Scholarly article on topic 'Liver PPARα is crucial for whole-body fatty acid homeostasis and is protective against NAFLD'

Liver PPARα is crucial for whole-body fatty acid homeostasis and is protective against NAFLD Academic research paper on "Biological sciences"

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Academic research paper on topic "Liver PPARα is crucial for whole-body fatty acid homeostasis and is protective against NAFLD"

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ORIGINAL ARTICLE

Liver PPARa is crucial for whole-body fatty acid homeostasis and is protective against NAFLD

Alexandra Montagner,1 Arnaud Polizzi,1 Edwin Fouché,1 Simon Ducheix,1 Yannick Lippi,1 Frédéric Lasserre,1 Valentin Barquissau,2,3 Marion Régnier,1 Céline Lukowicz,1 Fadila Benhamed,4,5,6 Alison Iroz,4,5,6 Justine Bertrand-Michel,2,3 Talal Al Saati,7 Patricia Cano,1 Laila Mselli-Lakhal,1 Gilles Mithieux,8 Fabienne Rajas,8 Sandrine Lagarrigue,9,10,11 Thierry Pineau,1 Nicolas Loiseau,1 Catherine Postic,4,5,6 Dominique Langin,2,3,12 Walter Wahli,1,13,14 Hervé Guillou1

► Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ gutjnl-2015-310798).

For numbered affiliations see end of article.

Correspondence to

Dr Hervé Guillou, INRA UMR1331, ToxAlim, Chemin de Tournefeuille, Toulouse 31027, France; herve.guillou@toulouse.inra.fr or

Prof. Walter Wahli Lee Kong Chian School of Medicine Nanyang Technological University The Academia, 20 College Road, Singapore 169856;

walter.wahli@ntu.edu.sg

Received 25 September 2015 Revised 28 December 2015 Accepted 4 January 2016 Published Online First 2 February 2016

► http://dx.doi.org/10.1136/ gutjnl-2016-31 1408

To cite: Montagner A, Polizzi A, Fouché E, et al. Gut 2016;65:1202-1214.

ABSTRACT

Objective Peroxisome proliferator-activated receptor a (PPARa) is a nuclear receptor expressed in tissues with high oxidative activity that plays a central role in metabolism. In this work, we investigated the effect of hepatocyte PPARa on non-alcoholic fatty liver disease (NAFLD).

Design We constructed a novel hepatocyte-specific PPARa knockout (Pparahep-/-) mouse model. Using this novel model, we performed transcriptomic analysis following fenofibrate treatment. Next, we investigated which physiological challenges impact on PPARa. Moreover, we measured the contribution of hepatocytic PPARa activity to whole-body metabolism and fibroblast growth factor 21 production during fasting. Finally, we determined the influence of hepatocyte-specific PPARa deficiency in different models of steatosis and during ageing.

Results Hepatocyte PPARa deletion impaired fatty acid catabolism, resulting in hepatic lipid accumulation during fasting and in two preclinical models of steatosis. Fasting mice showed acute PPARa-dependent hepatocyte activity during early night, with correspondingly increased circulating free fatty acids, which could be further stimulated by adipocyte lipolysis. Fasting led to mild hypoglycaemia and hypothermia in Pparahep-/- mice when compared with Ppara-/- mice implying a role of PPARa activity in non-hepatic tissues. In agreement with this observation, Ppara- - mice became overweight during ageing while Pparahep-/- remained lean. However, like Ppara-/- mice, Pparahep-/- fed a standard diet developed hepatic steatosis in ageing. Conclusions Altogether, these findings underscore the potential of hepatocyte PPARa as a drug target for NAFLD.

CrossMark

INTRODUCTION

Precise control of fatty acid metabolism is essential. Defective fatty acid homeostasis regulation may induce lipotoxic tissue damage, including hepatic steatosis.1 Peroxisome proliferator-activated receptors (PPARs) are transcription factors that serve as fatty acid receptors and help regulate gene expression in response to fatty acid-derived stimuli.2 PPARs act as ligand-activated receptors, controlling

Significance of this study

What is already known on this subject?

► Peroxisome proliferator-activated receptor a (PPARa) is a nuclear receptor expressed in many tissues and is responsible for several important metabolic controls, especially during fasting.

► PPARa is a target for the hypolipidemic drugs of the fibrate family.

► PPARa is less expressed in the liver of patients with non-alcoholic fatty liver diseases (NAFLD).

► Several PPAR-targeting molecules, including dual agonists, are currently under investigation for NAFLD treatment.

What are the new findings?

► Hepatocyte-restricted PPARa deletion impairs liver and whole-body fatty acid homeostasis.

► Hepatic PPARa responds to acute and chronic adipose tissue lipolysis.

► Hepatic PPARa regulates circadian fibroblast growth factor 21 (FGF21) and fasting-induced FGF21, and is partially responsible for the FGF21 increase in steatohepatitis.

► Hepatocyte-restricted PPARa deletion is sufficient to promote NAFLD and hypercholesterolaemia during ageing, but does not lead mice to become overweight.

How might it impact on clinical practice in

the foreseeable future?

