MOLECULAR AND CELLULAR BIOLOGY, Dec. 2009, p. 6257–6267 Vol. 29, No. 230270-7306/09/$12.00 doi:10.1128/MCB.00370-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Peroxisome Proliferator-Activated Receptor �/� (PPAR�/�) but NotPPAR� Serves as a Plasma Free Fatty Acid Sensor in Liver�†

Linda M. Sanderson,1,2 Tatjana Degenhardt,3 Arjen Koppen,4,5 Eric Kalkhoven,4,5 Beatrice Desvergne,6

Michael Muller,1,2 and Sander Kersten1,2*Nutrigenomics Consortium, TI Food and Nutrition, Nieuwe Kanaal 9A, 6709 PA Wageningen, The Netherlands1; Nutrition,

Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, Bomenweg 2,6703 HD Wageningen, The Netherlands2; Department of Biochemistry, University of Kuopio, 70211 Kuopio,Finland3; Departments of Metabolic and Endocrine Diseases, University Medical Centre Utrecht,

Utrecht, The Netherlands4; Netherlands Metabolomics Center, Leiden, The Netherlands5; andCentre Integrative Genomique, University of Lausanne, Lausanne, Switzerland6

Received 23 March 2009/Returned for modification 26 April 2009/Accepted 14 September 2009

Peroxisome proliferator-activated receptor � (PPAR�) is an important transcription factor in liver that canbe activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here weinitially set out to elucidate the similarities in gene induction between Wy14643 and fasting. Numerous geneswere commonly regulated in liver between the two treatments, including many classical PPAR� target genes,such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fastingindependently of PPAR�, including Lpin2 and St3gal5, suggesting involvement of another transcription factor.Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPAR�/� duringfasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPAR�. Binding of PPAR�/� to the Lpin2 andSt3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPAR�/�by plasma FFAs. Subsequent experiments using transgenic and knockout mice for Angptl4, a potent stimulantof adipose tissue lipolysis, confirmed the stimulatory effect of plasma FFAs on Lpin2 and St3gal5 expressionlevels via PPAR�/�. In contrast, the data did not support activation of PPAR� by plasma FFAs. The resultsidentify Lpin2 and St3gal5 as novel PPAR�/� target genes and show that upregulation of gene expression byPPAR�/� is sensitive to plasma FFA levels. In contrast, this is not the case for PPAR�, revealing a novelmechanism for functional differentiation between PPARs.

Hepatic lipid metabolism is governed by a complex interplaybetween hormones, transcription factors, and energy sub-strates, allowing for rapid adaptations to changes in metabolicneeds (21). According to the traditional view, energy substratessuch as fatty acids influence lipid metabolism by promoting fluxthrough a particular pathway via mass action. However, it hasbecome clear that energy substrates can also directly governthe transcription of enzymes involved in lipid metabolism viamechanisms analogous to those of many hormones. Indeed, itis now evident that glucose and fatty acids play a major regu-latory role in hepatic lipid metabolism via direct activation orinhibition of specific transcription factors, including carbohy-drate response element binding protein (6, 63), sterol responseelement binding protein 1 (SREBP1) (2, 41, 58, 61, 62), andperoxisome proliferator-activated receptor � (PPAR�) (38).

Although numerous transcription factors have been shownto be activated by fatty acids in vitro, recent data suggest thatPPAR� is dominant in mediating the effects of dietary fattyacids on gene expression in liver (48). PPAR� is a member ofthe superfamily of nuclear receptors and is closely related to

the other PPAR isoforms, �/� and � (32). Similar to severalother nuclear receptors, PPARs function as heterodimers withthe retinoid X receptor and bind to specific sequences on theDNA referred to as PPAR response elements (PPREs) (8, 11,26). Numerous studies have shown that fatty acids can directlybind to PPARs and activate DNA transcription (12, 17, 24, 28,31, 50). Binding of fatty acids changes the conformation of thePPAR protein (13, 23, 37, 60) and leads to recruitment ofcoactivator proteins (31, 48). Besides fatty acids and their de-rivatives, PPARs bind synthetic agonists, including the thia-zolidionediones, which serve as agonists for PPAR�, and thefibrates, which are PPAR� agonists (51).

Most of the information about the function of PPAR� inliver and its impact on target genes is based on studies thathave used high-affinity synthetic PPAR� agonists. These phar-macological studies have shown that PPAR� regulates a re-markably large number of genes, many of which are involved inhepatic lipid metabolism, thereby explaining the positive effectof synthetic PPAR� agonists on plasma lipid parameters (9,38). However, PPAR� did not evolve as a receptor for fibratesbut rather as a fatty acid sensor. Accordingly, the questionarises to what extent results from pharmacological studies re-flect the physiological function of PPAR�.

Physiological experiments using PPAR��/� mice haveshown that PPAR� is especially important for the adaptiveresponse to fasting. During fasting, the absence of PPAR�elicits a complex phenotype characterized by fatty liver, hypo-

* Corresponding author. Mailing address: Nutrition, Metabolismand Genomics Group, Wageningen University, Bomenweg 2, 6703 HDWageningen, The Netherlands. Phone: 31 317 485787. Fax: 31 317483342. E-mail: sander.kersten@wur.nl.

† Supplemental material for this article may be found at http://mcb.asm.org/.

� Published ahead of print on 5 October 2009.

6257

ketonemia, hypoglycemia, hypothermia, and elevated plasmafree fatty acid (FFA) levels (1, 19, 27, 34). Furthermore, thehepatic induction of numerous metabolic genes during fastingis abolished in PPAR��/� mice. While both pharmacologicaland physiological studies thus support a major role for PPAR�in hepatic lipid metabolism, evidence suggests that there isonly partial overlap between genes upregulated by PPAR�during fasting and genes upregulated by synthetic PPAR� ago-nists (45). One possible explanation is that PPAR� respondsdifferently to pharmacological compared to physiological acti-vation. Additionally, there may be a role for other PPARsubtypes. Besides PPAR�, PPAR�/� has been shown to be wellexpressed in hepatocytes (10, 22). However, the functional roleof PPAR�/� in hepatocytes and its physiological mechanismsof activation remain unknown.

