Nutrient Sensing Nuclear Receptors Coordinate Autophagy

Autophagy is an evolutionally conserved catabolic process that recycles nutrients upon starvation and maintains cellular energy homeostasis1–3. Its acute regulation by nutrient sensing signaling pathways is well described, but its longer-term transcriptional regulation is not. The nuclear receptors PPARα and FXR are activated in the fasted or fed liver, respectively4,5. Here we show that both regulate hepatic autophagy. Pharmacologic activation of PPARα reverses the normal suppression of autophagy in the fed state, inducing autophagic lipid degradation, or lipophagy. This response is lost in PPARα knockout (PPARα−/−) mice, which are partially defective in the induction of autophagy by fasting. Pharmacologic activation of the bile acid receptor FXR strongly suppresses the induction of autophagy in the fasting state, and this response is absent in FXR knockout (FXR−/−) mice, which show a partial defect in suppression of hepatic autophagy in the fed state. PPARα and FXR compete for binding to shared sites in autophagic gene promoters, with opposite transcriptional outputs. These results reveal complementary, interlocking mechanisms for regulation of autophagy by nutrient status.

Overlapping networks govern both acute and longer-term responses to nutrients. In the fasted liver, glucagon induces both rapid glycogen breakdown and transcriptional activation of gluconeogenesis 6 . Nutrient deprivation also acutely regulates nutrient reclamation by autophagy [7][8][9] . Among several transcription factors that have been linked to the control of autophagy at the transcriptional level, activation of the basic Helix-Loop-Helix transcription factor EB (TFEB) by fasting is the best described 10 .
Nutrient sensing nuclear receptors are key integrators of metabolic responses. PPARα is activated by fatty acids in the fasted state, promoting fatty acid oxidation 4,11,12 . In the fed state, FXR is activated by bile acids returning to the liver along with nutrients, suppressing gluconeogenesis 13 . We hypothesized that PPARα and FXR would directly regulate autophagy, and initially tested the impact of their pharmacologic activation in the murine hepatocyte cell line AML12 expressing a triple mRFP-GFP-LC3 fusion protein [14][15][16] . In autophagosomes, the combination of both RFP and GFP in the triple fusion yields yellow fluorescence, whereas autolysosomal delivery results in red. As expected, the yellow fluorescence in normally cultured cells was converted to red by acute nutrient deprivation ( Fig. 1, Extended Data Fig. 1f). The FXR agonist GW4064 17 prevented this process in the nutrient deprived cells, while the PPARα agonist GW7647 18 mimicked it in non-starved cells (Fig. 1, Extended Data Fig. 1f). Extensive cell based studies of LC3-II levels and other indicators of autophagy confirmed that these responses are dose and time dependent, and indicated that they are not dependent on altered mTORC1 activity, but are associated with a decrease in the inhibitory phosphorylation of Ulk1 19 in response to PPARα activation, and an increase in response to FXR activation ( Fig. 1b-c, Extended Data Fig. 2a-d). Indeed, GW7647 induces autophagic flux even in non-starved cells, whereas GW4064 suppresses it even in starved, or torin1 20 treated cells (Extended Data Fig. 1a-e). Activation of FXR by bile acids also suppresses LC3-II induction (Extended Data Fig. 2c).
In livers of chow fed or fasted wild-type C57BL/6J (WT), LC3-II levels were increased in the fasted state ( In fed or fasted mice treated with the FXR agonist, induction of LC3-II protein in the fasted state was suppressed in WT, but not FXR −/− livers (Fig. 2b, Extended Data Fig. 3d). LC3-II levels were unaffected by GW4064 in fed and fasted FXR −/− mice 22 (Fig. 2c). At the mRNA level, GW4064 completely suppressed the induction of LC3b in the fasted state (Extended Data Fig. 3c). As expected, GW4064 treatment also suppressed Cyp7a1, but induced SHP expression in both the fasted and fed states in WT, but not FXR −/− mice (Extended Data Fig.  2g, 3c). autophagosome formation were increased in fasted GFP-LC3 Tg/+ mice (Fig. 2c). The PPARα agonist also significantly increased puncta in the fed LC3 Tg/+ mice, but not in the PPARα −/− double mutants (Fig. 2c, Extended Data Fig. 3e). In the opposite direction, the FXR agonist strongly suppressed induction of puncta in the fasted LC3 Tg/+ mice, but not in the fed state, and had no effect in the FXR −/− double mutants (Fig. 2c, Extended Data Fig.  3e). The induction of puncta by fasting was significantly lower in the PPARα −/− ; GFP-LC3 Tg/+ mutants, whereas their number in the fed state was significantly increased in the FXR −/− ; GFP-LC3 Tg/+ mutants (Fig. 2c, Extended Data Fig. 3e). These partially defective responses in the double mutants demonstrate that PPARα is required for the full induction of autophagy by fasting, and FXR is required for its full suppression by feeding.
In agonist or vehicle treated fed and fasted livers, quantitation of autophagic vesicles by transmission electron microscopy confirmed an increase in response to GW7647 in the fed state, and a strong decrease in response to GW4064 in the fasted state (Fig. 3a). Moreover, fasted livers of PPARα −/− mice show compromised formation of autophagic vesicles but increased numbers and size of lipid droplets. Conversely, livers of fed FXR −/− mice show enhanced formation of autophagic vesicles (Extended Data Fig. 4a, c). Autophagosomes induced by the PPARα agonist frequently contained lipid droplets, suggesting an increase in lipophagy consistent with the role of this receptor in fatty acid oxidation (Extended Data Fig. 4b). This was confirmed in AML12 cells treated with or without oleate to induce lipid droplet formation, and then either starved or not and also treated with vehicle, Wy-14,643 or GW4064. Visualization of LC3 (red) revealed colocalization with BODIPY 493/503 (green) labeled lipid droplets in the starved cells, as expected, and also in the non-fasted, Wy-14,643 treated cells (Fig. 3b, Extended Data Fig. 3f). In accord with a functional role for the induction of lipophagy, specific knockdown of either Atg5 or Atg7 significantly blunted the increase in fatty acid oxidation in response to Wy-14,643 in basal and oleate treated AML12 cells, as indicated by ketone body production (Extended Data Fig. 3g). Similarly, both fasting and GW7647 induced serum β-hydroxybutryate levels in control Atg7 F/F mice 24 , and both responses were decreased in liver specific Atg7 knockouts (Extended Data Fig. 3h).
Direct transcriptional effects are the most likely explanation for the impact of both PPARα and FXR on autophagy. Initial results confirmed that total LC3 protein levels are increased and decreased in response to fasting and refeeding, and mRNA expression of LC3a and LC3b and several other autophagic genes is increased in the fasted state and decreased in the refed state (Extended Data Fig. 2e, f). Among a core panel of 63 autophagy-related genes (Supp. Table 1), 13 were both induced by GW7647 and repressed by GW4064 in WT mice, with both responses lost in relevant knockout mice (Fig. 4a, Extended data Fig. 5c), 11 more were responsive only to GW7647, while 4 responded only to GW4064 (Extended Data Fig.  5a, b).