► This work emphasises the relevance and potential of hepatic PPARa as a drug target for NAFLD.

target gene transcription. The three PPAR isotypes, PPARa, PPARp/S and PPARy, display specific tissue expression patterns and control different biological functions,3 but all bind lipids and control lipid homeostasis in different tissues, including the liver.2 A healthy liver does not accumulate lipids, but it plays central roles in fatty acid anabolism and export to peripheral organs, including white

adipose tissue for energy storage.4 During dietary restriction, hepatic fatty acid catabolism is also critical for using free fatty acids (FFAs) released from white adipose tissues. PPARa is the most abundant isotype in hepatocytes and is involved in many aspects of lipid metabolism,5 6 including fatty acid degradation, synthesis, transport, storage, lipoprotein metabolism and keto-genesis during fasting.7-9 In addition, PPARa controls glycerol use for gluconeogenesis9 as well as autophagy10 in response to fasting. Moreover, PPARa regulates the expression of the fibroblast growth factor 21 (FGF21) during starvation.11 12 In turn, FGF21 acts as an endocrine hormone targeting various functions including metabolic control.13 Finally, PPARa helps repress the acute-phase response and inflammation in the liver.14

Obesity can lead to organ and vascular complications.15 Non-alcoholic fatty liver disease (NAFLD), which are considered the hepatic manifestation of metabolic syndrome, range from benign steatosis to severe non-alcoholic steatohepatitis (NASH), potentially further damaging organs.16 Sustained elevation of neutral lipid accumulation (mostly triglycerides in hep-atocyte lipid droplets) initiates early pathological stages. Different fatty acid sources contribute to fatty liver development, including dietary lipid intake, de novo lipogenesis and adipose tissue lipolysis.4 In NAFLD, 60% of fatty acids accumulated in steatotic liver are adipose-derived.17

Preclinical18-21 and clinical22 studies highlight that PPARa influences NAFLD and NASH. Mice lacking PPARa develop steatosis during fasting,7 8 suggesting the importance of PPARa activity for using FFA released from adipocytes. However, PPARa is expressed and active in many tissues, including skeletal

muscles,23 adipose tissues,24 25 intestines,26 kidneys27 and

heart,28 which all contribute to fatty acid homeostasis. Therefore, it remains unknown whether the increased steatosis susceptibility in mice lacking PPARa depends on PPARa activity only in hepatocytes or also in other organs.

Here we investigated consequences of hepatocyte-specific Ppara deletion, focusing on effects on fatty acid metabolism in NAFLD, ranging from steatosis to steatohepatitis. We report the first evidence that adipocyte lipolysis correlates with and stimulates NAFLD when hepatocytes are lacking PPARa. Our data establish that hepatocyte-restricted Ppara deletion is sufficient to promote steatosis, emphasising this receptor's relevance as a drug target in NAFLD.

MATERIALS AND METHODS Animals

Generation of floxed-Ppara mice and of Ppara hepatocyte-specific knockout (Pparahep-/-) animals is described in online supplementary file 1.

In vivo experiments

In vivo studies followed the European Union guidelines for laboratory animal use and care, and were approved by an independent ethics committee.

Detailed experimental protocols are provided in online supplementary file 1.

Plasma analysis

Plasma FGF21 and insulin, respectively, were assayed using the rat/mouse FGF21 ELISA kit (EMD Millipore) and the ultrasensitive mouse insulin ELISA kit (Crystal Chem) following the manufacturer's instructions. Aspartate transaminase, alanine transaminase (ALT), total cholesterol, LDL cholesterol and HDL

cholesterol were determined using a COBAS-MIRA+ biochemical analyser (Anexplo facility).

Circulating glucose and ketone bodies

Blood glucose was measured using an Accu-Chek Go gluc-ometer (Roche Diagnostics). P-Hydroxybutyrate content was measured using Optium P-ketone test strips with Optium Xceed sensors (Abbott Diabetes Care).

Histology

Paraformaldehyde-fixed, paraffin-embedded liver tissue was sliced into 5 |j.m sections and H&E stained. Visualisation was performed using a Leica DFC300 camera.

Liver lipids analysis

Detailed experimental protocols are provided in online supplementary file 1.

Gene expression studies

Total RNA was extracted with TRIzol reagent (Invitrogen). Transcriptomic profiles were obtained using Agilent Whole Mouse Genome microarrays (4 x44k). Microarray data and experimental details are available in the Gene Expression Omnibus (GEO) database (accession number GSE73298 and GSE73299). For real-time quantitative PCR (qPCR), 2 mg RNA samples were reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Online supplementary file 2 presents the SYBR Green assay primers. Amplifications were performed using an ABI Prism 7300 Real-Time PCR System (Applied Biosystems). qPCR data were normalised to TATA-box-binding protein mRNA levels, and analysed with LinRegPCR.v2015.3.

Transcriptomic data analysis

Data were analysed using R (http://www.r-project.org). Microarray data were processed using Bioconductor packages (http://www.bioconductor.org, v 2.12)29 as described in GEO entry GSE26728. Further details are provided in online supplementary file 1.

Statistical analysis

Data were analysed using R (http://www.r-project.org). Microarray data were processed using bioconductor packages (http://www.bioconductor.org) as described in GEO entry GSE38083. Genes with a q value of <0.001 were considered differentially expressed between genotypes. Gene Ontology (GO) Biological Process enrichment was evaluated using conditional hypergeometric tests (GOstats package). For non-microarray data, differential effects were analysed by analysis of variance followed by Student's t-tests with a pooled variance estimate. A p value <0.05 was considered significant.