Here we initially set out to elucidate the similarities anddiscrepancies in gene regulation in liver between pharmaco-logical PPAR� activation by Wy14643 and physiologicalPPAR� activation by fasting. While our data reveal majoroverlap between the effects of Wy14643 and fasting, the dataalso indicate that a number of pharmacological PPAR� targetgenes are induced by fasting independently of PPAR�. Subse-quent analysis uncovered a role for PPAR�/� in hepatic generegulation and revealed different mechanisms of activation ofPPAR� versus PPAR�/� in mouse liver. Specifically, we foundthat upregulation of gene expression by PPAR�/� is sensitiveto plasma FFAs, while this is not the case for PPAR�.

MATERIALS AND METHODS

Materials. Tridocosahexaenoin was obtained from Nu-Chek-Prep, Inc. (Ely-sian, MN). SYBR green was purchased from Eurogentec (Seraing, Belgium).Protease inhibitor cocktail was purchased from Roche Diagnostics (Almere, TheNetherlands), sonicated salmon sperm DNA was from Invitrogen (Breda, TheNetherlands), and proteinase K was from Fermentas (St. Leon-Rot, Germany).All other chemicals were from Sigma (Zwijndrecht, The Netherlands).

Animals. Purebred Sv129 PPAR��/� mice (129S4/SvJae) and correspondingwild-type mice (129S1/SvImJ) were purchased from Jackson Laboratory (BarHarbor, ME). The PPAR�/��/� mice were on a mixed background (Sv129/C57BL/6) and have been previously described (42). The Angptl4�/�, Angptl4�/�,and transgenic mice were on a C57BL/6 background and have been previouslydescribed (30, 36, 39). Angptl4-transgenic mice overexpress Angptl4 in numer-ous tissues, including adipose tissue (36, 39), while the Angptl4�/� mice lackAngptl4 expression in all tissues (30). Only male mice were used, with 4 to 10mice per group. For the fasting experiment, food was withdrawn for 24 h startingat the onset of the light cycle.

PPAR� ligand testing. Wild-type and PPAR��/� mice were fasted for 4 h andthereafter given an intragastric gavage of 400 �l Wy14643 (10 mg/ml in 0.5%carboxymethyl cellulose; 160 mg/kg of body weight). The control treatment was400 �l of 0.5% carboxymethyl cellulose. Livers were collected after 6 h.

Oral lipid load testing. Wild-type and PPAR��/� mice were given an intra-gastric gavage of 400 �l synthetic triglyceride (tridocosahexaenoin) after a 4-hourfast. The control treatment was 0.5% carboxymethyl cellulose (400 �l). Liverswere collected 6 h after gavage.

PPAR�/� ligand testing. Wild-type mice were given a single oral gavage of 150�g GW501516 (6 mg/kg). Alternatively, PPAR��/� mice were fed 0.025% (wt/wt) L165041 mixed in food for 5 days (40 mg/kg).

Mice were anesthetized with a mixture of isoflurane (1.5%), nitrous oxide(70%), and oxygen (30%). Blood was collected by orbital puncture, after whichthe mice were sacrificed by cervical dislocation. Livers were dissected and di-rectly frozen in liquid nitrogen. For RNA analyses, tissue from the same part ofthe liver lobe was used.

The animal studies were approved by the Local Committee for Care and Useof Laboratory Animals at Wageningen University, The Netherlands, and theUniversity of Lausanne, Switzerland.

Affymetrix microarray. Total RNA from mouse liver was extracted withTRIzol reagent, purified, and DNase treated using the SV total RNA isolation

system (Promega, Leiden, The Netherlands). RNA quality measurements wereperformed on an Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam,The Netherlands) using 6000 Nano Chips in combination with the eukaryotetotal RNA Nano assay. RNA was judged as suitable for array hybridization onlyif samples showed intact bands corresponding to the 18S and 28S rRNA subunits,displayed no chromosomal peaks or RNA degradation products, and had anRNA integrity number above 8.0. Five micrograms of RNA was used for thecRNA synthesis mixture for one cycle (Affymetrix, Santa Clara, CA). Hybrid-ization, washing, and scanning of Affymetrix mouse genome 430 2.0 arrays/Affymetrix Nugo mouse arrays were carried out according to standard Affymetrixprotocols.

Packages from the Bioconductor Project were used for analyzing the scannedarrays (14). Arrays were normalized using quantile normalization, and expres-sion estimates were compiled using GC-RMA (GeneChip robust multiarrayaverage), applying the empirical Bayes approach (59). A nonspecific filtering stepwas applied to remove genes with low variation and included only those genesthat had an interquartile range across the samples of at least 0.25 on a log2 scale(55).

Gene set enrichment analysis (GSEA) was used to relate changes in geneexpression to functional changes between mice treated with the PPAR� agonistWy14643 for 6 h and mice fasted for 24 h. GSEA takes into account a broadcontext of physically interacting networks in which gene products function, in-cluding biochemical, metabolic, and signal transduction routes (53). Gene setswith a false discovery rate P value of �0.1 were considered significantly over-represented.

Plasma metabolites. Plasma was obtained from blood by centrifugation for 10min at 10,000 g. Plasma triglycerides and the glycerol concentration in cellculture medium were determined using kits from Instruchemie (Delfzijl, TheNetherlands). Plasma free fatty acids were determined using a kit from WAKOChemicals (Instruchemie, Delfzijl, The Netherlands).

Fat explants. Epididymal adipose tissue was excised and cut into 0.2- to0.3-cm3 pieces. The explants were subsequently incubated for 15 min at 37°C inDulbecco’s modified Eagle’s medium (DMEM) containing 1% lipid-free bovineserum albumin (BSA) and 1 mg/ml collagenase type 1. Fat cells were liberated bygentle stirring followed by centrifugation of the cell suspension for 1 min at400 g. Fat cells were isolated from the surface and washed once in phosphate-buffered saline (PBS). Subsequently, fat cells were incubated in DMEM con-taining 1% lipid-free BSA and 1 �M isoproterenol at 37°C with 5% CO2.Medium was collected at various time points and frozen for measurement of freefatty acids and glycerol (kits from Instruchemie, Delfzijl, The Netherlands).

