Scholarly article on topic 'Molecular mechanism of PPARα action and its impact on lipid metabolism, inflammation and fibrosis in non-alcoholic fatty liver disease'

Molecular mechanism of PPARα action and its impact on lipid metabolism, inflammation and fibrosis in non-alcoholic fatty liver disease Academic research paper on "Biological sciences"

CC BY-NC-ND
0
0
Share paper
Academic journal
Journal of Hepatology
OECD Field of science
Keywords
{PPARα / "Fatty acid oxidation" / Inflammation / Transrepression / Liver / Steatosis / NAFLD / NASH / Fibrosis}

Abstract of research paper on Biological sciences, author of scientific article — Michal Pawlak, Philippe Lefebvre, Bart Staels

Summary Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor belonging, together with PPARγ and PPARβ/δ, to the NR1C nuclear receptor subfamily. Many PPARα target genes are involved in fatty acid metabolism in tissues with high oxidative rates such as muscle, heart and liver. PPARα activation, in combination with PPARβ/δ agonism, improves steatosis, inflammation and fibrosis in pre-clinical models of non-alcoholic fatty liver disease, identifying a new potential therapeutic area. In this review, we discuss the transcriptional activation and repression mechanisms by PPARα, the spectrum of target genes and chromatin-binding maps from recent genome-wide studies, paying particular attention to PPARα-regulation of hepatic fatty acid and plasma lipoprotein metabolism during nutritional transition, and of the inflammatory response. The role of PPARα, together with other PPARs, in non-alcoholic steatohepatitis will be discussed in light of available pre-clinical and clinical data.

Academic research paper on topic "Molecular mechanism of PPARα action and its impact on lipid metabolism, inflammation and fibrosis in non-alcoholic fatty liver disease"

Review

°°EASL

JOURNAL OF HEPATOLOGY

CrossMark

Molecular mechanism of PPARa action and its impact on lipid metabolism, inflammation and fibrosis in non-alcoholic

fatty liver disease

Michal Pawlak, Philippe Lefebvre, Bart Staels*

European Genomic institute for Diabetes (EGID), FR 3508, F-59000 Lille, France; Université Lille 2, F-59000 Lille, France; Inserm UMR 1011,

F-59000 Lille, France; Institut Pasteur de Lille, F-59000 Lille, France

Summary

Peroxisome proliferator-activated receptor a (PPARa) is a ligand-activated transcription factor belonging, together with PPARy and PPARp/5, to the NR1C nuclear receptor subfamily. Many PPARa target genes are involved in fatty acid metabolism in tissues with high oxidative rates such as muscle, heart and liver. PPARa activation, in combination with PPARp/5 agonism, improves steatosis, inflammation and fibrosis in pre-clinical models of non-alcoholic fatty liver disease, identifying a new potential therapeutic area. In this review, we discuss the transcriptional activation and repression mechanisms by PPARa, the spectrum of target genes and chromatin-binding maps from recent genome-wide studies, paying particular attention to PPARa-regulation of hepatic fatty acid and plasma lipoprotein metabolism during nutritional transition, and of the inflammatory response. The role

of PPARa, together with other PPARs, in non-alcoholic steatohep-atitis will be discussed in light of available pre-clinical and clinical data.

© 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction

PPARa (NR1C1) is a ligand-activated nuclear receptor highly expressed in the liver, initially identified as the molecular target of xenobiotics inducing peroxisome proliferation in rodents [1]. Beside PPARa, the PPAR subfamily contains two other isotypes encoded by the PPARß/ä (NR1C2) and PPARy (NR1C3) genes, each displaying isoform-specific tissue distribution patterns and functions [2]. PPARa expression is enriched in tissues with high fatty

Keywords: PPARa; Fatty acid oxidation; Inflammation; Transrepression; Liver; Steatosis; NAFLD; NASH; Fibrosis. Received 28 June 2014; received in revised form 22 September 2014; accepted 26 October 2014

* Corresponding author. Address: INSERM UMR1011, Institut Pasteur de Lille, 1 rue du Professeur Calmette, BP245, 59019 LILLE Cédex, France. E-mail address: bart.staels@pasteur-lille.fr (B. Staels).

Abbreviations: PPAR, peroxisome proliferator-activated receptor; FAO, fatty acid oxidation; FA, fatty acid; APR, acute phase response; NASH, non-alcoholic steatohepatitis; NAFLD, non-alcoholic fatty liver disease; CVD, cardiovascular disease; LDL, low density lipoprotein; HDL-C, high density lipoprotein cholesterol; SPPARM, selective PPAR modulator; AF-1, activation function-1; MAPK, mitogen-activated protein kinase; DBD, DNA binding domain; PPRE, PPAR response element; DR-1, direct repeat-1; RXR retinoid X receptor; LBD, ligand binding domain; NCoR nuclear receptor co-repressor; PKC, protein kinase C; SUMO, small ubiquitin-like modifier; AF-2, activation function-2; LBP, ligand binding pocket; CBP, CREB-binding protein; SRC-1, steroid receptor coactivator-1 ; ACOX1, acyl-CoA oxidase 1; LTB4, leukotriene B4; 8(S)-HETE, 8(S)-hydroxyeicosatetraenoic acid; 8-LOX, 8-lipoxygenase; FATP-1, fatty acid transport protein-1; FAS, fatty acid synthase; 16:0/18:1-GPC, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine; ATGL, adipose triglyceride lipase; TG, triglyceride; HSL, hormone-sensitive lipase; EC50, half maximal effective concentration; GAL4, galactosidase 4; HAT, histone acetyltransferase; PBP, PPARa-binding protein; MED-1, mediator subunit 1; PPARaDJSS, PPARa mutant with selective transrepression activity; LXR, liver X receptor; DR-4, direct repeat-4; C/EBPa, CCAAT-enhancer-binding protein alpha; TBP, TATA-binding protein; GO, gene ontology; IL-6, interleukin-6; AP-1, activator protein 1; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; JNK, c-Jun N-terminal protein kinase; GR, glucocorticoid receptor; Fib, fibrinogen; SAA, serum amyloid A; Hg, haptoglobin; CRP, C reactive protein; STAT3, signal transducer and activator of transcription 3; Fib-p, fibrinogen-beta; GRIP-1/TIF-1, GR-interactin protein-1/transcription intermediary factor-2; ERR, estrogen-related receptor; SIRT-1, sirtuin-1; ERRE, ERR response element; LPS, lipopolysaccharide; TNF, tumor necrosis factor; ATP, adenosine triphosphate; LCFA, long-chain fatty acid; VCFA, very long-chain fatty acid; FAT/CD36, fatty acid translocase; L-FABP, liver fatty acid-binding protein; EHHADH, L-bifunctional enzyme; CPT, carnitine palmitoyltransferase; MCAD, medium-chain acyl-CoA dehydrogenase; LCAD, long-chain acyl-CoA dehydrogenase; VLCAD, very long-chain acyl-CoA dehydrogenase; let-7c, let-7 microRNA precursor; miRNA, microRNA; APO-AI, apolipoprotein-AI; HMGCS, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase; KD, ketogenic diet; FGF21, fibroblast growth factor 21; BDH, beta-D-hydroxybutyrate dehydrogenase; LPL, lipoprotein lipase; APO-CIII, apolipoprotein-CIII; ChREBP, carbohydrate-responsive element-binding protein; HNF-4, hepatocyte nuclear factor 4; FOXO1, forkhead box O1; APO-AII, apolipoprotein-AII; ChIP-seq, chromatin immunoprecipitation-sequencing; APO-AV, apolipoprotein-AV; SNP, single-nucleotide polymorphism; SREBP-1c, sterol regulatory element binding protein-1c; Acc1, acetyl-CoA carboxylase 1; Scd-1, stearoyl-CoA desaturase-1; PKA, protein kinase A; cAMP, cyclic adenosine monophosphate; mTORC1, mammalian target of rapamycin complex 1; PI3K, phosphoinositide 3-kinase; S6K2, protein S6 kinase 2; IR insulin resistance; AMPK, 5'-AMP-activated protein kinase; T2DM, type 2 diabetes mellitus; ICAM-1, intracellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; IL-1RA, interleukin-1 receptor antagonist; IkB, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor; MetS, metabolic syndrome; MCDD, methionine choline-deficient diet; CYP4A, cytochrome P450 4A; VNN1, vanin-1; ALMS1, Alstrom syndrome 1; HFD, high-fat diet; ALT, alanine aminotransferase; APO-E2, apolipoprotein-E2; ROS, reactive oxygen species; TGFp, transforming growth factor beta; COX-1, cyclooxygenase-1; AST, aspartate aminotransferase; yGT, gamma-glutamyl transpeptidase; LDLR, LDL-receptor; ALP, alkaline phosphatase; PUFA, polyunsaturated fatty acid.