RESULTS

Generation of hepatocyte-specific PPARa knockout mice

Progeny carrying the Pparaflox/flox alleles (figure 1A), referred to as floxed, were backcrossed in the C57Bl/6J background, and then crossed with albumin-Cre mice in the same genetic background, generating a hepatocyte-specific PPARa knockout (Pparaflox/floxalbumin-Cre+/-) referred to as Pparahep-/-( figure 1B). PPARa mRNA was not detected in livers from Pparahep-/- mice when compared with floxed and C57Bl6/J mice (figure 1C), suggesting that most hepatic PPARa expression is from hepatocytes. PPARa absence in hepatocytes did not alter mRNA expression of other PPAR isotypes (figure 1C).

Figure 1 Characterisation of the hepatocyte-specific peroxisome proliferator-activated receptor a (PPARa) knockout mouse model. (A) Schematic of the targeting strategy to disrupt hepatic Ppara expression. (B) PCR analysis of Ppara floxed (Pparahep +/+) and Albumin-Cre (Albumin-Cre+/-) genes from mice that are liver wild-type (WT), (Pparahep+/+) or liver knockout (Pparahep-/-) for Ppara using DNA extracted from different organs. (C) Relative mRNA expression levels of Ppara, Pparp/8 and Ppary from liver samples of WT, liver WT (Pparahep+/+), Ppara liver knockout

(Pparahep-/-) and Ppara knockout

(Ppara-1-) mice (n=8 mice per group). Data represent mean±SEM. ***p<0.005. FA, floxed allele; Flp, flippase; FRT, flippase recognition target; LoxP, locus of X-overP1; nd, not detected; PparaA, Ppara deletion; WT, the Albumin-Cre-/- allele.

Hepatocyte-autonomous effect of fenofibrate on PPARa activity

To determine whether PPARa response was hepatocyte-autonomous, we challenged wild-type (WT), floxed Pparahep+'+, Ppara-- and Pparahep-/- mice with the PPARa agonist fenofibrate. We measured mRNA expressions of PPARa target genes, including Cyp4a10 (figure 2A) and Cyp4a14 (figure 2B). Their expressions were strongly induced by fenofi-brate in WT and in floxed

Pparahep+/+

mice compared with Ppara-/- and Pparahep-/- mice. These samples were also used for pangenomic expression profiling through microarray analysis (figure 2C). Differentially expressed gene (DEG) analysis was subjected to hierarchical clustering, highlighting similar expression profiles between WT and floxed Pparahep+/+ mice within fenofibrate-treated or vehicle-treated groups. Whole-body Ppara-/- and Pparahep-/- mice were unresponsive to fenofibrate, suggesting that fenofibrate-induced hepatic changes were mainly

due to autonomous hepatocyte responses, not secondary to extrahepatic PPARa activation. GO biological function analysis revealed that fenofibrate upregulated lipid metabolism, and repressed immune and defence response, metabolic responses, and glycosylation and glycoprotein metabolism (figure 2C,

groups 1, 2, 6 and 7). However, untreated Ppara-/- and

Pparahep-/-

mice showed marked differences (figure 2C, groups 3, 4, 8 and 9). This implies that the absence of extrahepatic PPARa has a significant impact on the liver transcriptional profile and underscores the relevance of Pparahep-/- mice to define the hepatocyte autonomous role of the receptor in the control of liver function.

Hepatocyte PPARa activity is context-specific

The Pparahep-/- model was used to determine whether PPARa could drive hepatic regulations both in fasting-induced fatty acid catabolism as well as fatty acid anabolism during refeeding. The

Figure 2 Pharmacological peroxisome proliferator-activated receptor a (PPARa) activation using fenofibrate reveals hepatocyte-specific PPARa-dependent biological functions. Liver samples from wild-type (WT), PPARa knockout (Ppara--), liver WT (Pparo^ep+/+) and Ppara hepatocyte knockout (Pparctep-/-) mice treated with fenofibrate (Feno, +) or vehicle (-) by oral gavage for 14 days were collected. (A and B) The relative gene expression of two specific PPARa target genes Cyp4a10 (A) and Cyp4a14 (B) was measured by qRT-PCR. Data represent mean±SEM. **p<0.01, ***p<0.005. (C) Heat map representing data from a microarray experiment performed with liver samples. Hierarchical clustering is also shown, which allows the definition of nine gene clusters. Gene Ontology (GO) analysis of each cluster revealed significant biological functions (p<0.05). nd, not detected.

fasting-refeeding experimental design was validated by measuring glycaemia (figure 3A) and expression of fatty acid synthase (Fasn), which encodes the rate-limiting enzyme in lipogenesis (figure 3B). Both were low during fasting, intermediary in ad libitum-fed animals, and high in refed animals. Cyp4a14 (a well-known PPARa target) expression was low or undetectable in Pparahep-/- animals, and strongly upregulated with fasting in WT mice (figure 3C).

Next we evaluated the hepatic transcriptome expression pattern using microarrays. We performed hierarchical clustering (figure 3D). Most PPARa-dependent changes were observed in fasted mouse livers. Venn diagrams were used to show nutritional status-related PPARa-dependent changes (figure 3E). Among the significant DEGs, 3048 were related to fasting, 390 to ad libitum-fed animals and 156 to refed mice, suggesting context-specific PPARa activity. The results further highlighted