RNA isolation and quantitative reverse transcription-PCR. Total liver RNAwas isolated with TRIzol reagent (Invitrogen) according to the manufacturer’sinstructions. A NanoDrop ND-1000 spectrophotometer (Isogen Life Science,Ijsselstein, The Netherlands) was used to determine RNA concentrations. Onemicrogram of total RNA was reverse transcribed using iScript (Bio-Rad,Veenendaal, The Netherlands). cDNA was amplified on a Bio-Rad MyIQ oriCycler PCR machine using Platinum Taq DNA polymerase (Invitrogen). PCRprimer sequences were taken from the PrimerBank (56) and ordered fromEurogentec. Sequences of the primers used are available upon request.

Transactivation assay. Conserved PPREs were identified at 1,291 or 23,333nucleotides downstream of the transcriptional start site (TSS) of the mouseLpin2 or St3gal5 gene, respectively, using a published algorithm (20). A 201-nucleotide and 183-nucleotide fragment surrounding the putative PPRE withinthe Lpin2 and St3gal5 genes, respectively, was PCR amplified from mousegenomic DNA (strain C57BL/6) and subcloned into the KpnI and BglII sites ofthe pGL3 promoter vector (Promega, Leiden, The Netherlands). The reportervector (PPRE)3-TK-luciferase was included as a positive control. Reporter vec-tors were transfected into human hepatoma HepG2 cells together with an ex-pression vector (pSG5) for mouse PPAR�/�, in the presence or absence ofGW501516 (1 �M). A �-galactosidase reporter vector was cotransfected tonormalize for differences in transfection efficiencies. Transfections were carriedout using Nanojuice (Novagen, Nottingham, United Kingdom). Luciferase ac-tivity was measured 24 h posttransfection using the Promega luciferase assay kiton a Fluoroskan Ascent FL apparatus (Thermo Labsystems, Breda, The Neth-erlands). �-Galactosidase activity was measured in the cell lysate by a standardassay using 2-nitrophenyl-�-D-galactopyranoside as a substrate.

For the GAL4 transactivation assay, the human osteosarcoma cell line U2OSwas maintained in DMEM Glutamax containing 10% fetal calf serum (Invitro-gen), 100 �g of penicillin/ml, and 100 �g streptomycin/ml (Invitrogen). Forluciferase reporter assays, cells were seeded in 24-well plates and transientlytransfected using Lipofectamine 2000 (Invitrogen) according to the manufactur-er’s protocol. Each well was cotransfected with 1 �g of the 5Gal4-E1BTATA-pGL3 reporter, 10 ng of pcDNA3-GAL4-hPPAR�DBD, and 2 ng of pCMV-

6258 SANDERSON ET AL. MOL. CELL. BIOL.

Renilla (Promega, Madison, WI). The next day, medium was replaced withmedium containing ligands. At 24 h after the incubation, cells were harvested inpassive lysis buffer (Promega) and assayed for luciferase activity according to themanufacturer’s protocol (Promega dual-luciferase reporter assay system) and forRenilla luciferase activity to correct for transfection efficiency. The relative lightunits were measured by a Centro LB 960 luminometer (Berthold Technologies,Bad Wildbad, Germany). Experiments were performed in triplicate.

Chromatin immunoprecipitation (ChIP) assay. It is becoming increasinglyapparent that most nuclear receptor binding sites, including PPREs, are notfound in proximity of the annotated TSS of a gene but are often located quitedistant (4, 33, 43). Nuclear receptors bound to such distal sites likely contact thebasal transcription machinery via DNA looping. Binding of PPAR to distantPPREs can thus be demonstrated by showing cross-linking of PPAR to the TSS(5, 49).

Wild-type and PPAR��/� mice on an Sv129 background were fed or fasted for24 h (n 3). Transgenic mice overexpressing Angptl4 (Angptl4-Tg), wild-typemice (Angptl4�/�), and homozygous knockout mice (Angptl4�/�) (n 3/group)were fasted for 24 h. At the end of the fasting period, mice were killed by cervicaldislocation and livers were extracted. Livers were cut in smaller pieces anddirectly put in PBS containing 1% formaldehyde. Cross-linking was stopped after15 min by adding glycine to a final concentration of 0.125 M for 5 min at roomtemperature. After a short centrifugation to collect the liver pieces, two washingsteps with ice-cold PBS were carried out. Livers were homogenized and there-after centrifuged. After supernatant was removed, liver homogenate was resus-pended in lysis buffer (1% sodium dodecyl sulfate [SDS], 10 mM EDTA, 50 mMTris-HCl, pH 8.1, protease inhibitors), and the lysates were sonicated with aBioruptor (Diagenode, Liege, Belgium) to achieve a DNA length of 300 to 1,000bp. After removal of cellular debris by centrifugation, supernatants were diluted1:10 in ChIP dilution buffer (150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 20mM Tris-HCl, pH 7.5, protease inhibitors). Chromatin was incubated over-night at 4°C with 2 �g antibody, 25 �l BSA (10 mg/ml), and 2.4 �l sonicatedsalmon sperm (10 mg/ml). Antibodies used were anti-PPAR� (sc-9000), anti-PPARGC1� (sc-13067), and anti-PPAR�/� (sc-7197), all of which were obtainedfrom Santa Cruz Biotechnology (Heidelberg, Germany). Immunocomplexeswere collected with 25 �l MagaCell protein A magnetic beads (Isogen LifeScience) for 1 h at room temperature and subsequently washed sequentially with700 �l of the following buffers: twice with ChIP wash buffer 1 (150 mM NaCl, 1%Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8, protease inhibitors), oncewith ChIP wash buffer 2 (500 mM NaCl, 1% Triton X-100, 2 mM EDTA, 0.1%SDS, 20 mM Tris-HCl, pH 8, protease inhibitors), once with ChIP wash buffer 3(250 mM LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH8), and two times with TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8). Elutionof immunocomplexes was carried out in 250 �l of elution buffer (10 mM EDTA,0.5% SDS, 25 mM Tris-HCl, pH 7.5) at 64°C for 30 min. After collection ofsupernatant, elution was repeated with 250 �l elution buffer at room tempera-ture for 2 min. After combining the supernatants, cross-linking was reversed at64°C overnight with 2.5 �l proteinase K (20 mg/ml) for digestion of any remain-ing proteins. Genomic DNA fragments were recovered by phenol-chloroformextraction with phase lock gel (Eppendorf, Wesseling-Berzdorf, Germany), fol-lowed by salt-ethanol precipitation. Samples were diluted in sterile H2O andanalyzed by quantitative PCR.