Table 1. Functional analysis ofPPARa structural domains.

Domain PTM Function

N-term A/B AF-1 MAPK-dependent phosphorylation at Ser 6, 12 and 21 Ligand-dependent/independent activation function Target gene specificity

C DBD Binding to PPRE Interaction with cJun

D Hinge region PKC-dependent phosphorylation at Ser 179 and 230 SUMOylation at lysine 185 Providing NR structure flexibility Potentiating NCoR recruitment

C-term E/F LBD/AF-2 SUMOylation at lysine 358 Ligand binding specificity

Interaction with RXR and p65

Interactions with multiple co-regulators e.g., CBP/p300 and SRC/p160

PPARa displays a classical NR canonical architecture. PPARa domains (from A to F) fulfil distinct functions by providing interaction surfaces with other TFs, co-regulators and ligands, thus contributing to specific PPARa transcriptional regulation. PPARa undergoes several post-translational modifications (PTM) that markedly impact receptor function (details in the text).

acid oxidation (FAO) rates such as liver, heart, skeletal muscle, brown adipose tissue, and kidney, although it is also expressed in many tissues and cells including the intestine, vascular endothelium, smooth muscle and immune cells such as monocytes, macrophages and lymphocytes [3]. PPARa is a nutritional sensor, which allows adaptation of the rates of fatty acid (FA) catabolism, lipogenesis and ketone body synthesis, in response to feeding and starvation [4]. PPARa is a transcriptional regulator of genes involved in peroxisomal and mitochondrial p-oxidation, FA transport and hepatic glucose production, the latter being rodent-specific [5]. PPARa negatively regulates pro-inflammatory and acute phase response (APR) signalling pathways, as seen in rodent models of systemic inflammation, atherosclerosis and non-alcoholic steatohepatitis (NASH) [6,7].

Dyslipidemia and chronic inflammation are frequent features of non-alcoholic fatty liver disease (NAFLD), likely explaining the association between cardiovascular disease (CVD) and NAFLD. However, there is currently no approved NAFLD treatment. In patients with atherogenic dyslipidemia, fibrates acting as synthetic PPARa agonists, lower plasma triglycerides and small dense low density lipoprotein (LDL) particles, and raise high density lipoprotein cholesterol (HDL-C) levels. Fibrates reduce major cardiovascular events, especially in patients with high triglyceride and low HDL-C [8]. Thus PPARa agonists may potentially be useful in the management of NAFLD and co-morbidities such as CVD. PPARa activation, in combination with PPARp/5 agonism, improves steatosis, inflammation and fibrosis in rodent models of NASH [9]. Thus, selective and potent PPARa modulators (SPPARMs) and dual PPAR agonists constitute promising strategies for the treatment of NAFLD. In this review, novel mechanistic insights into PPARa action, in hepatic lipid metabolism, under different nutritional states, and its role in liver inflammation and fibrosis are presented. We also summarize the (pre) clinical findings on PPAR agonists under development for NAFLD treatment.

Functional analysis of PPARa structure

Canonical structure of PPARa

The human and mouse PPARa genes, respectively on chromosome 22 and chromosome 15, encode 468 amino acid polypeptides with 91% homology. In both species, the coding DNA sequence spans the 3' region of exon 3, exons 4-7, and the 5'

extremity of exon 8 [10]. PPARa has a canonical nuclear receptor organization with six domains starting from the N-terminal A/B to the C terminus F domain (Table 1). These domains integrate intracellular signals to control the transcriptional activity of multiple target genes. The A/B domain contains the AF-1 region providing basal, ligand-binding-independent and -dependent activity, which can be potentiated by MAPK phosphorylation of serines 6, 12, and 21 [11]. Comparative studies of chimeric PPARa/p/y proteins identified the AF-1 region as a determinant of isotype-specific target gene activation [12]. The A/B domain is connected to the DNA binding domain (DBD), harboring two zinc-fingers, which binds PPAR response elements (PPREs), localized in gene regulatory regions and organized as direct repeats of two hexamer core sequences AGG(A/T)CA, separated by one nucleotide (DR-1). PPARa/p/y bind PPREs uniquely as heterodi-mers with retinoid X receptor (RXR)a/p/y [13]. The A/T rich motif upstream of the DR-1 provides a polarization signal of the PPAR-RXR heterodimer, and may confer isotype-binding specificity. Accordingly, PPARs interact with 5'-extended hexamers, whereas RXR binds to the downstream motif of the response element [14]. The hinge region (domain D) is a highly flexible domain linking the DBD (domain C) and the ligand binding domain (LBD). The structural integrity of the hinge region conditions the interaction of PPARa with nuclear receptor corepres-sors, such as NCoR, in the unliganded conformation [15]. The hinge region is a target for post-translational modifications, such as phosphorylation catalyzed by PKC on serines 179 and 230. SUMOylation also targets the hinge domain of human PPARa at lysine 185 and potentiates NCoR recruitment [16,17]. The C-terminal LBD is the only domain of PPARa whose structure ^cu

has been solved by X-ray crystallography [18]. Similar to PPARy and PPARp/5, the PPARa LBD is composed of a helical sandwich flanking a four-stranded (1-sheet and contains the AF-2 helix. The 1400 A3 volume of the PPARa ligand binding pocket (LBP) is only slightly different than the total volume of the 1600 PPARy and 1300 A3 PPARp/5 LBPs [19,20]. Nevertheless, the PPARa LBP is more lipophilic and less solvent-exposed than the LBPs of the other PPARs, hence allowing the binding of more saturated FA. In contrast to PPARy, the PPARa AF-2 helix is more tightly packed against the LBD core when complexed with an agonist [21]. Crystallography identified tyrosine 314 as the main determinant of isotype ligand-specificity [18]. The AF-2 domain undergoes ligand-dependent conformational changes, thereby directing various co-activators such as CBP/p300 and SRC-1, carrying LXXLL motifs (L-leucine, X-any amino acid), to a hydrophobic cleft on

the PPARa LBD surface, thus promoting the formation ofan active transcriptional complex. The AF-2 domain may also play a role in ligand-dependent gene repression. Agonist binding unmasks lysine 358 in the LBD for SUMOylation, hence conferring repressive activity to PPARa [22].