Figure 3 Hepatocyte-specific peroxisome proliferator-activated receptor a (PPARa) function is dependent on nutritional status. Wild-type (WT) and PPARa liver knockout (Pparahep-/-) male 8-week-old mice were fed ad libitum, fasted for 24 h, or fasted for 24 h and refed for 24 h. All mice were killed at ZT14, and sera and livers were collected. (A) Quantification of circulating glucose levels. (B, C) Relative mRNA expressions of Fasn (B) and Cyp4a14 (C) in liver samples quantified by qRT-PCR. Data represent mean±SEM. *p<0.05, **p<0.01, ***p<0.005. (D) Heat map was performed based on average gene expression levels from WT (n=12 (6 WT and 6 Pparcihep+/+)) and from Pparahep-/- (n=6). (E) Venn diagram and associated Gene Ontology (GO) function analysis (p<0.05), GO categories corresponding to functions down in the absence of PPARa are in bold, GO categories corresponding to functions up in the absence of PPARa are in regular font.

that fasting, rather than feeding or refeeding, triggered the broader PPARa-dependent hepatocytic response, with most upregulated genes related to metabolism (figure 3E). However, the expression of several genes was identified as PPARa dependent regardless of the nutritional condition tested (fasting, but also feeding and refeeding). These genes are mostly downregu-lated in the absence of PPARa and pathway analysis highlights

their involvement in mitochondrial fatty acid catabolism (see online supplementary file 3).

Biological function analyses revealed that both transcriptional activation and repression were PPARa sensitive (figure 3E). The functions of PPARa-sensitive repressions (GO categories up in Pparahep-/- mice) varied with context, and included GO categories not directly related to metabolism, including acute-phase

response (fed), translation (refed) and protein glycosylation (fasted).

Hepatocyte PPARa is required for liver and whole-body fatty acid homeostasis in fasting

We next used Pparotep-/- mice to determine the contribution of hepatocyte PPARa, and compared it with Ppara-/- and WT mice. We measured FFA and p-hydroxybutyrate (ketonaemia) levels in fasted and non-fasted mice (figure 4A). Plasma FFA was elevated in fasting mice of all three genotypes, but was significantly higher in and Ppara ' mice compared with controls. Fasting strongly increased ketone body levels in WT mice and to a lesser degree in Pparahep-/-This suggests that hepatic PPARa is required for FFA disposal and for p-hydroxybutyrate production. Correspondingly, fasting Pparahep-/- and Ppara ' mice showed elevated hepatic triglycerides and cholesterol esters (figure 4B), and substantial centri-lobular steatosis (figure 4C), confirming that hepatic PPARa expression is required for fasting-induced FFA catabolism. PPARa absence led to defective expressions of PPARa target genes (figure 4D), including those involved in fatty acid catabolism and processing in lipid droplets (figure 4E). As a consequence of PPARa deficiency in hepatocytes, mice exhibit a distinct fasting-induced fatty acid profile with a significant increase in oleic acid (C18:1n-9) and linoleic acid (C18:2n-6) when compared with WT mice (see online supplementary file 4).

Hepatocyte-specific Ppara deletion impairs constitutive and fasting-induced FGF21 expression

FGF21 is a hepatokine mainly produced by the liver. We examined liver Fgf21 mRNA expression (figure 5A) and plasma FGF21 levels (figure 5B) in fed and fasted animals. We identified a constitutive expression peak during the day (ZT8) in both groups, and a fasting-triggered night-time peak (ZT16). In Pparahep-/- mice, we examined whether fasting-induced FGF21 expression/production was strictly dependent on PPARa hepatic activity. Ppara-1- and Pparctep-1- mice showed very low plasma FGF21 protein at ZT8 or at ZT16 with fasting (figure 5C).

Since FGF21 has been shown to reduce steatosis and lipotoxic lipids13 30 we questioned whether the absence of FGF21 determines fasting-induced steatosis observed in Pparahep-/- and Ppara-1- mice. FGF21 expression was rescued by adenoviral delivery both in and in Ppara ' mice (figure 5D).

Comparable expression of FGF21 (figure 5E) was obtained in liver of WT, Pparahep-/- and in Ppara ' mice. FGF21-sensitive genes such as G6pd and Scdl showed significantly different expression in response to FGF21 overexpression (figure 5E). However, FGF21 only reduced hepatic triglycerides and cholesterol esters in WT mice, but not in

mice (figure 5F, G). These results indicate that the fasting-induced steatosis occurring in Pparctep-/- and in Ppara-1 - mice does not depend on the lack of FGF21. This is in line with our observations that FGF21- and PPARa-sensitive target genes are different (see online supplementary file 5A). Moreover, it is also consistent with the observation that FGF21 overexpression does not rescue the expression of PPARa target genes and conversely that PPARa-sensitive regulations occur in Fgf21-1- mice (see online supplementary file 5B, C).

In addition to their defective fatty acid catabolism, Ppara--mice are hypoglycaemic and hypothermic during fasting.7 Because FGF21 is important for glucose homeostasis and for thermogenesis,13 we investigated the role of hepatocyte PPARa in controlling fasting glycaemia and body temperature. Both

Pparahep-/- and Ppara ' mice were hypoglycaemic and hypothermic compared with WT mice during fasting. However, this phenotype was much stronger in fasted Ppara-/- mice compared with fasted Pparotep-/- mice (figure 5H-J), indicating that extra-hepatic PPARa strongly influenced whole-body glucose homeo-stasis and temperature independent of hepatocytic PPARa activity and FGF21 production.