Primers were from Eurogentec and designed to cover the TSS of the followinggenes: Aldh3a2 (forward [F], 5�-CAGGTGAGGGAGCACAGTAC-3�; reverse[R], 5�-CGCTTGGCTCTTTTCTGAAG-3�); Cpt2 (F, 5�-GCCAGTCACGCAACAGCAG-3�; R, 5�-TAGTTTAGAGACCGCTTCCG-3�); Lpin2 (F, 5�-CCGTCTTGTGATTGGGCAGG-3�; R, 5�-GAAGGAAACTCACCAGAATCC-3�);St3gal5 (F, 5�-GCCTTCCACTATCTAATCACG-3�; R, 5�-GTGTCCGCTCTGCCGACTG-3�); Rplp0 (F, 5�-CGAGGACCGCCTGGTTCTC-3�; R, 5�-GTCACTGGGGAGAGAGAGG-3�).

FAO cell culture. Rat hepatoma FaO cells were grown in DMEM containing10% fetal bovine serum, 100 U/ml penicillin, and 100 �g/ml streptomycin. Cellswere incubated with albumin only (control), albumin-bound oleic or linoleic acid(100 �M; molar ratio for FA:BSA of 3.5:1), or GW501516 (100 nM) for 4 h,followed by RNA isolation and quantitative reverse transcription-PCR.

Cofactor recruitment assay. Nuclear receptor PamChip arrays (PamGene,s’Hertogenbosch, The Netherlands) were used as described previously (29).Upon binding a ligand, PPAR�/� undergoes a conformational change whichpromotes the formation of a cofactor binding pocket, subsequently allowinginteraction with the so-called LxxLL motif within some coregulators. The Pam-Chip arrays consist of 53 peptides encompassing the LxxLL motifs of 22 differentcoregulator proteins. Briefly, the arrays were incubated with glutathione S-transferase-tagged PPAR�/� ligand binding domain (LBD) (Invitrogen, Breda,The Netherlands) in the presence and absence of ligand (GW501516 at 400 nM;

oleic and linoleic acids at 125 �M). Quantification of the interaction betweenPPAR�/� and coregulators was made using Alexa 488-conjugated anti-glutathi-one S-transferase rabbit polyclonal antibody (Invitrogen).

Microarray accession numbers. All microarray experimental results have beendeposited into the Gene Expression Omnibus database under accession numberGSE 8396 and GSE 17863.

RESULTS

Overlap in gene regulation between pharmacological andphysiological PPAR� activation. PPAR� in liver can be acti-vated pharmacologically by using synthetic agonists such asWy14643 or physiologically by fasting. To assess the similaritiesand discrepancies in gene regulation between these two stim-uli, we compared microarray data from livers of mice treatedwith the synthetic PPAR� agonist Wy14643 for 6 h and liversof mice subjected to 24 h of fasting. GSEA showed greatsimilarity and overlap in top-regulated pathways between fast-ing and Wy14643 treatment, almost all of which correspondedto pathways of lipid metabolism (Fig. 1A). Much less overlapwas observed at the individual gene level (Fig. 1B). Neverthe-less, a substantial number of genes upregulated by Wy14643were also induced by fasting. Many of these genes representclassical PPAR� target genes involved in fatty acid catabolism,such as Acox1, Cpt2, Aldh3a2, Acot8, Ehhadh, and Hmgcs2.Consistent with an important role of PPAR�, induction ofclassical PPAR� target genes by fasting was abolished inPPAR��/� mice (Fig. 1C and D). In contrast, a number ofWy14643-responsive genes could be identified that were moresignificantly upregulated by fasting in PPAR��/� mice than inwild-type mice, suggesting PPAR�-independent regulationduring fasting (Fig. 1C and D). Overall, these data indicatethat targets of pharmacological PPAR� activation exhibit di-verse responses following physiological PPAR� activation byfasting, being either up- or downregulated and showing a vari-able dependence on PPAR�.

To explore the possible mechanism underlying the moresignificant induction by fasting of a number of pharmacologicalPPAR� targets in PPAR��/� mice compared to wild-typemice, two representative genes were investigated in more de-tail: Lpin2 and St3gal5. Remarkably, in contrast to classicalPPAR� targets Cpt2 and Aldh3a2 (Fig. 2A and C), inductionof Lpin2 and St3gal5 by fasting and dietary fatty acids waslargely maintained in PPAR��/� mice (Fig. 2B and D). Theseresults imply that the effects of fasting and dietary fatty acidson hepatic expression of Lpin2 and St3gal5 may be partiallymediated by a transcription factor other than PPAR�. On thecontrary, effects of fasting and dietary fatty acids on Cpt2 andAldh3a2 are entirely mediated by PPAR�. It should be men-tioned that the expression profiles of Cpt2 and Aldh3a2 are rep-resentative of a large set of classical PPAR� targets (Fig. 1D).