To define the basis for PPARα induction of autophagy-related genes, we determined the mouse liver PPARα cistrome with or without GW7647. The most significantly enriched binding motif among identified peaks was the known PPARα-RXR binding site (DR1), as expected, and nearly all of the peaks were absent in the PPARα −/− cistromes (Extended Data Fig. 6a, 7a, c). PPARα binding sites on core autophagy machinery gene loci as well as many regulatory and effector genes were confirmed by standard PPARα ChIP-qPCR analysis (Extended Data Fig. 6e, 7). Many of the peaks on these genes (Extended Data Fig.  7a), and a further panel of key autophagy components including Pink1, Optn, Vps11 and Becn1 ( Supplementary Fig. 1) were further enhanced by GW7647 treatment, demonstrating that PPARα agonist treatment not only generates new binding sites, but also further increases PPARα recruitment to specific target genes (Extended Data Fig 6d).
The PPARα cistrome shows that at least 124 of an extended 230 autophagy-related gene list have at least one receptor binding peak within 20 kb from the transcription start site, while a FXR 25 cistrome shows 61 potential targets (Extended Data Fig. 6d). A hypergeometric test confirmed that both are highly enriched relative to a random gene pool (Extended Data Fig.  6d). Intriguingly, approximately 27% of agonist induced PPARα binding peaks overlapped with 72% of agonist induced FXR binding peaks (Extended Data Fig. 6c). Consistent with this, peaks for PPARα and FXR directly overlap at 41 out of the 230 extended autophagic target genes, as exemplified by LC3a (Fig. 4b) and LC3b (Extended Data Fig. 8a). This was unexpected, based on their very different direct (PPARα -DR1) and inverted (FXR -IR1) repeat consensus binding sites. However, FXR has also been reported to bind DR1 sites in the ApoCIII 26 and ApoA 27 promoters, where it acts as a ligand-dependent transcriptional repressor, raising the prospect that it could repress autophagic gene expression via a similar mechanism.
Quantitative assessment confirmed that GW7646 treatment increased PPARα binding to peaks containing a single DR1 site in the LC3a and LC3b enhancers (Fig. 4c) and the ApoA1 promoter (Extended Data Fig. 8a, b), and increased RNA polymerase II recruitment to the genes. Thus, in the fed state, the increased PPARα binding was able to overcome the relatively silent chromatin state and decrease FXR occupancy for the LC3a and Acox1 DR1 sites. In the opposite direction, GW4064 did not have much impact on the already silent chromatin in the fed state, in accord with the functional studies (Figs. 2, 3). In the transcriptionally active fasted state, however, the FXR agonist significantly increased FXR binding, decreased RNA polymerase II recruitment, and also decreased PPARα binding to all 3 elements (Fig. 4c, Extended Data Fig. 8b). Overall, these results closely match the functional results and indicate that pharmacologic activation of each nutrient sensing nuclear receptor can overcome the chromatin state imposed by the opposite nutrient signal (fed for PPARα and fasted for FXR). They also indicate that PPARα and FXR compete for binding to single sites in some chromatin states.
To test this, we focused on the DR1 motifs in the regulatory region of the LC3a and LC3b genes. Cell-based reporter assays with promoter constructs including only the DR1 motifs confirmed the opposite responses, with the PPARα agonist further inducing, and the FXR agonist suppressing luciferase expression relative to the increased basal transactivation observed with both PPARα or FXR alone (Extended Data Fig. 8d). In the presence of both receptors, GW4064 suppressed both basal and Wy-14,643 induced expression. Similar results were also observed using a conventional PPAR response element reporter construct (3 × PPRE-Luc) (Extended Data Fig. 8c). Mutation of the LC3a or LC3b DR-1 elements blocked all responses to PPARα, as expected, but only diminished the responses to FXR (Extended Data Fig. 8d). The basis for this residual response, which cannot be correlated with direct FXR or FXR/RXR binding in vitro, is unclear. The ability of GW4064 to inhibit PPARα dependent transactivation of the LC3b DR1 motif is dependent on corepressors, since it was blocked by siRNA knockdown of either SMRT or SHP (Extended Data Fig. 8e). Consistent with these functional results, GW7647 treatment increased p300 coactivator recruitment to the DR1 region of LC3a and LC3b enhancers and increased histone H4 acetylation, an active chromatin mark. In contrast, GW4064 treatment increased NCoR and SMRT corepressor binding and the repressive H3K27trimethyl mark at the same DR1 regulatory region, particularly in the fasted state (Extended Data Fig. 9). Overall, we conclude that PPARα and FXR directly compete for binding to the same DR-1 elements at autophagic, and likely additional promoters, with opposite transcriptional outputs.
This mechanism is distinct from, but complementary to the inhibition of CRTC2 recruitment described in the accompanying manuscript. Thus, CRTC2 was not significantly recruited to the LC3a DR1 enhancer, as expected, but we confirmed the ability of GW4064 treatment to decrease CRTC2 occupancy to its proximal promoter site in the fasted state (Extended Data Fig. 10a).
These results define novel mechanisms for regulation of autophagy by nutrient sensing nuclear receptors (Extended Data Fig. 10b). Pharmacologic activation of either PPARα or FXR robustly impacts hepatic autophagy. GW7467 activates it even in the fed state, which is associated with an increase in lipophagy. GW4064 potently suppresses the process in the fasted state. The significantly blunted autophagic responses of PPARα and FXR knockouts to fasting or feeding, respectively, strongly confirm the physiologic relevance of the pharmacologically defined responses.
Both nuclear receptors directly induce or repress extensive panels of autophagy-related genes in accord with the overall responses. However, the impact of the synthetic agonists appears greater than the magnitude of the transcriptional responses, suggesting a role for less direct effects. We did not observe effects on mTOR, the dominant upstream regulator of autophagy. However, there were alterations in the inhibitory phosphorylation of Ulk1 S757 that would be consistent with such effects. In addition, induction of the transcriptional repressor SHP is a well-described mechanism for FXR dependent suppression of genes of bile acid biosynthesis 28,29 . However, preliminary results indicate that the suppressive effects of GW4064 are maintained in SHP −/− mice (data not shown).
These results reveal intersecting and complementary genomic circuits in which PPARα and FXR respectively induce and suppress hepatic autophagy (Extended Data Fig. 10b). They further integrate autophagy, which is often associated with more extreme nutrient stresses, with more physiologic nutrient responses. They also establish PPARα and FXR as potential targets for therapeutic modulation autophagy, which may be useful for liver or other metabolic tissues and could impact the pathogenesis of a wide range of human diseases.