Fasting-enhanced hepatocytic PPARa activity is time-restricted and sensitive to adipocyte lipolysis

We next tested the kinetics of other fasting-induced hepatic PPARa activity in vivo. We used several measures of PPARa activity, including Fgf21 (figure 5A) and Vaninl, Cyp4a10, Cyp4a14 and Fsp27 mRNAs (figure 6A), since these genes were most sensitive to fasting and to fenofibrate, and were strictly PPARa dependent (see online supplementary files 6-10A). Plasma FFA and glucose levels were also measured during fasting (figure 6B). FFA were markedly increased in the early night (ZT14-ZT16). The FFA pattern was correlated with the PPARa mRNA expression profile and expressions of Fgf21, Vaninl, Cyp4a10, Cyp4a14 and Fsp27 (figures 5A and 6A). This strongly suggested that FFA released from adipocytes during fasting-influenced hepatic PPARa expression and activity without inflammatory response since hepatic Tnfa mRNA expression was not sensitive to fasting. We further determined that acute treatment of fasted mice with the p3-adrenergic receptor agonist CL316243 enhanced circulating FFA levels in WTand Pparahep-/- mice (figure 6C), and increased expressions of Fgf21, Cyp4a14, Vanin1, Cyp4a10 and Fsp27 in WT mice but not

Pparahep-/-

mice (figure 6D) without inducing Tnf a in response to fasting or in response to CL316243 (see online supplementary file 10C and D). These data support a role for acute adipocyte lipolysis as a signal for hepatocyte PPARa activity during fasting.

Hepatocyte PPARa is required for protection in steatohepatitis

We next examined whether the hepatocytic PPARa response to chronic lipolysis occurred during methionine-deficient and choline-deficient diet (MCD)-induced weight loss. In rodents, this diet rapidly promotes lipolysis in adipocytes, resulting in steatohepatitis. On the MCD diet, mice of each genotype showed weight loss (figure 7A), steatosis (figure 7B), and increased hepatic triglycerides, cholesterol esters (figure 7C) and plasma ALT (figure 7D). Compared with WT, Pparahep-/- and Ppara-/- mice showed greater steatosis and liver damage, suggesting a more severe MCD diet-induced phenotype without hepatocyte PPARa. MCD also induced increased expressions of Cyp4a14 and Vanin1 in WT mice, but not

Pparahep-/-

Ppara-1- mice (figure 7E). Fgf21 mRNA (figure 7E) and circulating FGF21 (figure 7F) were increased through a mechanism that is partly dependent on hepatic PPARa. Overall, hepatocyte-specific Ppara deletion aggravated MCD diet-induced liver damage, correlating with defective PPARa-dependent pathway upregulation in response to chronic lipolysis.

Additionally, we questioned whether hepatocyte PPARa may also be required for the protection of the liver during early hits in steatosis such as those occurring in response to short-term exposure to a high-fat diet (HFD). Over 2 weeks of HFD, mouse liver accumulated hepatic triglycerides and cholesterol esters. Importantly, this steatosis was twice higher in

Pparahep-/-

mice than in WT mice, and was further elevated in Ppara-/-mice (see online supplementary file 11). Altogether, these data suggest that hepatic PPARa is essential in hepatoprotection.

Figure 4 Fasting is the major inducer of hepatic peroxisome proliferator-activated receptor a (PPARa) activity. Wild-type (WT), hepatocyte-specific PPARa knockout (Pparahep- ) and total PPARa knockout (Ppara-/-) mice were fed ad libitum or fasted for 24 h and then killed. (A) Quantification of plasma free fatty acids (FFAs) and ketone bodies (ketonaemia). (B) Hepatic triglycerides and cholesterol esters hepatic levels. (C) Representative pictures of H&E staining of liver sections. Scale bars, 100 mm. (D) Relative mRNA expression levels of Ppara, Cyp4a14 and Vnn1 in liver samples determined by qRT-PCR. (E) Quantification of mRNA expression of Acox1, Hmgcs2, Acadl, Fsp27 and Plin5 by qRT-PCR. Data shown as mean±SEM. *p<0.05, **p<0.01, ***p<0.005.

Hepatocyte PPARa deficiency leads to steatosis and hypercholesterolaemia but not excess weight gain in ageing mice

Lastly, we questioned the long-term consequences of hepatocyte-specific Ppara deletion during ageing. More specifically, since PPARa is broadly expressed in metabolic tissues, we aimed at clarifying whether the steatosis that develops in aged whole-body Ppara-/- mice is due to the hepatocytic defect in PPARa activity. WT, Pparahep-/- and Ppara-/- mice were fed a standard diet over 1 year. Ppara-/- mice, but not

Pparahep-/-

mice, grew overweight with ageing (figure 8A-C). Both Pparahep -/- and Ppara-/- mice showed spontaneous centrilobular stea-tosis (figure 8D), elevated hepatic triglycerides and hepatic cholesterol esters (figure 8E), as well as hypercholesterolaemia (see figure 8F online supplementary file 12) without hyperglycaemia (figure 8G). Overall, hepatocyte-specific PPARa deficiency was sufficient to induce spontaneous steatosis and disrupt whole-body fatty acid as well as cholesterol homeostasis, but did not affect weight gain and diabetes during ageing.

DISCUSSION

NAFLD are a spectrum of diseases presenting a major public health concern that is strongly linked with obesity. Most accumulated hepatic fatty acids in NAFLD come from increased non-esterified FFA in the fasting state.17 Thus, it is essential to

define the mechanisms by which the liver adapts to this influx. FFA processing largely involves the fatty acid oxidative pathway, coupled to ketogenesis allowing the liver to use lipids,31 which is critical during fasting and requires transcriptional regulation of rate-limiting enzymes.32

Whole-body Ppara-1- mice show impaired coping with prolonged fasting, resulting in defective fatty acid oxidation and steatosis, hypoglycaemia and hypothermia. However, PPARa also contributes to metabolic homeostasis through expression in other tissues. Here we investigated the impact of hepatocyte-specific PPARa deletion on liver physiology and lipid metabolism in vivo. To our knowledge, this is the first report that selective PPARa deletion in hepatocytes (Pparahep-/-) was sufficient to promote hepatic steatosis.