PPAR�/� as an alternative transcription factor to PPAR�in mouse liver during fasting. One obvious candidate alterna-tive transcription factor is PPAR�/�, which is well expressed inliver (10, 16). Supporting regulation of Lpin2 and St3gal5 byPPAR�/�, the PPAR�/� agonists GW501516 and L165041 sig-nificantly induced Lpin2 and St3gal5 mRNA (Fig. 3A). Toestablish whether Lpin2 and St3gal5 are direct PPAR targetgenes, we identified a conserved PPRE within the Lpin2 andSt3gal5 genes (Fig. 3B) and cloned a genomic region encom-passing the PPRE in front of a luciferase reporter to perform

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transactivation assays. GW501516 significantly increased re-porter activity for the Lpin2 and St3gal5 genomic regions,which was further enhanced by cotransfection with PPAR�/�(Fig. 3C). In subsequent ChIP experiments, PPAR�/� as wellas PPAR� could be cross-linked to the TSS of the Lpin2 andSt3gal5 genes, at least in the fasted state, which provided evi-dence for the presence of a distant functional PPRE (Fig. 3D).These data suggest that Lpin2 and St3gal5 genes represent directPPAR target genes. Interestingly, while fasting increased bind-ing of both PPAR� and PPAR�/� to the Lpin2 and St3gal5genes, fasting increased binding of only PPAR� to the Aldh3a2and Cpt2 genes (Fig. 3D). No binding of PPAR� and PPAR�/�to the negative control gene Rplp0 was observed. All togetherthese data suggest that Lpin2 and St3gal5 are dual targets ofPPAR� and PPAR�/�, whereas Aldh3a2 and Cpt2 are exclu-sive targets of PPAR�. In agreement with this notion, induc-

tion of Lpin2 and St3gal5 by fasting was partially abolished inPPAR�/��/� mice (Fig. 3E).

Given the more pronounced induction of Lpin2 and St3gal5by fasting in PPAR��/� versus wild-type mice, we speculatedthat either expression of PPAR�/� may be upregulated inPPAR��/� mice as a compensatory mechanism or that ligandactivation of PPAR�/� is enhanced in PPAR��/� mice. Whilewe could not detect a change in PPAR�/� mRNA inPPAR��/� mice (data not shown), consistent with the secondscenario, plasma FFA levels were markedly elevated in fastedPPAR��/� mice (Fig. 3F), which was associated with markedinduction of PPAR�/� binding to the Lpin2 and St3gal5 pro-moter (Fig. 3D). These data suggest that in the absence ofPPAR� plasma FFAs can induce Lpin2 and St3gal5 expressionvia PPAR�/�. It should be noted that levels of plasma FFAsduring fasting are unaltered in PPAR�/��/� mice (Fig. 3G).

FIG. 1. Hepatic genes activated by Wy14643 and fasting show a variable dependence on PPAR�. Livers from wild-type and PPAR��/� micetreated with the PPAR� agonist Wy14643 for 6 h or fasted for 24 h were used for gene expression profiling (n 4 to 5 mice per group). (A) Overlapin top-regulated pathways between Wy14643 treatment and fasting according to gene set enrichment analysis. Gene sets with a false discovery rateP value of �0.1 were considered significant. (B) Overlap of upregulated genes between Wy14643 treatment and fasting (criteria for inclusion: P� 0.01 and a change of �1.5-fold). (C) Scatter plot showing the effects of fasting in genes significantly upregulated by Wy14643. The y axis andx axis show the effects of fasting in wild-type and PPAR��/� mice, respectively. Red dots represent classical PPAR� target genes, while blue dotsare Wy14643-responsive genes that are more significantly upregulated by fasting in the PPAR��/� mouse compared to wild type. (D) Heatmapshowing the changes (n-fold) of genes compared to the wild-type control/fed state. (Upper panel) Classical PPAR� target genes, showing aPPAR�-dependent increase in gene expression upon Wy14643 treatment as well as fasting. Genes in the lower panel exhibit a PPAR�-dependentinduction upon Wy14643 treatment but are induced independently of PPAR� upon fasting.

6260 SANDERSON ET AL. MOL. CELL. BIOL.

Circulating FFAs activate PPAR�/� but not PPAR� in mouseliver. Importantly, recent evidence suggests that PPAR� inliver cannot be (ligand) activated by plasma FFAs, while it canbe activated by fatty acids synthesized de novo (3). To study theactivation of PPAR� and PPAR�/� by plasma FFAs, we mod-ulated fasting plasma FFA levels by taking advantage of aunique transgenic model system based on whole-body overex-pression or inactivation of the mouse Angptl4 gene, whichencodes a prolipolytic factor involved in lipid metabolism (39,52). Previously, intravenous injection of Angptl4 was shown tocause an immediate increase in plasma FFA (65). In support,Angptl4 increased release of glycerol from 3T3-L1 adipocytes(Fig. 4A).

Consistent with a prolipolytic effect of Angptl4, Angptl4overexpression in mice was associated with a significant in-crease in release of fatty acids and glycerol from adipose tissue,whereas the opposite was observed in Angptl4�/� mice (Fig.4B). In agreement with these data, fasting plasma FFA levelswere increased or decreased upon Angptl4 overexpression orinactivation, respectively (Fig. 4C). In fact, the fasting-inducedincrease in plasma FFA was entirely blunted in Angptl4�/�

mice. Plasma triglyceride levels were increased or decreasedupon Angptl4 overexpression or inactivation (Fig. 4D), respec-tively, reflecting the well-documented inhibitory effect of Angptl4on lipoprotein lipase activity (25). Finally, the defective lipol-ysis in Angptl4�/� and Angptl4�/� mice was supported by theabsence of changes in adipocyte cell size upon fasting, in con-trast to Angptl4-Tg and wild-type mice (Fig. 4E). These resultscorroborate the stimulatory effect of Angptl4 on adipose tissuelipolysis, which we exploited to study the effect of plasma FFAson hepatic gene expression.

If hepatic PPAR� is activated by plasma FFAs, expression ofclassical PPAR� target genes during fasting would be expected tobe proportional to the plasma FFA level throughout the variousAngptl4 mouse models. Remarkably, rather than going down,

gene expression of classical PPAR� targets Aldh3a2, Cpt2, andothers was stable or went up as plasma FFAs declined (Fig. 5A;see also Fig. S1A in the supplemental material). Expression ofPpar� itself, which is auto-regulated, followed a very similar pat-tern (Fig. 5B), suggesting that regulation of classical PPAR�targets is determined by PPAR� expression level. Supportingthe use of the Angptl4 mouse models to study hepatic generegulation by FFA, hepatic expression of Srebp1, which isknown to be suppressed by fatty acids, negatively correlatedwith plasma FFA concentration (data not shown). In combi-nation with previously published data (3), these data stronglysuggest that PPAR� is not activated by plasma FFA in mouseliver.