Endogenous and synthetic PPARa agonists

PPARa ligands are FA derivatives formed during lipolysis, lipogenesis or FA catabolism. Substrates of the first rate-limiting peroxisomal p-oxidation enzyme, acyl-CoA oxidase 1 (ACOX1), likely are PPARa agonists. Consistently, disruption of ACOX1 in mice results in increased peroxisome proliferation, hepatocarci-noma and elevated PPARa target gene expression [23,24]. Eicosanoid derivatives, including the chemoattractant LTB4 and 8(S)-HETE, the murine 8-LOX product from arachidonic acid, are thought to be endogenous PPARa agonists [25]. The oxidized phospholipid fraction of oxidized LDL enhances PPARa transcrip-tional activity and induces its target gene, FATP-1, in human primary endothelial cells [26]. Liver-specific knockout of fatty acid synthase (FAS), an enzyme catalysing the synthesis of FA, resulted in hypoglycemia and liver steatosis when mice were fed a fatdepleted diet, which was reversed by dietary fat or a synthetic PPARa agonist, identifying products of FAS-dependent de novo lipogenesis as PPARa activators [27]. Mass spectrometry analysis on purified hepatic PPARa revealed the presence of 16:0/ 18:1-GPC bound to its LBD in mice expressing hepatic FAS, but not in liver-specific FAS knockout mice, identifying this phospho-lipid as a FAS-dependent lipid intermediate and endogenous PPARa ligand [28]. Adipose triglyceride lipase (ATGL)-dependent hydrolysis of hepatic intracellular TG also yields lipid PPARa ligands [29]. In line, overexpression of hepatic hormone-sensitive lipase (HSL) and ATGL triggers PPARa-dependent FAO gene expression and ameliorates hepatic steatosis [30].

A range of synthetic PPARa agonists, differing in species-specific potencies and efficacies, have been identified. Fibrates such as gemfibrozil, fenofibrate and ciprofibrate, are clinically used in the treatment of primary hypertriglyceridemia or mixed dyslipi-demia [8]. However, fibrates are weak PPARa agonists with limited clinical efficacy [31]. Moreover, the potency of synthetic PPARa agonists may differ between the human and mouse receptor, as measured by using the PPARa-GAL4 transactivation system, i.e., fenofibrate (mouse receptor, EC50 = 18,000 nM vs. human receptor, EC50 = 30,000 nM), bezafibrate (EC50 = 90,000 nM vs. 50,000 nM, respectively) and Wy14,643 (EC50 = 630 nM vs. 5000 nM, respectively) [32]. This may contribute to interspecies differences in response to PPARa agonists that are detailed in the following sections of this review. Potent and selective PPARa modulators (SPPARMs), such as K-877 (EC50 = 1 nM) and GFT505 (EC50 = 6 nM for PPARa), a dual PPARa/5 agonist, are currently under development for the treatment of atherogenic dyslipidemia and NAFLD, respectively [31-33]. The therapeutic potential of novel PPAR agonists on NAFLD is further discussed in this review.

exhibit HAT activity, and other co-activators forming the transcriptionally active PPARa-interacting cofactor complex [34]. Such interactions are not seen with a PPARa AF-2 domain deleted mutant [35]. Disruption of the Pbp/Med1 gene showed its essential role in PPARa-dependent gene regulation. PBP/MED1 stabilizes and directs a large transcription initiation complex containing numerous co-activators and RNA polymerase II to the DNA-bound PPAR-RXR heterodimer [36] (Fig. 1A). However, RXR homodimers may bind DR-1 PPREs independent of PPARa and induce PPARa target gene transcription through a co-activator-dependent mechanism [37]. Recently, using a PPARa mutant (PPARaDISS), which lacks PPRE-binding activity but maintains interactions with RXR and transcriptional co-regulators, we showed that PPARa-driven transactivation depends on PPRE binding in vitro, in human hepa-toma HepG2 cells and in vivo in Ppara-deficient mice with liver-specific PPARaDISS expression [35].

Genome-wide transcriptomic and PPARa chromatin binding maps

Genome-wide localization and activity-occupancy studies revealed that induction of PPARa target gene expression by PPARa agonists is associated with increased binding of PPARa to chromatin, rather by strengthening affinity and stability of existing interactions, than creating de novo ligand-inducible binding regions [38]. Interestingly, almost half of the PPARa-binding regions in human hepatoma cells are located within introns, whereas only 26% of them are localized in close vicinity (<2.5 kb) of the transcription start site [39]. In addition, genome-wide profiling of liver X receptor (LXR), RXR, and PPARa in the mouse liver showed overlapping chromatin binding regions of LXR-RXR and PPARa-RXR het-erodimers. Nevertheless, only a few percent of LXR and PPARa binding sites contain consensus DR-4 and DR-1 elements, respectively [38]. De novo motif analysis showed co-enrichment of PPARa-binding regions in C/EBPa and TBP motifs, suggesting that PPARa may influence gene expression through the formation of complexes with other transcription factors [39]. Interestingly, PPARa chromatin binding mapping, combined with transcripto-mics in primary human hepatocytes treated with the synthetic PPARa agonist Wy14,643, showed that genes whose promoter regulatory regions are directly bound by PPARa via PPREs, are on average more strongly upregulated than genes in which PPARa binds to the DNA indirectly [40]. Comparative transcriptomic studies in primary hepatocytes treated with Wy14,643 revealed only partial overlap of up- (~20%) or downregulated (~12%) genes upon PPARa activation, between humans and mice [41]. Nevertheless, searching for enriched biological themes, in human and mouse sets of regulated genes by gene ontology (GO) classification, showed a 50% conservation in over-represented GO categories, mostly corresponding to lipid metabolic pathways [41]. Importantly, the glycolytic and gluconeogenic pathways were specifically upregulated in mice, whereas xenobiotic metabolism and apolipoprotein synthesis pathways rather in human hepatocytes [41,42].