PPARa is targeted by several fibrate drugs,33 and by pan-agonists for PPAR isotypes21 that are currently in clinical trials for NASH treatment. Using Pparahep-/- mice, we demonstrated an autonomous transcriptional response of hepatocytes to fenofibrate, indicating that fibrates' effects on the liver gene expression are largely independent from those in extrahepatic tissues. Moreover, liver gene expression profiles markedly differed between untreated Ppara-/- and Pparahep-/- mice, suggesting that extrahepatocytic PPARa activity substantially influenced the hepatic transcriptome.

Food restriction induces PPARa activity, and endogenous PPARa ligand production requires hepatic lipogenesis, which

Figure 5 Hepatocyte and extrahepatocyte peroxisome proliferator-activated receptor a (PPARa) regulate fibroblast growth factor 21 (FGF21), glycaemia and body temperature during fasting. (A and B) Eleven-week-old male mice of the C57Bl/6J background were fed ad libitum or fasted for 24 h, and were killed around the clock from ZT0 to ZT24. (A) Fgf21 mRNA was quantified by qRT-PCR. (B) Quantification of circulating FGF21 levels by ELISA. (C) Twelve-week-old wild-type (WT), PPARa-hepatocyte knockout (Pparctep-/-) and PPARa knockout (Ppara-/-) male mice were fed ad libitum or fasted for 16 h and blood was collected at ZT8 (ZT8 fed) or at ZT16 (ZT16 fasted). FGF21 plasma level was determined by ELISA. (D-G) Male mice of WT, Ppardhep-/- and Ppara-/- genotypes were infected with an adenoviral construct containing cDNA of Fgf21 or an empty vector. Mice were sacrificed after a 24 h fasting period at ZT14. (D) Quantification of circulating FGF21 levels by ELISA. (E) Fgf21, G6pd and Scd1 mRNAs were quantified by qRT-PCR. (F) Quantification of hepatic cholesterol esters and triglycerides. (G) Representative pictures of H&E staining of liver sections. Scale bars, 100 mm. (H) Plasma glucose level was monitored over a 24 h fasting period from ZT0 to ZT24 in WT, Ppard'ep-/- and Ppara-/-mice. (I, J) Plasma glucose (I) and body temperature (J) were determined at ZT0 in fed mice or at ZT0 in mice fasted for 24 h. Data are shown as mean±SEM. *p<0.05, **p<0.01, ***p<0.005.

Figure 6 Hepatocyte peroxisome proliferator-activated receptor a (PPARa) activity is induced by adipose tissue lipolysis. (A and B) Liver samples were collected from male wild-type (WT) C57Bl/6J mice that were fed ad libitum (black curve) or fasted (blue curve) over 24 h. (A) Hepatic mRNA expression levels of Ppara, Cyp4a14, Vnn1, Cyp4a10, Fsp27 and Tnfa were quantified by qRT-PCR. (B) Plasma glucose and free fatty acids (FFA) were measured. (C and D) WT and PPARa hepatocyte-specific knockout (Ppard'ep-/-) mice were treated with the p3-adrenergic receptor agonist CL316243 at ZT6 and then killed at ZT14. (C) Quantification of plasma FFA. (D) Relative mRNA expression levels of Fgf21, Cyp4a14, Vnn1, Cyp4a10 and Fsp27 were measured by qRT-PCR. Data are shown as mean±SEM. *p<0.05, **p<0.01, ***p<0.005.

increases upon feeding.34 35 Thus, PPARa may be important during fasting-induced lipid catabolism and in the response to anabolic fatty acid-derived signals. Our data revealed the context dependency of PPARa hepatocytic activity defined by DEGs. This activity was clearly the highest during fasting.

During fasting, hepatocyte-specific PPARa deletion resulted in steatosis, increased plasma FFA and impaired ketone bodies. This supports the concept that FFA released from adipose stores during fasting may activate PPARa for hepatic use. Accordingly, we found that

Pparahep-/-

mice accumulate high oleic and

Figure 7 Liver peroxisome proliferator-activated receptor a (PPARa) deficiency aggravates non-alcoholic steatohepatitis in response to a methionine-deficient and choline-deficient diet (MCD). Wild-type (WT), PPARa hepatocyte knockout (Pparahep-/-) and PPARa knockout (Ppara-/-) mice were fed a MCD or a control diet for 2 weeks and were killed at ZT8. (A) Body weight gain was measured over 2 weeks. (B) Representative pictures of H&E staining on liver sections. Scale bar, 100 mm. (C) Quantification of hepatic triglycerides and cholesterol esters. (D) Alanine transaminase activity level in plasma. (E) Hepatic mRNA expression levels of Cyp4a14, Vnn1 and Fgf21. (F) Plasma levels of fibroblast growth factor 21 (FGF21). Data are shown as mean±SEM. *p<0.05, **p<0.01, ***p<0.005.

linolenic acids in the liver during fasting (see online supplementary file 4), which is in agreement with the fact that both of them are the main fatty acids stored in the white adipose tissues of mice fed a chow diet.36 Importantly, we found a high correlation between the kinetics of circulating FFA increase and expression of PPARa and several of its target genes. Moreover, treatment with a p3-adrenergic receptor agonist further enhanced this response in vivo through PPARa but did not induce detrimental FFA-sensitive response driven by toll-like receptor 4 (TLR4). This is likely due to the mixture of FFA released from the adipose stores. Indeed, fatty acids that accumulated in the liver of