While plasma FFAs seemingly do not activate hepaticPPAR�, data presented above suggested that plasma FFAsinduce Lpin2 and St3gal5 expression by activating PPAR�/�, atleast when PPAR� is absent. In order to explore activation ofhepatic PPAR�/� by plasma FFA, we first determined thatfatty acids are able to bind and activate PPAR�/� in vitro. Ina transactivation assay using the GAL4-LBDPPAR�/� fusion,oleic and linoleic acids markedly induced luciferase activity,indicating activation of PPAR�/� (Fig. 5C). Next we studiedthe effects of oleic and linoleic acids on the interaction betweenPPAR�/� and peptides corresponding to specific coregulator-nuclear receptor binding regions (29). Both fatty acids andGW501516 promoted the interaction between PPAR�/� andnumerous coregulator peptides, which demonstrated their bind-ing to PPAR�/� (Fig. 5D). Finally, oleic and linoleic acidssignificantly induced expression of the PPAR�/� marker geneAdfp in hepatoma cells (Fig. 5E). These data demonstrate thatfatty acids directly activate PPAR�/�. To assess activation ofPPAR�/� in vivo by circulating FFAs in the presence ofPPAR�, we determined binding of PPAR�/� to the Lpin2 andSt3gal5 genes in the Angptl4 mouse models by using ChIP.Importantly, independent of PPAR�/� gene expression levels,

FIG. 2. Lpin2 and St3gal5 are induced during fasting independently of PPAR�. Livers from wild-type and PPAR��/� mice fasted for 24 h ortreated with tridocosahexaenoin (DHA) for 6 h were used for gene expression profiling (n 4 to 5 mice per group). (A and B) Gene expressionof classical PPAR� targets Aldh3a2 and Cpt2 (A) and Lpin2 and St3gal5 (B) in fed and fasted wild-type and PPAR��/� mice. (C and D) Geneexpression of Aldh3a2 and Cpt2 (C) and Lpin2 and St3gal5 (D) after treatment with the dietary fatty acid tridocosahexaenoin. Error bars representstandard errors of the means. *, significantly different according to Student’s t test (P � 0.05).

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which remained constant (Fig. 5F), binding of PPAR�/� to theLpin2 and St3gal5 genes was proportional to the plasma FFAconcentration and mimicked fasting Lpin2 and St3gal5 expres-sion levels (Fig. 5G and H). In contrast, binding of PPAR� tothe Lpin2 and St3gal5 genes was minimal and did not followthe plasma FFA concentration (Fig. 5G). Again, no binding ofPPAR� and PPAR�/� to the negative control gene Rplp0 wasobserved. These data suggest that PPAR�/� can be activatedby plasma FFAs. Other Wy14643-induced genes whose expres-sion followed the plasma FFA concentration independent ofPPAR� included lipid droplet proteins 2310076L09Rik (MLDP),and S3-12, as well as Slc16a5 and Gadd45b, suggesting they

might represent targets of PPAR�/� as well (see Fig. S1B inthe supplemental material).

PPAR� target genes may be upregulated during fasting viainduction of PGC1�. If elevated plasma FFAs cannot ac-count for the induction of classical PPAR� activation duringfasting, the question arises what other mechanism may beresponsible. One possibility is increased coactivator expres-sion. The coactivator PPAR coactivator 1� (PGC1�) plays amajor role in the liver during fasting by upregulating genesinvolved in gluconeogenesis and fatty acid oxidation/keto-genesis, mediating activation by several transcription fac-tors, including PPAR� (47, 54, 64). In agreement with pre-

FIG. 3. PPAR�/� as alternative transcription factor to PPAR� in mouse liver. (A) Lpin2 and St3gal5 expression in livers of wild-type mice (n 5) treated with the PPAR�/� agonist GW501516 for 6 h or PPAR��/� mice (n 5) treated with the PPAR�/� agonist L165041 for 5 days. Errorbars represent standard errors of the means. (B) PPREs conserved between mouse and human were identified 1,291 bp and 23,333 bp downstreamof the TSS of the Lpin2 and St3gal5 genes. (C) HepG2 cells were transfected with a PPAR�/� expression vector and a simian virus 40 reportervector containing 201-nucleotide and 183-nucleotide fragments with the putative PPREs within the Lpin2 and St3gal5 genes, respectively. Thereporter vector (PPRE)3-TK-luciferase served as a positive control. Luciferase and �-galactosidase activities were determined 24 h after exposureof the cells to 1 �M GW501516. Error bars represent standard errors of the means. (D) Chromatin was extracted from livers of fed or 24-h-fastedwild-type and PPAR��/� mice (n 3 per group). ChIP was performed with antibodies against PPAR� and PPAR�/� on the TSS of Lpin2, St3gal5,Aldh3a2, Cpt2, and Rplp0. Rabbit IgG was used as a specificity control. Gray bars, fed state; black bars, 24-h fasted state. Error bars representstandard deviations. (E) Expression of Lpin2 and St3gal5 in livers of fed and 24-h-fasted wild-type and PPAR�/��/� mice (n 4 to 5 per group).Relative induction by fasting is indicated. Error bars represent standard errors of the means. (F and G) Plasma FFA levels in wild-type andPPAR��/� mice (F) or wild-type and PPAR�/��/� mice (G) sacrificed in a fed or 24-h-fasted state. Error bars represent standard errors of themeans. *, significantly different according to Student’s t test (P � 0.05).

6262 SANDERSON ET AL. MOL. CELL. BIOL.

vious data (64), expression of Pgc1a went up significantlyduring fasting (Fig. 6A). Importantly, fasting markedly en-hanced binding of PGC1� to the TSS of the PPAR� targetgenes Aldh3a2 and Cpt2, which was abolished in PPAR��/�

mice (Fig. 6B). No binding of PGC1� to Rplp0 was ob-served. These data suggest that upregulation of Pgc1�mRNA may contribute to induction of classical PPAR�target genes during fasting via increased PPAR�-dependentbinding of PGC1� to gene promoters.

