged with vehi-cle or Wy14643 for 7 days, and sacrificed without earlier fasting. (A) Hepatic lipid contents. (B) Plasma metabolites. (C) Representative Oil RedO of liver frozen sections. (D) Relative expression of selected transcripts. *P� 0.05; **P� 0.01, Wy14643 versus vehicle; ††P� 0.01, Ad-shFsp27 versus Ad-shSCR.

1234 LANGHI AND BALD�AN HEPATOLOGY, April 2015

activation was again effective in reducing plasmaTAG, compared to vehicle (Fig. 5A), but did notimpact liver TAG contents (Fig. 5B,C). Importantly,Fsp27 silencing alone was sufficient to reduce hepaticTAG levels, and when combined with Wy14643, ledto further reduction of TAG contents (Fig. 5B,C).Remarkably, hepatic TAG in HFD-fed mice treatedwith both shFsp27 and Wy14643 were comparable tothose from control chow-fed animals (compare Figs.5B and 4B). These effects were specific, given that nochanges were noted in liver FFA or cholesterol (Fig.5B). Analysis of selected transcripts in the same liversdid not reveal significant changes in gene expression,except for Fsp27 (Fig. 5D). Together, data in Fig. 5suggest that PPARa agonists improve diet-inducedhepatosteatosis only if the induction of Fsp27 isprevented.

Loss of Fsp27 Activity Potentiates FAO UponPPARa Activation in Primary Hepatocytes. Wehypothesized that induction of CIDEC/Fsp27 counter-acts the effect of PPARa on FAO. To test this pro-posal, we transduced hepatocytes with shSCR orshFsp27 vectors and cultured them in media supple-mented with 0.3 mmol/L of oleic acid (to promoteTAG accumulation), followed by treatment with vehi-cle or GW7647. Data in Fig. 6A show that shFsp27robustly decreased Fsp27 expression without alteringexpression of other transcripts. Consistent with the invivo data above (Figs. 4 and 5), simultaneous Fsp27silencing and GW7647 treatment significantly reducedcell TAG contents, compared to either treatment alone(Fig. 6B).

Cells cultured in parallel were used for metaboliclabeling experiments. Consistent with previousreports,13 Fsp27 silencing resulted in acceleratedTAG turnover (Fig. 6C). Data in Fig. 6D show that,as expected, GW7647 induced the catabolism ofFFA in control cells; Fsp27 silencing alone, on theother hand, had no effect. However, combination ofboth GW7647 and shFsp27 resulted in acceleratedFFA b-oxidation, compared to GW7647 alone (Fig.6D). Additional experiments showed that Fsp27silencing did not change fatty acid uptake or de novolipogenesis (Supporting Fig. 3). Collectively, theseresults suggest that loss of CIDEC/FSP27 activitypotentiates the stimulatory effects of PPARa onFAO.

Discussion

This report highlights the critical role of CIDEC/FSP27 in liver TAG homeostasis under both physio-

logical and pathological conditions and provides evi-dence that disruption of FSP27 activity interferes withTAG storage and reverses hepatosteatosis in mice fedan HFD. Consistent with a recent report from Xuet al.,21 Fsp27b was the only isoform expressed in liver(even in HFD-fed mice) and primary hepatocytes,whereas Fsp27a was abundant in adipose tissue (datanot shown). CIDEC/FSP27 was originally described asan adipocyte-specific LD protein that promoted intra-cellular lipid accumulation.5-8 Further studies demon-strated that Fsp27 is also expressed in livers ofgenetically and diet-induced obese mice in a PPARc-dependent manner,4,12-14 as well as early during fastingthrough the canonical PKA-CREB pathway.15 Furtherstudies showed that, in adipocytes, FSP27 interactswith PLIN1 at the contact sites of nascent LDs tofacilitate lipid transfer and formation of unilocularLDs.22,23 Other investigators showed that FSP27 alsoacts as a lipolytic barrier by interacting with adiposetriglyceride lipase and preventing lipid utilization.24