Pparahep-/-

mice during fasting were mostly oleic (C18:1n-9) and linoleic acids (C18:2n-6), and not only saturated fatty acids such as palmitic acid (C16:0). Interestingly, it has been shown that palmitic acid cannot activate TLR4 in the presence of unsaturated FFA.37

Overall, our data highlight hepatic PPARa activity regulation by fatty acids released from adipocytes. This contrasts with the previous evidence that PPARp/5 rather than PPARa may act as a FFA sensor.38 However, our data support the possibility that this adipose-derived signal is time-restricted and specifically efficient in early night. Moreover, other pathways likely influence PPARa activity by providing ligands.34 35 39 40 Several insulinsensitive signalling mechanisms influence hepatic PPARa, and adipocyte lipolysis is insulin sensitive.41 Thus, insulin may coordinate hepatic PPARa, both through cell-autonomous mechanisms and adipocyte lipolysis inducing interorgan communication mediated by FFA release. Our findings also correspond with the recent evidence that adipocyte lipolysis may regulate hepatic Fgf21.42 Circulating FGF21 was strictly dependent on hepatocytic PPARa activation during fasting. Most circulating FGF21 is liver-derived43 and Ppara-1- mice

A Body weight ß Body weight Q

E Hepatic triglycerides Hepaticcholesterolesters F Plasma cholesterol TotalHDL TotalLDL G Glycaemia

Figure 8 Mice deficient in hepatic peroxisome proliferator-activated receptor a (PPARa) develop spontaneous hepatic steatosis during ageing. Wild-type (WT), PPARa hepatocyte knockout (Pparahhp-/-) and PPARa knockout (Ppara-'-) mice were fed a chow diet for 51 weeks. All mice were killed at ZT16 in a non-fasted state. (A) Body weight gain was followed over time. (B) Comparison of body weight between weeks 11 and 50. (C) Representative pictures of 52-week-old mice of the three genotypes. (D) Representative images of H&E staining of liver sections. Scale bar, 100 mm. (E) Quantification of hepatic triglycerides and cholesterol esters. (F) Measurement of plasma total cholesterol, HDL cholesterol and LDL cholesterol. (G) Fasting glycaemia. Data are shown as mean±SEM. *p<0.05, **p<0.01, ***p<0.005.

show very little FGF21.11 12 Other transcription factors can also regulate hepatic Fgf21 expression44-48 and PPARa is also expressed in extrahepatic tissues. Our findings in Pparahep-/-mice showed very little FGF21 without hepatic PPARa in both fed and fasted states. Ppara-/- mice are hypoglycaemic and hypothermic during fasting7 and FGF21 is known for its endocrine effect on glucose homeostasis and thermogenesis.13 However, compared with fasted Ppara-/- mice, fasted Pparahep-1- mice showed reduced hypoglycaemia and hypothermia while FGF21 was equally absent in both models. This indicates that extrahepatocytic PPARa strongly influenced whole-body glucose homeostasis and temperature independently of hepatocyte PPARa and FGF21 production during fasting. In addition, while FGF21 prevents steatosis in different mouse models13 30 and FGF21 reduces hepatic lipids in WT mice, its overexpression is not sufficient to protect from lipid accumulation in Pparahep-/- and in Ppara-/- mice. Therefore, the absence of FGF21 is not the primary cause for the steatosis observed in Pparahep-/- mice.

Lack of hepatic PPARa impairs the liver's ability to use FFA from acute lipolysis, resulting in steatosis. MCD diet-induced weight loss49 50 also correlated with hepatic PPARa activity, suggesting that chronic lipolysis elevates hepatocytic PPARa activity in non-fasted mice. In agreement with the findings in whole-body PPARa-deficient mice,20 our data demonstrated that the absence of hepatocytic PPARa was sufficient to increase MCD diet-induced liver damage. FGF21 expression/circulating levels

increased in steatohepatitis, supporting the possibility that elevated FGF21 may reflect liver stress without fasting. This MCD diet-induced FGF21 increase was not strictly PPARa-dependent, consistent with the findings that amino acid deprivation induces hepatic FGF21 expression presence led

to greater FGF21 increase, and may contribute to hepatoprotec-tion from lipotoxic lipid accumulation.30

MCD diet is widely used for preclinical NASH studies. However, it has many limitations, including the important weight loss that occurs in mice fed such diet. Therefore, we also tested the role of hepatocyte PPARa in lipid homeostasis in response to a short-term HFD feeding, which is sufficient to initiate early neutral lipid accumulation that may promote NAFLD. Pparahep-/- mice showed marked increase in hepatic steatosis in response to 2 weeks of HFD feeding (see online supplementary file 11) suggesting that hepatocyte PPARa plays a dual role in exogenous (dietary) as well as in endogenous (released from adipocyte lipolysis) fatty acid homeostasis.