Mechanism of PPARa-dependent transactivation

Formation of transcriptionally active multiprotein PPARa complexes

Ligand-activated PPARa recruits numerous co-activator proteins, including members of the CBP/p300 and SRC/p160 family, which

Models of PPARa transcriptional repression

PPRE-independent transcriptional repression

PPARa negatively regulates pro-inflammatory signalling pathways via protein-protein interactions, a tethering mechanism extensively studied in vitro and in mouse models of acute

PPRE-dependent

JOURNAL OF HEPATOLOGY

PPRE-independent

Complement C3

ACOX1 CPT-I DBDi IL-1RA lKBa

PPARa PPARa

Hinge / Hinge / .

^^ -- DBD .. ,

NFkB RE Ap-1 RE

Hinge DBD

Hinge DBD

Fibrinogen ß

NFkB re

C/EBPß RE

PPRE AGG(ATT)CA N AGG(ATT)CA

Fig. 1. Models of PPARa transcriptional regulation. Several models of PPARa transcriptional regulation have been proposed, via which PPARa modulates expression of its target genes as well as pro-inflammatory transcription factors and acute phase response genes. (A) Formation of the PPRE-dependent ligand-activated transcriptional complex containing PPARa-RXR heterodimer, co-activators, HAT, PBP/MED1 and the transcriptional preinitiation complex (PIC). (B) PPRE-dependent inhibition of NFkB transcriptional activity. Upon ligand activation, DNA-bound PPARa directly interacts with p65 to abolish its binding to an NFkB response element (NRE) in the complement C3 promoter. (C) PPARa directly interacts with pro-inflammatory transcription factors cJun and p65 to negatively regulate their target genes by a mechanism that is thought to be PPRE-independent. (D) Simultaneous ligand-activation of GR and PPARa leads to the enhanced repression of TNF-induced IL-6 transcriptional activity, by the mechanism that stems from a direct GR-PPARa physical interaction. (E) PPARa downregulates fibrinogen p transcriptional activity via ligand-dependent mechanisms, engaging physical interaction between PPARa and GRIP-1/TIF-2.

inflammation. Ligand-activated PPARa represses cytokine-induced IL-6 gene expression via interference with AP-1 and NFkB signalling pathways. PPARa-driven transrepression involves direct physical interactions between PPARa, the p65 Rel homology domain, and the N-terminus JNK-responsive part of cJun (Fig. 1C) [43]. Moreover, synergistic transrepression of NFKB-driven gene expression occurs upon simultaneous activation of PPARa and glucocorticoid receptor (GR), a well-characterized NFkB repressor (Fig. 1D) [44]. However, PPARa and GR transrepress distinct but overlapping sets of genes in vascular endothelial cells [45]. PPARa activation downregulates hepatic APR genes, such as Fib, Saa, and Hg in rodents, and CRP in human hepatocytes. Mechanistically, PPARa downregulates mRNA and protein levels of GP80 and GP130, components of the IL6-recep-tor, thus disrupting the STAT3 and cJun signalling pathways involved in the APR [6]. Similarly, in the liver, fibrates downregu-late IL-6-stimulated Fib-p expression via PPARa-dependent titration of GRIP-1/TIF-2, thus interfering with C/EBPp activity

(Fig. 1E) [46]. Another mechanism of PPARa-dependent tran-scriptional repression occurs in the control of ERR-driven mitochondrial respiration and cardiac contraction, where a PPARa-SIRT1 complex binds directly to a single hexameric ERRE motif, thus competitively downregulating ERR target genes [47,48]. Recently, we showed that hepatic PPARa represses cytokine- and LPS-induced inflammatory responses in vitro and in vivo, independently of direct DNA binding [35].

PPRE-dependent transcriptional repression

Recently, a novel PPRE-dependent model of transcriptional regulation has been proposed, through a negative crosstalk between PPARa and p65, diminishing complement C3 promoter transcrip-tional activity in a human hepatoma cell line. Ligand-dependent activation of PPARa inhibits TNF-mediated upregulation of complement C3 through the physical interaction between PPRE-bound PPARa and p65, to abolish p65 binding to the upstream

NFkB response element on the complement C3 promoter (Fig. 1B) [49]. In line with these observations, genome-wide studies revealed the presence of STAT-PPAR binding motifs within ligand-inducible PPARa binding regions of downregulated genes. This suggests a direct negative crosstalk between PPRE-bound PPARa and pro-inflammatory transcription factors [39].

Key Points 1

Transcriptional regulation by PPARa

• Transactivation: PPARa recognizes and binds to PPREs located in the regulatory regions of its target genes

• Transrepression: PPARa directly or indirectly interacts with transcription factors to block their transcriptional activity

• PPARa target genes related to fatty acid oxidation are regulated mainly in a PPRE-binding dependent manner

• Expression of pro-inflammatory genes can be repressed by PPARa either via PPRE-binding dependent or PPRE-binding independent mechanisms. Further studies are needed to understand the mechanisms of PPRE-independent PPARa activities

Regulation of fatty acid metabolism by PPARa

PPARa-regulated FA transport and oxidation

humanized mice, whereas the incidence of hepatocellular carcinoma was 71% in wild-type mice [65]. Mechanistically, murine but not human PPARa downregulated the expression of let-7C, an miRNA targeting the c-myc oncogen [66]. Moreover, long-term treatment of hyperlipidemic patients with either gemfibrozil or fenofibrate showed no effect on peroxisomal proliferation and hepatocyte hyperplasia, as assessed by light and electron microscopy of liver biopsies [67,68]. Importantly, a meta-analysis of long-term randomized controlled trials demonstrated neutral effects of fibrate treatment on cancer [69].

PPARa and ketogenesis

During fasting, hepatic FAO increases, yielding acetyl-CoA which is further converted into ketone bodies. Ligand-activated PPARa upregulates mitochondrial HMGCS, a rate-limiting enzyme of ketogenesis, which catalyses condensation of acetyl-CoA and ace-toacetyl-CoAto generate HMG-CoAand CoA [70]. The mild pheno-type of Ppara-deficient mice fed ad libitum became more pronounced during fasting, being characterized by impaired FAO, lipid accumulation in liver and heart as well as hypoglycemia and an inability to augment ketone body synthesis [71,72]. Moreover, high-fat, low-carbohydrate ketogenic diet (KD)-feeding increased hepatic mRNA expression and plasma levels of FGF21, in parallel with PPARa induction [73]. Fgf21 knock-down in KD-fed mice impaired hepatic expression of FAO genes (Acoxl, Cpt-I) and ketogenesis (Hmgcs, Bdh), indicating that FGF21 is required for the activation of these metabolic pathways [73]. Further studies identified FGF21 as a direct PPARa target gene, induced, in mice and humans, in response to fasting and upon PPARa ligand administration [73,74].

FA are transported in cells by membrane-associated FATPs [50]. FATP1, which catalyses ATP-dependent esterification of LCFA and VCFA into acyl-CoA derivatives, is a direct PPARa target gene [51,52]. Another plasma membrane FA transporter, FAT/CD36, is positively regulated by PPARa ligands [53]. Functional PPREs were identified within the promoter of the intracellular lipid trafficking L-Fabp [54]. Direct protein-protein interaction were reported between PPARa and L-FABP, suggesting that L-FABP may channel PPARa ligands to the receptor [55,56]. Consistently, a positive correlation between L-FABP protein and PPRE-driven gene transcription was observed in human hepatoma HepG2 cells, treated with PPARa agonists [57].