Primary hepatocyte culture
8 to 10-week-old male GFP-LC3 Tg/+ mice were anesthetized with Avertin (Sigma Aldrich, # T48402, 240 mg/kg body weight) by intraperitoneal (i.p.) injection. The liver perfusion were done by injecting butterfly needle into the portal vein and providing the following solutions sequentially: 50 mL of EBSS (ATLANTA biologicals, # B31350) supplemented with 10 mM HEPES (Invitrogen, # 24020-117) and 0.5 mM EGTA and then 50 mL of HBSS supplemented with 100 U/mL of Collagenase IV (Invitrogen, # 17101-015), and 0.05 mg/mL of Trypsin inhibitor (Invitrogen, # 17075-029). The perfused liver was carefully taken out, put onto a petri dish, added 25 mL of hepatocyte wash media (Invitrogen # 17704-024), and massaged with two cell scrapers until the liver has become apart with only connective tissue left behind. Dissociated cells were passed through funnel with mesh into 50 mL of falcon tube. After centrifugation at 600 rpm for 2 min, cell pellet was resuspended in hepatocyte wash media, which were carefully overlaid percoll solution (final 40%) premixed with 150 mM of NaCl. After centrifugation at 600 rpm for 10 min, harvested cell pellet was washed twice with hepatocyte wash media, and then suspended in Williams' E medium (Invitrogen, # 12551-032) supplemented with 10% FBS, 1% penicillin/ streptomycin, ITS, and glutaMAX (Invitrogen, #35050-061). All drug treatments or starvation experiments were performed 4-6 h after the initial plating on the culture dishes.