Consistent with a role in mediating LD growth,Fsp27–/– mice were lean and protected from diet-induced obesity and insulin resistance (IR),8,9 whereasFsp27 overexpression promoted TAG accumulation inboth adipocytes and hepatocytes.13 These studies showthat Fsp27 is a critical mediator for LD homeostasisand suggest that reduced Fsp27 expression accelerateslipid utilization. In agreement with that proposal,investigators reported that gastric bypass surgery25 andcaloric restriction26 reduced both hepatic TAG con-tents and CIDEC expression in obese subjects. Impor-tantly, a lipodystrophic patient was reported carrying ahomozygous nonsense mutation in CIDEC.10 Intrigu-ingly, this patient also showed dyslipidemia, hepatos-teatosis, IR, and diabetes.10 This clinical profile istypical of deleterious lipodystrophies27 and contrastswith the lean, healthy phenotype of Fsp27–/– mice.The reasons for the discrepancies between the mouseand human loss-of-function models remain obscure,but the presence of the 185-amino-acid truncatedCIDEC (78% of full length) in this patient mightaffect intracellular lipid homeostasis and confound theliver phenotype. Nevertheless, these studies show that,under pathological conditions, CIDEC/Fsp27 is highlyinduced in the liver, where its expression correlateswith lipid accumulation, and suggest that CIDECmight be an attractive therapeutic target to managepatients with hepatosteatosis.

We present data to support the proposal thatCIDEC/Fsp27 is a direct transcriptional target ofPPARa. Consequently, we demonstrate that bothWy14643- and GW7647-induced expression of Fsp27

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are lost in livers and hepatocytes, respectively, ofPPARa–/– mice. Importantly, this regulation is con-served in human Huh7 hepatocytes. Further evidenceof PPARa targeting can be found in earlier observa-tions that the Wy14643 compound was able to up-regulate CIDEC/Fsp27 in both human28 andmouse28,29 primary hepatocytes, as well as in trans-genic mice expressing a constitutively active PPARa.30

This is in contrast with results by Vila-Brau et al.,15 inwhich incubation of HepG2 cells with Wy14643 failedto induce CIDEC expression. The reasons for this dis-crepancy are unknown. Nevertheless, herein we estab-lished that PPARa binds and activates the CIDEC/Fsp27 promoter by a conserved PPRE that had beenpreviously characterized to drive PPARc responsive-ness.13 Interestingly, although several studies reported

Fig. 6. Fsp27 silencing and PPARa activation accelerate FAO. Mouse primary hepatocytes were transduced with scrambled (Ad-shSCR, closedbars) or anti-Fsp27 (Ad-shFsp27, open bars) and cultured in media supplemented with 0.3 mmol/L oleate and vehicle or 1 mmol/L GW7647for 24 hours. (A) Relative expression of selected transcripts. (B) Intracellular TAG contents. (C) Additional cells were pulsed with [14C]-oleateovernight, then transduced with adenovirus and chased in media containing Triacsin-C. Lipid extracts were resolved on thin-layer chromatographyplates, autoradiographed, and TAG spots (left panel) scraped and counted by scintillation. (D) FAO capacity calculated from release of [3H]-waterinto culture medium. See Experimental Procedures in the Supporting Information for details on metabolic labeling experiments. Data from 3 inde-pendent experiments (n 5 4); *P� 0.05; **P� 0.01, GW7647 versus dimethyl sulfoxide; †P� 0.05, ††P� 0.01, Ad-shFsp27 versus Ad-shSCR.

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that PPARc was necessary for elevated expression ofFsp27 in livers of obese, insulin-resistant mice,4,12,13

treatment with rosiglitazone failed to induce hepaticFsp27 expression in lean mice.13 These latter resultsare likely explained by the very low expression ofPPARc in nonsteatotic livers11 and question the rele-vance of PPARc signaling in normal hepatic physiol-ogy. Our data show that only agonists for PPARa, butnot for PPARd or PPARc, alter expression of Fsp27 inmouse primary hepatocytes.