PPARa controls gene expression levels of the rate-limiting enzymes of peroxisomal p-oxidation, including ACOX1 and EHHADH, most pronouncedly in rodents [41]. In rodents and primates, FA transport across the mitochondrial membrane is triggered by PPRE-dependent regulation of CPT-I and CPT-II, which proteins are localized in the outer and inner mitochondrial membrane respectively [58-60]. Moreover, PPARa regulates the critical reaction of mitochondrial p-oxidation by directly controlling MCAD, LCAD, and VLCAD expression levels [61,62].

Enhanced expression of peroxisomal genes involved in lipid metabolism is related to the induction of peroxisome proliferation by PPARa agonists, which may contribute to tumorigenesis in rodents [63]. A comparative study between mouse and human PPARa expressed in Ppara-deficient mice revealed that Wy14,643 induces mouse liver peroxisomal proliferation in a receptor species-independent manner [64]. However, long-term Wy14,643 treatment induced liver tumors only in 5% of PPARa

PPARa in the regulation of hepatic lipid and plasma lipoprotein metabolism

Molecular insights into the lipid normalizing effects of PPARa

In rodent models, the reduction of plasma TG-rich lipoprotein upon PPARa activation is related to enhanced FA uptake, conversion into acyl-CoA derivatives, and further catabolism via the p-oxidation pathways. Moreover, the TG-lowering action ofPPARa is also due to increased lipolysis via induction of lipoprotein lipase (LPL), which catalyses the hydrolysis of lipoprotein TG into free FA and monoacylglycerol. PPARa controlled LPL mRNA through binding to a PPRE in the human and mouse LPL gene promoters [75]. Furthermore, PPARa enhanced LPL activity indirectly by decreasing mRNA levels and secretion of hepatic APO-CIII, an LPL inhibitor [76]. Interestingly, glucose induced APO-CIII transcription in hepatocytes through a mechanism involving the transcription factors ChREBP and HNF-4 [77]. Conversely, hepatic expression of APO-CIII was inhibited by insulin through insulin-dependent phosphorylation of FOXO1, resulting in its displacement from the nucleus and inability to drive APO-CIII transcriptional activity [78]. In hepatocytes, inhibition of APO-CIII transcription by fibrates was the consequence of multiple cooperative mechanisms including PPARa-driven displacement of HNF-4 from the APO-CIII promoter, inhibition of FOXO1 activation of APO-CIII transcription via the insulin-responsive element, and inhibition of glucose-stimulated APO-CIII expression [76,79].

In humans, fibrates increase plasma HDL-C by stimulating the synthesis of its major apolipoproteins, APO-AI and APO-AII.

However, species-differences exist between humans and rodents with respect to apolipoprotein regulation by PPARa. A functional PPRE is present in the human, but not rodent APO-AI promoter, as illustrated by increased human APO-AI production in humanized Apo-AI transgenic mice upon treatment with fibrates [80]. In contrast, APO-AI and HDL-C levels are elevated in Ppara-deficient mice and fibrate treatment decreases Apo-AI mRNA in wild-type animals [81,82]. In the human and mouse liver, APO-AII expression is induced by PPARa. Hepatic human APO-AII gene transcription is induced by PPARa through interaction with a PPRE localized within the APO-AII promoter region. A functional PPRE could not be identified within the mouse Apo-AII promoter [83]. However, based on available data from genome-wide PPARa binding map, we inspected through promoter regions of hepatic mouse Apo-AII for the presence of PPARa ChIP-seq peaks [38] and identified a PPARa binding also in the mouse Apo-AII proximal promoter, 100 bp downstream of the transcription start site (our unpublished data). Similar species-specific transcriptional regulation modes are observed for APO-AV, which enhances LPL activity, by PPARa [84,85]. Studies using human LPL transgenic/ Apo-AV-deficient mice and human APO-AV transgenic/Lpl-deficient mice support the hypothesis that APO-AV reduces TG levels by trafficking VLDL and chylomicrons to proteoglycan-bound LPL for lipolysis [86,87]. In vitro and in vivo studies comparing wild-type versus transgenic humanized APO-AV mice revealed that human, but not mouse APO-AV expression is induced in the liver by PPARa agonists [88,89]. These findings are consistent with the identification of a functional PPRE in the human, but not mouse Apo-AV promoter [88,89]. In humans, rare SNPs in the APO-AV promoter region are associated with paradoxical decreases in plasma HDL-C and APO-AI in response to fibrates, whereas SNPs within the APO-AV gene are associated with enhanced lipid response to fibrate and statin therapy [90-93]. Thus, unexpected responses to fibrate treatment in some individuals may be due to genetic variations in PPARa target genes, such as APO-AV.

PPARa and hepatic lipogenesis

Besides its ability to orchestrate lipoprotein metabolism, PPARa also controls, directly or indirectly, lipogenic pathways in the liver. Lipogenesis is the metabolic pathway allowing FA synthesis when dietary carbohydrates are abundant. Dietary regulation of hepatic lipogenic genes is under control of the insulin-dependent transcription factors SREBP-1c and ChREBP [94]. PPARa agonists enhance human SREBP-lc transcriptional activity through PPARa interacting with a DR-1 element in the human SREBP-lc promoter. Consistently, PPARa binding to the human SREBP-lc promoter is demonstrated in vitro and in vivo, in human primary hepatocytes [95]. In mouse livers, the SREBP-1c target genes Fas, Accl, and Scd-1 are positively regulated by PPARa agonists [96,97]. Nevertheless, neither Srebp-lc nor its downstream targets have been identified as direct PPARa target genes in mice, with the exception of Scd-1, which contains a PPRE in its promoter [97]. In mice, fibrates increase the protein levels of the mature hepatic form of SREBP-1c, by increasing the rate of proteolytic cleavage of its membrane-bound precursor form, without changing Srebp-lc mRNA levels [98]. The insulin-dependent enhancement of SREBP-lc transcription requires the participation of LXR and SREBP-1c itself [99]. Moreover, via LXR-binding sites in the human and mouse Srebp-lc promoter, LXR agonists induce its

transcriptional activity [95,100]. PPARa can also indirectly modulate SREBP-lc transcription via cross-regulation with the LXR signaling pathway. In mice, PPARa is required for the LXRa-dependent response of SCD-1 and FAS to insulin in re-fed conditions, suggesting a potential role for PPARa in the synthesis of endogenous LXRa ligands [101]. In human primary hepatocytes, PPARa agonists, cooperatively with insulin and LXR agonists, induce lipogenic gene expression, such as FAS and ACCl [95].