BODIPY and immunofluorescence assay in cells
AML12 cells were plated on two-chamber Lab-tek II dishes at 30% to 50% confluent cells per chamber. The following day, cells were treated with Veh (0.1% DMSO), or 10 µM Wy, or 1 µM GW4064 in complete medium with or without 125 µM Oleate for 24 h. Veh or GW4064-treated cells were starved for 2 h in HBSS medium prior to fixation of cells. Cells were washed twice with PBS followed by fixation with 3% PFA for 20 min at room temperature. After fixation, cells were washed four times with PBS followed by incubation with blocking buffer (1.5 g glycine, 3 g BSA, and 2 ml 0.5% (w/v) saponin in 100 ml PBS) for 45 min. Cells were incubated with rabbit anti-LC3b antibody (1:400 dilution in antibody diluent: 100 mg BSA, 2 ml 0.5% saponin in 100 ml PBS) for overnight at 4°C. Cells were washed four times (10 min each) with PBS followed by incubation with the secondary antibody (Alexa fluor 594 goat anti-rabbit IgG, 1: 200 dilution in antibody diluent) and BODIPY 493/503 (1 µg/ml concentration) for 1 h at room temperature. After that, cells were washed four times with PBS followed by DAPI staining with mounting solution. Cell images were obtained using a confocal microscope (NIKON A1Rsi dual scanner). Orange and yellow dots were defined as a lipophagic vacuole, which was quantified at least in 30 cells per condition using ImageJ software.

RNA purification and qPCR analysis
Total RNA was isolated from snap-frozen liver tissues using Trizol Reagent (Invitrogen #15596-026) and prepared for the cDNA using QuantiTect reverse transcription kit (Qiagen # 205311). Hepatic gene expression was determined by qPCR using FastStart SYBR Green master (ROX) (Roche # 04673484001). mRNA levels were normalized by the 36B4 gene. qPCR primer information for autophagy-related genes was listed in Table S1 and all other primers were purchased from Qiagen.