Is hepatic PPARa activation the critical driver for theremarkable elevation in Fsp27 during fasting or inresponse to HFD? Our data comparing WT andPPARa–/– mice show that, although the response toHFD was significantly attenuated in PPARa–/– mice,compared to control animals, it was not completelyabrogated (Supporting Fig. 4). Additionally, Fsp27 wasstill induced during fasting in chow-fed PPARa–/– mice(Supporting Fig. 4). We reason that additional factors,besides PPARa, control hepatic expression of Fsp27under fasting and in response to HFD. Hence, we pro-vide evidence of a compensatory increase in PPARcexpression in livers of both WT and PPARa–/– mice fedthe HFD (Supporting Fig. 4), which likely accounts forinduction of hepatic Fsp27 in those mice. On the otherhand, the PKA-CREB pathway is likely sufficient topromote induction of Fsp27 during early fasting.15 Weconclude that, despite being a bona-fide PPARa target,CIDEC/Fsp27 can still be induced by alternative mecha-nisms. However, and crucially, our work suggests thatCIDEC expression should be induced in the human liverupon pharmacological PPARa activation.

Synthetic PPARa activators, such as fibrates, arecommonly used to treat dyslipidemias and are usefulin curbing plasma TAG.11 Although fibrates have beenshown to reduce and/or prevent fatty liver in diet-induced murine models of NAFLD,31,32 othersreported an increase in liver TAG content after PPARaactivation.19,33,34 Efficacy of PPARa agonists onhuman NAFLD or nonalcoholic steatohepatitis(NASH) also remains controversial, given that moststudies failed to show improved liver histology ordecreased hepatic TAG contents in subjects takingfibrates.35 However, from a mechanistic perspective, it isimportant to consider that hepatic PPARa targets notonly FAO genes (i.e., CPT1A, medium-chain acyl-CoAdehydrogenase, and ACOX), but also genes involved inLD formation (PLIN2, PLIN5, and FSP27; accordingto previous work17-19 and this report). It is tempting tospeculate that these two sets of targets act in concert tomodulate intracellular lipid utilization/storage and pre-

vent lipotoxic effects. We hypothesize that fibrates mightbe more efficient in promoting hepatic lipid clearancewhen used in combination with agents that preventinduction of LD-related genes. Our data provide sup-port for this hypothesis: We show that blocking Fsp27induction upon PPARa activation potentiates the effectof GW7647 on FAO in hepatocytes, and that Wy14643and shFsp27 synergize to decrease hepatic TAG contentsin both chow- and HFD-fed mice. Importantly, the ben-eficial effect of Fsp27 silencing on ameliorating hepatos-teatosis is not dependent upon further induction ofFAO genes, compared to mice treated with fibratesalone, but it is likely the result of increased TAG turn-over, which promotes FFA partitioning toward oxida-tion. In other words, loss of FSP27 activity increases theavailability of substrates for FAO, not the amounts oractivities of FAO genes. We conclude that hepaticCIDEC/FSP27 acts as a brake for PPARa-stimulatedFAO by limiting the release of substrates available for b-oxidation. Our results suggest that specific PPARa mod-ulators (SPPARMs) that were able to selectively inducethe expression of target genes involved in FAO, but notin LD biogenesis, might be potent therapeutic tools todecrease TAG in livers of NAFLD and NASH patients.

Acknowledgment: The authors thank Erin Touch-ette for outstanding technical support with in vivoexperiments, Dr. David Ford for helping with tissuelipidomics analysis and interpretation, Dr. MaureenDonley for help with statistical analysis, and membersof the Bald�an laboratory for helpful discussion. ABdedicates this article to Mordisquitos (2011-2014). Weshall grieve not, rather find solace in the memories ofour time together. We will meet again at RainbowBridge.

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Author names in bold designate shared co-firstauthorship.

Supporting Information

Additional Supporting Information may be found athttp://onlinelibrary.wiley.com/doi/10.1002/hep.27607/suppinfo.

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