Key Points 2

PPARa-dependent activities in mice and humans

Fatty acid metabolism and ketogenesis are the most conserved PPARa-regulated biological processes between mice and humans

Regulation of the glycolysis-gluconeogenesis pathway by PPARa agonists occurs in mice, but not in men

Xenobiotic metabolism and apolipoprotein synthesis pathways are specifically controlled by PPARa agonism in human hepatocytes

Peroxisomal proliferation genes are induced upon activation of both human and mouse PPARa, however, humans are protected from fibrate-induced tumorigenesis

Hepatic PPARa activity switches in the fed-to-fasted transition states

PPARa coordinates different pathways of de novo lipid synthesis in the fed state, to supply FA for storage as hepatic TG, for periods of starvation. During fasting, when the organism switches to the utilization of FA, deriving either from the liver or from peripheral tissues, PPARa also shifts its activity to promote FA uptake and p-oxidation, thus yielding substrates for ketone body synthesis to provide energy for peripheral tissues (Fig. 2). The adjustment of PPARa transcriptional activity in the adaptation to fasting/feeding transition can be potentially brought about by kinases controlled by different nutritional states and phosphorylating PPARa or its regulatory proteins.

Several kinases, including PKA, PKC, and MAPK, have been shown to modify PPARa transcriptional activity (see also Table 1), although many studies were performed in vitro, and thus lack physiological translation to the coordinated responses to different nutritional signals in the living organism. However, insulin-activated MAPK and glucose-activated PKC stimulate PPARa transactivation in HepG2 cells [16,102], suggesting that MAPK-and PKC-dependent phosphorylations may promote PPARa activity in the post-prandial state. Conversely, in fasting, glucagon induces cAMP and cAMP-dependent kinase PKA activity [103]. PKA-mediated phosphorylation potentiates ligand-dependent PPARa activation and increases expression of FAO genes in mouse primary hepatocytes [104].

Studies performed in mice hint that mTORC1 also plays a role in switching PPARa activities during the feeding/fasting transition as well as in pathophysiological conditions. In the fed state, when mTORC1 is activated by the insulin-dependent PI3K pathway, NCoR1 is partitionned in the cytoplasm and the nucleus of hepatocytes, thus repressing PPARa target gene expression

Fasting

Intestine

^Glucocorticoids

Adipose tissue

ß-oxidation -— AcCoA----

Ketone bodies

Glucose

Energy Peripheral tissues

Fig. 2. Molecular switch of PPARa activity in the fed-to-fasted state. Augmented postprandial glucose levels lead to increased production and secretion of insulin by p-cells, which acts on the liver to induce glucose uptake and glycolysis, yielding acetyl-CoA (AcCoA), and enhances FA synthesis. Insulin stimulates PPARa phosphorylation via PKC and enhances its transcriptional activity, whereas insulin-activated mTORC1 blocks PPARa activity by promoting nuclear localization of NCoR Lipogenesis yields fatty acid-derivatives operating as PPARa ligands. During fasting, stress hormones such as adrenaline and glucocorticoids are synthesized together with glucagon. Glucagon sustains gluconeogenesis through a stimulatory effect on hepatic gluconeogenic precursor uptake as well as on the efficiency of gluconeogenesis within the liver. Moreover, glucagon increases cAMP levels triggering PKA-dependent PPARa phosphorylation and activity. Fasting leads to decreased mTOR1C activation and stimulation of PPARa-dependent FAO and ketogenesis. The lipolytic release of adipose tissue fatty acids raises plasma levels of free fatty acids (FFA) that are subsequently stored in the liver as TG. ATGL-dependent hydrolysis of hepatic intracellular TG provides lipid ligands for PPARa activation. PPARa activation leads to increased p-oxidation rates directly and via FGF21 activation to provide substrates for ketone body synthesis and gluconeogenesis, thus maintaining energy sources for peripheral tissues. During prolonged fasting, high intracellular AMP levels induce AMPK to stimulate energy production by PPARa-driven FAO.

[105]. Inhibition of mTORC1 and its downstream effector S6K2 during fasting promotes a cytoplasmic relocalization of NCoR1, hence increasing ketogenesis via PPARa derepression [105,106].

Interestingly, S6K2 phosphorylation is elevated in ob/ob mice, a model of obesity and insulin resistance (IR) [106]. The ability of FAS to synthesize phospholipids, acting as endogenous PPARa

ligands, depends on its subcellular localization and post-transla-tional modifications [107]. Insulin-dependent phosphorylation of cytoplasmic FAS by mTORC1 limits PPARa ligand generation, whereas membrane-associated FAS, producing lipids for energy storage and export, is less susceptible to phosphorylation. Conversely, in the fasting state, de-phosphorylated cytoplasmic FAS is in a permissive state, allowing the generation of endogenous PPARa ligands, thus activating PPARa-target genes [107].

Hepatic PPARa activity can also be stimulated by AMPK, a sensor of the intracellular energy state activated by high AMP-to-ATP ratios, i.e., during fasting [108]. In contrast, glucose represses PPARa gene expression via AMPK inactivation in pancreatic p-cells [109,110], although it is unknown whether a similar mechanism occurs in the liver. Adiponectin, an insulin-sensitizing adipokine, increases FAO gene expression via AMPK-dependent PPARa activation [111]. Serum adiponectin is decreased in obesity and T2DM [112], which may contribute to an impaired PPARa activity in these pathologies.

PPARa in acute and chronic liver inflammation

PPARa and acute hepatic inflammation

PPARa exerts anti-inflammatory activities in murine models of systemic inflammation. PPARa agonism specifically attenuates the IL-6-induced APR in vitro and in vivo, by downregulating hepatic expression levels of Saa, Hg, and Fib-a, -p and -y [6]. Similar inhibitory effects of PPARa agonists on IL-1 p- and IL-6-induced APR were observed in mice with liver-restricted Ppara expression [113]. By contrast, treatment with IL-1 p decreases expression of liver PPARa and its target genes, suggesting a negative crosstalk between IL-1 p-induced inflammation and hepatic FAO regulation [114]. In line with these observations, LPS-induced APR is counteracted by fibrates in Ppara-deficient mice with liver-specific reconstituted Ppara [113]. Interestingly, pre-treatment with a PPARa agonist markedly prevents the LPS-induced increase of plasma IL-1, IL-6, and TNF, and the expression of adhesion molecules, such as ICAM-1 and VCAM-1 in the aorta, suggesting that liver PPARa controls, in a yet undefined manner, the systemic inflammatory response [113]. The anti-inflammatory effects of hepatic PPARa may also derive from its ability to upregulate anti-inflammatory genes, such as Il-lra and IxBa, a cytoplasmic inhibitor of NFkB, suggesting a possible cooperation between PPARa-dependent transactivation and transrepression to turn on anti-inflammatory pathways [115,116].

PPARa action in pre-clinical models of NAFLD

NAFLD is a chronic liver disease, which affects 10-24% of the population and is associated with IR and the MetS [117]. The pathology initiates with hepatic steatosis, which in some individuals progresses toward NASH, fibrosis, cirrhosis and finally liver failure. The ability of PPARa to counteract different stages of NAFLD has been studied in animal models, which partially replicate the human pathology [118].