DISCUSSION

It has been clearly established that PPAR� governs thefasting-induced upregulation of numerous genes involved inhepatic fatty acid oxidation, many of which are direct PPAR�target genes (19, 27, 34). However, it has remained unclearwhether elevated plasma FFAs themselves are responsible forthe induction of hepatic fatty acid catabolism via enhancedligand activation of PPAR� (19, 27, 34). Recently, using mice

FIG. 4. Angptl4 stimulates adipose tissue lipolysis. (A) Glycerol concentration in medium of 3T3-L1 cells treated for 30 min with isoproterenolor with concentrated conditioned medium of HEK293 cells transfected with mAngptl4. Control cells were treated with conditioned medium ofnontransfected HEK293 cells. Error bars represent standard errors of the means. *, significantly different according to Student’s t test (P � 0.05).(B) Increase in fatty acid and glycerol concentrations in medium of adipose tissue explants from transgenic mice overexpressing Angptl4 (Tg),wild-type (�/�), and homozygous knockout (�/�) mice. Values are corrected for weight of explants. (C and D) Plasma FFAs (C) and triglycerides(D) in transgenic mice overexpressing Angptl4 (Angptl4-Tg) and wild-type (Angptl4�/�), heterozygous (Angptl4�/�), and homozygous (Angptl4�/�)mice fed or in a 24-h-fasted state (n 5). Gray bars, fed state; black bars, 24-h-fasted state. Error bars represent standard errors of the means.Different letters indicate statistically significant differences (Student’s t test; P � 0.05). (E) Eosin and hematoxylin staining of epididymal adiposetissue.

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FIG. 5. Plasma FFAs do not activate hepatic PPAR�. Transgenic mice overexpressing Angptl4 (Angptl4-Tg) or wild-type (Angptl4�/�), heterozygous(Angptl4�/�), and homozygous knockout (Angptl4�/�) mice were sacrificed in the fed state or after a 24-h fast (n 5). Results show hepatic gene expressionof classical PPAR� targets Aldh3a2 and Cpt2 (A), Ppar� (B), and Ppar�/� (F). Gray bars, fed state; black bars, 24-h-fasted state. Error bars represent standarderrors of the means. *, P � 0.05. (C) Fatty acids activate PPAR�/� in a transactivation assay using a GAL4-LBDPPAR�/� fusion. Fatty acids were used at 125�M or 250 �M (normal or bold plus sign). (D) A nuclear receptor PamChipH assay was used to measure the interaction between PPAR�/� and immobilizedpeptides corresponding to specific coregulator-nuclear receptor binding regions in the presence and absence of fatty acids (125 �M). (E) Fatty acids (100 �M)upregulated expression of PPAR�/� target Adfp in rat FaO hepatoma cells. (G) Chromatin was extracted from livers of 24-h-fasted transgenic mice overex-pressing Angptl4 (Tg) and wild-type (�/�) and homozygous Angptl4 knockout (�/�) mice (n 3 per group). ChIP was performed with antibodies againstPPAR� and PPAR�/� on the TSS of Lpin2, St3gal5, and Rplp0. Rabbit IgG was used as a specificity control. Error bars represent standard deviations. Differentlowercase letters indicate statistically significant differences (Student’s t test; P � 0.05). (H) Hepatic gene expression of Lpin2 and St3gal5. Gray bars, fed state;black bars, 24-h-fasted state. Error bars represent standard errors of the means. Different lowercase letters indicate statistically significant differences (Student’st test; P � 0.05).

6264

with liver-specific inactivation of the Fasn gene, Chakravarthyet al. showed that unlike dietary fatty acids and de novo-synthesized fatty acids, circulating FFAs fail to activate hepaticPPAR� (3). In the present study, by using mice differentiallyexpressing Angptl4 we arrived at essentially the same conclu-sion.

Importantly, our data also suggest that in contrast to PPAR�,hepatic PPAR�/� can be activated by plasma FFA, which ac-counts for the plasma FFA- and fasting-dependent upregula-tion of several genes in wild-type and PPAR��/� mice, includ-ing Lpin2 and St3gal5. Indeed, we demonstrated that Lpin2and St3gal5 expression and binding of PPAR�/� to the Lpin2and St3gal5 promoter closely mirror plasma FFA levels. Therole of PPAR�/� in gene regulation by plasma FFA duringfasting was substantiated by the observation that induction ofLpin2 and St3gal5 by fasting is reduced in PPAR�/��/� mice.

In contrast to plasma FFAs, evidence abounds indicatingthat dietary fatty acids are able to activate PPAR� (40, 44, 46).Recently, it was shown that the effects of dietary fatty acids onhepatic gene expression are quantitatively almost entirely me-diated by PPAR� (48). Additionally, the present data suggestthat dietary fatty acids can also activate PPAR�/�, as inductionof Lpin2 and St3gal5 by dietary fat was entirely or partiallymaintained in PPAR��/� mice.

It may be argued that the lack of effect of declining plasmaFFAs on hepatic PPAR� activation may be because PPAR�,in contrast to PPAR�/�, is already saturated with fatty acids atlow plasma FFA levels, thus allowing no further activation.Previously, it has been shown that fatty acids bind to PPAR�/�with an about 5- to10-fold-lower affinity than to PPAR� (60).However, as treatment with synthetic agonists clearly results inmore pronounced PPAR� activation compared to fasting (44),the argument of PPAR� saturation is only tenable if we as-sume fatty acids act as partial agonists that do not elicit fullPPAR� activity compared to synthetic agonists. Saturation ofPPAR� is also not supported by the findings reported byChakravarthy et al. (3).