Histology and immunofluorescence
Liver tissues were harvested and fixed in 4% PFA in PBS overnight at 4°C. The tissues were treated with 15% sucrose in PBS at 4°C for at least 4 hr and then with 30% sucrose in PBS at 4°C for overnight. Tissue samples were embedded in Tissue-Tek OCT (Sakura Finetek, # 4583) and stored at −70°C. The samples were sectioned at 5∼7 µm thickness, air-dried for 30 min, and stored at −20°C until use (Comparative Pathology Laboratory at Baylor College of Medicine). Images were taken on a confocal microscope (NIKON A1Rsi dual scanner). GFP-LC3 puncta were quantified in three independent visual fields from at least three independent mice using ImageJ software.

PPARα ChIP-seq
For ChIP, frozen liver tissues were ground in liquid nitrogen and cross-linked in 1% Formaldehyde for 20 min, followed by quenching with 1/20 volume of 2.5 M glycine solution, and washes twice with 1xPBS. Cell lysis and chromatin fragmentation were performed by sonication in ChIP dilution buffer (50 mM HEPES, 155 mM NaCl, 1.1% Triton X-100, 1% SDS, 0.11% sodium deoxycholate, 1 mM EDTA, and complete protease inhibitors tablet, pH 7.5). PPARα proteins were immunoprecipitated using PPARα antibody, cross-linking was reversed overnight at 65°C, and DNA was isolated using phenol/ chloroform/isoamyl alcohol. Precipitated DNA was analyzed by qPCR. ChIP was performed independently on liver samples from fed WT or PPARα −/− mice treated with vehicle or GW7647 (5 mg/kg, twice a day), and the precipitated DNA or input DNA samples were pooled. 10 ng of the pooled DNA was then amplified according to ChIP Sequencing Sample Preparation Guide provided by Illumina, using adaptor oligo and primers from Illumina, enzymes from New England Biolabs and PCR Purification Kit and MinElute Kit from Qiagen. Deep sequencing was performed by the Functional Genomics Core (J.Schug and K. Kaestner) of the Penn Diabetes Endocrinology Research Center using the Illumina Genome Analyzer IIx, and sequences were obtained using the Solexa Analysis Pipeline.

ChIP-seq data analysis
Solexa sequencing reads were mapped to reference genome mm9 using Bowtie 32 . Peak calling was performed with MACS14 using default settings 33 . Peak heights were normalized to the total number of uniquely mapped reads and displayed in UCSC genome browser 34 as the number of tags per 10 million tags. De novo motif analysis was performed with MEME 35 using 200 bp regions centered at the summit of each peak. To analyze the enrichment of autophagy-related genes near PPARα binding sites, All genes containing a PPARα binding site (with or without PPARα motif DR1) within 20 kb from the TSS were compared with a list of autophagy-related genes [36][37][38][39][40][41][42][43] (Table S1). P-values were calculated with hypergeometric test.

Transmission electron microscopy
Anesthetized mice were perfused with PBS for 3 min, followed immediately by perfusion with 2% PFA + 2.5% glutaraldehyde in 0.1 M Millonig's phosphate buffer (pH 7.4). Liver tissue was quickly removed and minced in a large drop of cold fixative, then transferred to vials of cold fix and fixed over night in 4°C. After washing three times in 0.1 M Millonig's phosphate buffer, the tissue was post-fixed at room temperature in 0.1 M Millonig's phosphate buffer containing 1% OsO4 for 1 h. Post-fixation was followed by 3 rinses, 5 min each, of 0.1 M Millonig's phosphate buffer, after which all tissues were dehydrated through a gradient series of ethanol, beginning with two 10 min changes of 50% ethanol and ending with three 20 min changes of 100% ethanol from a freshly opened bottle. Liver tissue was en bloc stained with saturated uranyl acetate in 50% ethanol for 1 h during the 50% ethanol dehydration stage. After complete dehydration, the tissue was infiltrated over a period of 2 days with progressively concentrated mixtures of plastic resin and 100% ethanol. Infiltration continued until the tissue reached pure resin. The tissue was then given 3 changes of pure, freshly made Spurr's plastic resin (Spurr, A.R., (1969) J. Ultrastruct. Res. 26, 31-42) for 3 h each, after which the liver tissue was embedded in 00 BEEM capsules (Glauert, A. M., ed: Practical Methods in Electron Microscopy. Ro. 143-144. North-Holland American Elsevier, 1975.) and placed in a 68°C oven overnight. Thin sections of approximately 70 nm were obtained using an RMC MT6000-XL ultramicrotome (RMC. Inc, AZ, USA) and a Diatome Ultra45 diamond knife (DiATOME, USA), and collected on 150 hex-mesh copper grids. The sections were counter-stained with Reynold's lead citrate (J. Cell Biol. 17:208-12, 1963) for 4 min. Dry samples were examined on a Hitachi H7500 transmission electron microscope (Hitachi, Japan) and images were captured using a Gatan US1000 digital camera (Gatan, 1995) with Digital Micrograph (Gatan, v1.82.366) software.