Administration of an methionine choline-deficient diet (MCDD) to rodents leads to the development of steatohepatitis, histologically similar to human NASH. However, MCDD does not induce peripheral IR, normally observed in human NASH. Ppara-deficiency in MCDD-fed mice provokes more severe

steatosis and hepatitis [7]. In wild-type mice, PPARa agonism normalizes histological changes by preventing intrahepatic lipid accumulation, liver inflammation, and fibrosis [119]. Pharmacological activation of PPARa increases CYP4A-driven ro-oxidation as well as peroxisomal and mitochondrial b-oxidation, leading to enhanced hepatic lipid turnover. Moreover, synthetic PPARa agonists decrease the number of activated macrophages and stellate cells in the liver, and lower the expression of fibrotic markers [7]. In rodents, PPARa appears to be expressed mainly in hepatocytes [120], suggesting that the hepatoprotective effects of fibrates in rodents likely occur via PPARa within liver parenchy-mal cells (Fig. 3). We showed that the hepato-specific expression of the DNA-binding disabled PPARaDISS protects from MCDD-induced inflammation and liver fibrosis, without affecting FAO genes and lipid accumulation in the liver [35]. Hepatoprotective effects of PPARa agonism can also occur via the regulation of hepatic Vnnl expression [121], since Vnnl-deficiency links hepatic steatosis in response to fasting and changes the expression of inflammation and oxidative stress genes [122]. The role of ATGL-dependent intracellular TG hydrolysis, to generate endogenous PPARa agonists with anti-inflammatory potential, was recently demonstrated in Atg/-deficient mice [123], which display increased susceptibility to LPS- and MCDD-induced hepatic inflammation due to impaired PPARa signaling. The hepatic phe-notype of At/g-deficient mice is partially improved upon treatment with a synthetic PPARa agonist. The foz/foz (ALMS1 mutant) mouse model of Alstrom syndrome spontaneously exhibits a strong metabolic phenotype hallmarked by severe obesity, hyperinsulinemia and T2DM [124-126]. In this genetic background, PPARa activation reverses HFD-induced hepatocel-lular injury, liver inflammation and improves insulin sensitivity [127]. Similarly, Ppara-deficiency promotes HFD-induced hepatic TG, macrophage infiltration and elevates plasma levels of ALT and SAA [128]. In contrast to the observation that PPARa activation improves insulin sensitivity [129], Ppara-deficient mice are protected from HFD-induced IR, as assessed by glucose tolerance test and euglycemic-hyperinsulinemic clamps in fasted mice [129,130]. Similar tests performed in non-fasted Ppara-deficient mice, however, show no protection from IR compared to wildtype mice [131]. These contradictions can result from the impaired response to fasting in Ppara-deficient mice, in which the inability to oxidize FA leads to a preferential glucose use and depletion of glycogen stores [132].

The development of early stages of NASH was studied in the humanized APO-E2 knock-in (APO-E2KI) mouse. In this model, the Apo-E gene has been substituted for the human APOE2 allele under the control of the endogenous mouse promoter, faithfully mimicking mouse endogenous APO-E tissue distribution and expression levels. The reduced affinity of hAPO-E2 for the LDL-receptor leads to a plasma lipoprotein profile similar to that occurring in human type III hyperlipoproteinemia [118]. APO-E2-KI mice fed a western diet rapidly develop a phenotype characterized by steatosis and inflammation. Interestingly, macrophage infiltration in the liver precedes lipid accumulation. This is in contradiction with the concept that NASH pathogenesis always stems from initial liver steatosis, which leads to inflammation [133]. In accordance, clodronate liposome-induced depletion of residual liver macrophages (Kupffer cells) reduces hepatic TG content in HFD-fed wild-type mice [114]. Western diet-fed Ppara-deficient/APO-E2-KI mice manifest exacerbated liver steatosis and inflammation compared to wild-type APO-E2-KI mice,

Synthetic agonist

Hyperglycemia Hyperinsulinemia

FIBROSIS

Fig. 3. Hepatoprotective effects of fibrates: examples from rodent models of NAFLD. Development of NASH is provoked by different risk factors, such as Western-type diet, physical inactivity and genetic predispositions that often lead to insulin resistance and T2DM. Exaggerated food intake leads to FA synthesis via hepatic lipogenesis pathways. Enhanced TG storage in the liver (steatosis) provokes uncontrolled lipid peroxidation that generates reactive oxygen species (ROS) and cytotoxic aldehydes. Hepatocyte damage leads to increased inflammatory signaling (IL-1, TNF), acute phase response (APR) and recruitment of circulating (Mu) and residual macrophages (KC). All of these mechanisms can directly induce apoptosis, necrosis and TGFp-dependent activation of hepatic stellate cells (HSC) that are the main source of extracellular matrix protein in liver, thus contributing in fibrosis progression. In several mouse models of NAFLD, fibrate-activated PPARa counteracts different stages of NAFLD by promoting FAO and hampering pro-inflammatory response. Moreover, fibrate treatment induces catalase (CAT) expression thus diminishing H2O2 levels in the liver. Hepatic cirrhosis is associated with endothelial dysfunction and impaired intrahepatic hemodynamics that may lead to liver failure. Fibrates improve and ameliorate hepatic vascular resistance by reducing cyclo-oxygenase-1 (COX-1) protein expression.

indicative of a protective role of PPARa against NASH [134]. Consistently, in primary hepatocytes isolated from APO-E2-KI mice, HFD induces an aberrant histone H3K9me3 and H3K4me3 meth-ylation profile of the promoter of Ppara, which correlates with decreased Ppara mRNA expression [135]. In APO-E2-KI mice expressing PPARa, fibrates inhibit NASH due to their inhibitory effects on pro-inflammatory genes and the increase in lipid catabolism in the liver [133,134]. Among the ROS, hydrogen peroxide is a major agent activating TGFp and collagen production by hepatic stellate cells [136,137]. The anti-fibrotic action of synthetic PPARa agonists was demonstrated in a rat model of thioac-etamide-induced liver cirrhosis. PPARa directly upregulates catalase expression, thus ameliorating hydrogen peroxide detoxification and protecting hepatocytes from oxidative stress [138]. Moreover, fibrates improve endothelial dysfunction and ameliorate intrahepatic hemodynamics in CCl4 cirrhotic rats, at least in part, by reducing COX-1 protein expression [139].