Alternatively, it is conceivable that fatty acids are present inhepatocytes in distinct pools which have different activitiestoward PPAR� and PPAR�/�. In this context, it should berealized that dietary fatty acids present in chylomicron rem-nants are internalized differently compared to plasma FFAs.Whereas the former are liberated after endosomal and lysoso-

mal degradation of cholesteryl esters and triglycerides, plasmaFFAs are likely internalized via diffusion as well as via specificfatty acid transport proteins, including CD36. The third con-tributor to the hepatic fatty acid pool is de novo lipogenesis, aprocess which occurs in the cytosol. It is presently unclear towhat extent these three sources of fatty acids undergo similarmetabolic fates. Recent studies support the existence of dis-tinct hepatic fatty acid pools that are differentially shuttled intovarious metabolic pathways, including oxidation and incorpo-ration into very-low-density lipoprotein (VLDL) triglycerides(67). For example, there is evidence that fatty acids generatedby de novo lipogenesis only marginally contribute to VLDLtriglycerides, in contrast to plasma FFAs (15). Our present andprevious data suggest that in terms of gene regulation, a similartype of segregation occurs between the three sources of fattyacids (3). The mechanism underlying the differential activity offatty acids from distinct pools toward PPAR� and PPAR�/�remains unknown. One could hypothesize a role for fatty acidbinding proteins (FABPs). It can be speculated that FABP1,which has been shown to interact with PPAR� (57), picks uplipoprotein-derived fatty acids and shuttles them to PPAR�,whereas another FABP expressed in liver such as FABP2 mayselectively bind free fatty acids coming from plasma and shuttlethem to PPAR�/�.

An important lingering question is that if plasma FFAs donot activate hepatic PPAR� during fasting, what mechanismaccounts for activation of PPAR�-dependent gene regulationduring fasting? Previously, a role for PGC1� in fasting-depen-dent upregulation of hepatic mitochondrial fatty acid oxidationand ketogenesis was shown (47, 54). Our ChIP analysis indi-cates enhanced recruitment of PGC1�, which itself is upregu-lated by fasting, to classical PPAR� target genes during fasting.Accordingly, activation of PPAR� by fasting may be driven bythe increase in PGC1� expression, although an important rolefor other coactivators cannot be excluded. Recently, it wasshown that PGC1� cooperates with BAF60a (SMARCD1) toactivate transcription of PPAR� target genes involved in per-oxisomal and mitochondrial fatty acid oxidation genes (35).

While our data suggest that PPAR�/� mediates the effects ofplasma FFAs on a small set of genes in the liver, the overallimportance of PPAR�/� in hepatic gene regulation by FFAsremains unclear. The same is true for the actual functionalrole of PPAR�/� in liver. Presently, combined transcriptom-

FIG. 6. PPAR� activation during fasting may be mediated by PGC1� upregulation. (A) Expression of Pgc1a in livers of fed or fasted wild-typemice (n 5). Fasting statistically significantly induced gene expression of Pgc1� (P � 0.05). Error bars represent standard errors of the means.(B) Chromatin was extracted from livers of fed and 24-h-fasted wild-type and PPAR��/� mice (n 3 per group). ChIP was performed withantibodies against PGC1� on the TSS of Aldh3a2 and Cpt2 and the negative control gene Rplp0. Rabbit IgG was used as a specificity control. Graybars, fed state; black bars, 24-h-fasted state. Error bars represent standard deviations. Fasting significantly induced binding of PGC1� in wild-typebut not PPAR��/� mice (P � 0.05).

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ics and metabolomics analyses of livers of PPAR��/� andPPAR�/��/� mice are under way to gain more understandingabout the role of PPAR�/� in the liver and to determine theextent to which PPAR� and PPAR�/� regulate distinct sets ofgenes and govern distinct metabolic pathways, especially underphysiological circumstances.

In this study we have used variable expression of the Angptl4gene to create variations in fasting plasma FFA levels in mice.Angptl4 is a potent inhibitor of lipoprotein lipase and hepaticlipase and decreases uptake of lipoprotein remnants by theliver, thereby decreasing hepatic uptake of dietary fatty acids(36). In addition, it stimulates adipose tissue lipolysis, as shownby the acute increase in plasma FFA upon injection of recom-binant Angptl4 (65) and by elevated plasma FFAs and glycerollevels in mice overexpressing Angptl4 (39). The prolipolyticeffect of Angptl4 is supported by recent data in humans (52). Inthe present paper, Angptl4 markedly induced glycerol releasefrom 3T3-L1 adipocytes. Activation of lipolysis by Angptl4 wasfurther substantiated by the altered release of fatty acids andglycerol from adipose tissue explants from Angptl4-Tg andAngptl4�/� mice, as well as by the lack of an increase in FFAduring fasting in Angptl4�/� mice. As a consequence, hepaticVLDL production is reduced in Angptl4�/� mice (7). By in-hibiting lipoprotein lipase and stimulating adipose tissue lipol-ysis, Angptl4 promotes switching of hepatic fatty acid uptakefrom remnant-derived fatty acids toward plasma FFAs (39).Importantly, the variations in plasma FFAs in the Angptl4mouse models are specifically elicited by fasting, permittingstudy of the impact of differential plasma FFAs on hepaticgene expression during fasting.

While in vivo and in vitro studies using synthetic PPAR�agonists are extremely relevant to assess the toxicological andpharmacological impacts and significance of PPAR�, it is un-clear to what extent they report on the physiological role ofPPAR� in liver. Our results reveal that several genes upregu-lated following pharmacological PPAR� activation are not in-duced by PPAR� under physiological conditions such as fast-ing, or are induced by fasting independently of PPAR� but aredependent on PPAR�/�. It is well known that in reporterassays PPAR� and PPAR�/� (and PPAR�) can activate thesame genes, suggesting that all PPARs share an intrinsic abilityto transactivate any given PPAR target gene. The present dataon Lpin2 and St3gal5 are consistent with the notion that in vivothe dominant receptor in the regulation of a particular PPARtarget is context dependent and importantly may differ be-tween pharmacological and physiological stimuli. Genes otherthan Lpin2 and St3gal5 that have been shown to be activated byboth PPAR� and PPAR�/� include Adfp (18), G0s2 (66), andPdk4 (5). Overall, the data imply that studies using high-affinitysynthetic PPAR agonists are not perfectly suited to assess thefunctions of PPARs during normal physiology.

ACKNOWLEDGMENTS

We thank Laeticia Lichtenstein, Janna van Diepen, Karin Mudde,Bianca Knoch, Shohreh Keshtkar, Jose van den Heuvel, Jenny Jansen,and Mechteld Grootte-Bromhaar for laboratory analysis. We alsothank Anja Koster (Eli Lilly) for the gift of Angptl4�/� animals.

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