Molecular cloning and Cell-based luciferase reporter assay
The oligonucleotides encompassing 3 copies of DR1 motifs found in mouse LC3a or LC3b enhancer region were annealed and treated with Klenow. Purified DNAs were cloned into pTK-Luc plasmid by a serial digesting with SacI and BglII. Successful cloning was confirmed by DNA sequencing analysis. Oligonucleotide sequences used for luciferase reporter assays were listed in Table S3. For the luciferase assay, AML12 cells were cultured in 24 well plates. Transient transfections were performed using Lipofectamine 2000. Cell were transfected with 200 ng of reporter constructs, 100 ng of CMX, CMX-mouse PPARα, or CMX-human FXR, and 50 ng of CMV-β-galactosidase. pCDNA3.1 was added to prepare the total DNA to 500 ng per well. After 4 h transfection, cells were treated with or Veh (0.1% DMSO), 10 µM Wy, or 1 µM GW4064 in media containing charcoal-stripped serum for 20 h before performing luciferase and β-galactosidase assay. Luciferase activity was normalized with β-galactosidase activity. Normalized values from vehicle-treated cells transfected with empty CMX plasmid were set as fold 1. generate control littermates (Atg7 F/F ) and hepatocyte-specific Atg7 null (Alb-Cre/+; Atg7 F/F ) mice. 8-week-old male Atg7 F/F and Alb-Cre/+; Atg7 F/F mice were orally gavaged with either vehicle (DMSO in 4:1 of PEG-400 and Tween 80) or GW7647 (5 mg/kg body weight) twice a day (first injection: 12:00 am, second injection: 12:00 pm). Mice were fed ad libitum or fasted for 24 h from 6:00 PM (day 1) to 6:00 PM (day 2) and then scarified to collect blood and livers. Serum β-hydroxybutyrate concentrations were normalized with mouse liver weights.

Statistical analysis
Sample size for experiments was determined empirically based on preliminary experiments to ensure appropriate statistical power. Age, sex, and weight-matched mice were randomly assigned for the treatments. No animals were excluded from statistical analysis, and the investigators were not blinded in the studies. Values are expressed as mean ± s.e.m. and error bars (s.e.m.) for all results were derived from biological replicates rather than technical replicates. Significant differences between two groups were evaluated using a two-tailed, unpaired t-test, which was found to be appropriate since groups displayed a normal distribution and comparable variance. P value < 0.05 was considered statistically significant.
Quantification of autophagic flux shown in Fig. 1a. Numbers of autolysosomes (ALs) induced by 2 h starvation + Veh were set as 100%. Numbers of autolysosomes = RFP positive vesicles -GFP positive vesicles. 30 cells were counted per condition (**P < 0.01 vs No starvation + Veh). Data represent mean±s.e.m. Statistics by two-tailed t-test.   Fed or fasted WT mice were orally gavaged with Veh, GW7647, or GW4064 twice a day. Hepatic ChIP-qPCR analysis with indicated antibodies (p300, NCoR1, SMRT, acetyl-H4, and H3K27me3) was used to determine recruitments of coregulators and subsequent alterations of epigenetic marks induced by PPARα/FXR genomic competition for DR1 found in the enhancer region of LC3a and LC3b genes. (n=3 per group, *P < 0.05, **P < depicting transcriptionally activating or suppressive nutrient sensing NR, PPARα or FXR, respectively, which coordinates autophagy in liver. PPARα or FXR activation competes each other for binding to response elements found in autophagy-related genes.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.