Previous studies have shown that Ppara-/- mice show a significant alteration of systemic lipid metabolism that leads to hepatic steatosis in ageing mice. Since PPARa is active in skeletal muscles,23 adipose tissues,24 25 intestines,26 kidneys27 and heart,28 which all contribute to fatty acid homeostasis, it is impossible to determine whether the spontaneous steatosis that occurs in ageing Ppara-/- mice originates from a defect in the hepatocytic PPARa activity. This led us to investigate

ageing-related differences between Ppara-/- and Pparahep-/-

Figure 9 Overview of hepatocyte-specific peroxisome proliferator-activated receptor a (PPARa)-regulated genes involved in fatty acid metabolism. This figure was designed based on transcriptome analysis of PPARa-dependent gene expression in hepatocytes. Genes listed in regular font are induced by fenofibrate and by fasting in wild-type (WT) but not in Pparahep-/- mice. Genes in italics are repressed by fenofibrate and by fasting in WT but not in Pparahep- mice. Genes referenced in bold are downregulated in PparOhep-/- compared with WT mice, whatever the conditions.

mice. During ageing, Ppara - mice became overweight and developed steatosis, mice only suffered stea-

tosis. Therefore, neither obesity nor hyperglycaemia, which are both known to promote NAFLD,15 16 is responsible for the steatosis observed in mice with hepatocyte-specific PPARa deletion.

Furthermore, both Ppara--- and Pparctep--- ageing mice were hypercholesterolaemic. This is likely due to the dysregulation of apolipoproteins gene expression as well as cholesterol transport (Abcg8) as revealed in microarray analysis (see online supplementary file 12A). It is also possible that the cholesterol biosynthesis pathway driven by SREBP-2 may be dysregulated in the absence of PPARa since some of the SREBP-2 genes are elevated in Ppara- - and/or in Pparahep-/- mice (see online supplementary file 12B). Therefore, this suggests that drugs that activate hepatocytic PPARa will likely influence whole-body fatty acid and cholesterol homeostasis.

Altogether, our extensive analysis performed in Pparahep-/-mice has allowed us to extend the evidence for the central role of PPARa in hepatocyte fatty acid homeostasis (figure 9). PPARa is strikingly essential to many aspects of fatty acid homeostasis including degradation through oxidative pathways. Our work provides the first demonstration that hepatocyte-specific PPARa deletion impairs whole-body fatty acid homeo-stasis during fasting, MCD and HFD feeding as well as in ageing. These findings underscore the central role of PPARa in the clearance of dietary fatty acids and of FFA released from adipocytes, the major source of lipid accumulation in NAFLD. These data highlight the relevance of PPARa as a drug target for NAFLD treatment.

Author affiliations

1INRA UMR1331, ToxAlim, University of Toulouse, Toulouse, France

2INSERM UMR 1048, Institute of Metabolic and Cardiovascular Diseases, Toulouse,

France

3University of Toulouse, UMR1048, Paul Sabatier University, France 4INSERM U1016, Cochin Institute, Paris, France 5CNRS UMR 8104, Paris, France

6University of Paris Descartes, Sorbonne Paris Cité, Paris, France

7INSERM/UPS-US006/CREFRE, Service d'Histopathologie, CHU Purpan, Toulouse, France

8INSERM U855, University of Lyon, Lyon, France 9INRA UMR1348 Pegase, Saint-Gilles, France 10Agrocampus Ouest, UMR1348 Pegase, Rennes, France "Université Européenne de Bretagne, France

"Laboratory of Clinical Biochemistry, Toulouse University Hospitals, Toulouse, France 13Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

14Center for Integrative Genomics, University of Lausanne, Genopode Building, Lausanne, Switzerland

Acknowledgements We thank all members of the EZOP staff, particularly Colette Bétoulières for her careful and outstanding help from the early start of this project. We thank Aurore Dequesnes and Laurent Monbrun for their excellent work on plasma biochemistry. We thank Christine Salon and Florence Capilla for their excellent work on histology. We thank the staff from the Genotoul: Anexplo, Get-TriX and Metatoul-Lipidomic facilities. The authors wish to thank Professor Daniel Metzger, Professor Pierre Chambon (IGBMC, Illkirch, France) and the staff of the Mouse Clinical Institute (Illkirch, France) for their critical support in this project. We thank Professor Didier Trono (EPFL, Lausanne, Switzerland) for providing us with the Albumin-cre mice. We thank Professor David Mangelsdorf (Howard Hughes Medical Institute, Dallas, TX) and Professor Steven Kliewer (UT Southwestern, Dallas, TX) for providing us with the FGF21-deficient mice. We thank Alice Marmugi and Géraldine Michel for their technical assistance. We thank Professor Bertrand Cariou and Professor Bart Staels for constructive discussions.

Contributors AM initiated the project, designed experiments, performed experiments, analysed the data and wrote the paper. AP, EF, SD, YL, FL, MR, CL, FB and AI contributed to design experiments, perform experiments and to analyse the data. VB designed and performed a critical experiment. JB-M, TAS, PC and LL provided critical analysis and technical support. SL contributed to analyse the data. GM, FR and TP provided critical materials and contributed to design the project. NL, CP and DL critically contributed to design the project and supervised experiments. WW provided critical reagents, designed the project, analysed the data and wrote the paper. HG designed the project, performed experiments, analysed the data and wrote the paper.

Funding This work was funded by grants from the Human Frontier Science Program (HFSP) (WW), by Start-Up Grants from the Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore (to WW), by SFN (to HG), by ANRs 'Crisalis' (to CP and HG) by 'Obelip' (to DL, CP, AM and HG). DL is a member of the Institut Universitaire de France. AM, DL, WW and HG were supported by Région Midi-Pyrénées.

Competing interests None declared.

Provenance and peer review Not commissioned; externally peer reviewed.

Data sharing statement Gene expression array raw data are deposited in GEO as indicated in the manuscript.

Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/ licenses/by-nc/4.0/

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