PPARa agonism in NAFLD therapy

Few clinical pilot studies were performed to assess the impact of fibrates, which improve atherogenic dyslipidemia, on the evolution of NASH. Fenofibrate treatment (48 weeks) of 16 patients

with biopsy-confirmed NAFLD reduces the proportion of patients with elevated ALT, AST and yGT plasma levels and histologically-assessed hepatocellular ballooning [140]. However, the grade of steatosis, inflammation and fibrosis is not significantly changed upon fenofibrate treatment, in this relatively small, phenotypi-cally heterogeneous cohort [140]. Short-term bezafibrate treatment (2-8 weeks), combined with diet and exercise, of donors for liver transplantation with steatosis decreases macrovesicular steatosis [141]. Treatment of NASH patients (4 weeks) with gemfibrozil lowers ALT, AST, and yGT plasma levels [142]. Treatment with clofibrate (12 months) of 16 patients with NASH does not improve either ALT, AST and yGT or histologically assessed stea-tosis, inflammation and fibrosis [143]. However, serum TG does not decrease in these hypertriglyceridemic patients, casting doubts on the treatment efficacy. Larger randomized studies evaluating the action of novel PPARa agonists with SPPARM activity, on a broad spectrum of liver pathologies and combining several methods of NAFLD assessment, are still to be performed to unequivocally assess their efficacy. Moreover, despite numerous reports of beneficial effects of fibrates in mice, species-specific differences may exist in the response to PPARa agonism [32]. The relatively weak potency of the currently used PPARa agonists in humans can be additionally affected by the lower

expression level of PPARa in the human compared to mouse liver [144,145]. Importantly, we found that hepatic Ppara expression decreases with progressive stages of liver fibrosis in patients with NASH (our unpublished data). Thus, novel PPARa agonists with greater potency and efficacy may prove to be more useful in the treatment of NAFLD. Amongst these, K-877 manifests greater efficacy than fibrates in terms of TG-lowering activity. Moreover, K-877 raises plasma FGF21 levels in Ldlr-deficient mice fed a Western diet [31]. Consistently, Phase II clinical trials showed better efficacy of K-877 treatment on fasting plasma TG and HDL-C, in individuals with atherogenic dyslipidemia, in comparison to fenofibrate [31]. These data suggest that K-877 could be a novel treatment option to tackle the residual cardiovascular risk. So far no data are available on the effects of K-887 on NAFLD. Recently, GFT505 was shown to counteract multiple stages of NAFLD, as assessed in several animal models of NASH and fibrosis [9,146], effects likely due to the combined activation of the PPARa and S receptors. GFT505 exerts preventive effects on liver steatosis and inflammation, induced in APO-E2-KI mice by a Western-diet and in db/db mice by an MCDD. Furthermore, GFT505 exerts anti-fibrotic activities on CCl4-induced fibrosis in rats [9]. In phase II clinical trials, GFT505 treatment decreases plasma concentrations of ALT, yGT, and ALP, in MetS patients [9]. Considering its ability to improve peripheral insulin sensitivity and lower plasma FFA levels, likely via PPARS activation, in abdominally obese patients, as well as its TG lowering/HDL increasing activity in subjects with combined dyslipidemia, GFT505 is a promising drug candidate for the treatment of diseases linked to IR, such as T2DM and NASH [146,147].

Key Points 3

PPARa activities in NASH and in liver fibrosis

• PPARa deficiency leads to exaggerated lipid accumulation in the liver

• Pharmacological PPARa activation decreases liver steatosis by increasing FAO gene expression

• PPARa agonism diminishes chronic liver inflammation and fibrosis independent of its effect on liver steatosis

• The dual PPARa/5 agonist GFT505 is currently tested in a phase lib trial for the therapy of NASH in metabolic syndrome and type 2 diabetes

Perspectives

Genome-wide approaches have shown that PPARa is a master regulator of FA metabolism and ketogenesis in the liver [41]. The ability of PPARa agonists to counteract steatohepatitis and fibrosis appears prominent in murine models of NAFLD, which can be explained by the fact that PPARa expression is more abundant in the mouse compared to human liver and may further decrease with NASH progression (our unpublished data). Moreover, commonly used fibrates are relatively low activators of human PPARa. Thus potent and highly specific PPARa agonists, such as K-877 and the dual PPARa/S agonist GFT505, have appeared as promising therapies for CVD or NAFLD, respectively. Nevertheless, further clinical studies are required to determine the effectiveness and safety of such SPPARMs in humans. Since

the anti-inflammatory and anti-fibrotic activities of PPARa seem to be dissociable from its effect on liver steatosis in mice [35], more potent, possibly selective transrepression-triggering PPARa agonists could be designed in the future, based on virtual drug screening and transcriptomics. A better understanding of PPARa regulation by different nutritional signals in healthy individuals and in MetS patients will allow the design of specific pharmacological therapies, simultaneously targeting different NASH-triggering factors. Moreover, to improve NASH, dietary strategies, such as n-3 PUFA supplementation may be considered to ameliorate steatosis and inflammation, by a mechanism that may partially rely on PPARa activation [148,149]. However, the efficacy of n-3 PUFA in the treatment of NASH in human subjects remains to be demonstrated.

Conflict of interest

BS is an advisor of Genfit SA. References

[1] Issemann I, Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 1990;347: 645-650.

[2] Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ, et al. Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc Natl Acad Sci U S A 1994;91:7355-7359.

[3] Lefebvre P, Chinetti G, FruchartJC, Staels B. Sorting out the roles of PPAR alpha in energy metabolism and vascular homeostasis. J Clin Invest 2006;116:571-580.

[4] Hashimoto T, Cook WS, Qi C, Yeldandi AV, Reddy JK, Rao MS. Defect in peroxisome proliferator-activated receptor alpha-inducible fatty acid oxidation determines the severity of hepatic steatosis in response to fasting. J Biol Chem 2000;275:28918-28928.

[5] Xu J, Xiao G, Trujillo C, Chang V, Blanco L, Joseph SB, et al. Peroxisome proliferator-activated receptor alpha (PPARalpha) influences substrate utilization for hepatic glucose production. J Biol Chem 2002;277: 50237-50244.

[6] Gervois P, Kleemann R, Pilon A, Percevault F, Koenig W, Staels B, et al. Global suppression of IL-6-induced acute phase response gene expression after chronic in vivo treatment with the peroxisome proliferator-activated receptor-alpha activator fenofibrate. J Biol Chem 2004;279:16154-16160.

[7] Ip E, Farrell GC, Robertson G, Hall P, Kirsch R Leclercq I. Central role of PPARalpha-dependent hepatic lipid turnover in dietary steatohepatitis in mice. Hepatology 2003;38:123-132.

[8] Staels B, Maes M, Zambon A. Fibrates and future PPARalpha agonists in the treatment of cardiovascular disease. Nat Clin Pract Cardiovasc Med 2008;5:542-553.

[9] Staels B, Rubenstrunk A, Noel B, Rigou G, Delataille P, Millatt LJ, et al. Hepatoprotective effects of the dual peroxisome proliferator-activated receptor alpha/delta agonist, GFT505, in rodent models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Hepatology 2013;58:1941-1952.

[10] Gearing KL, Crickmore A, Gustafsson JA. Structure of the mouse peroxisome proliferator activated receptor alpha gene. Biochem Biophys Res Commun 1994;199:255-263.

[11] Barger PM, Browning AC, Garner AN, Kelly DP. P38 mitogen-activated protein kinase activates peroxisome proliferator-activated receptor alpha: a potential role in the cardiac metabolic stress response. J Biol Chem 2001;276:44495-44501.

[12] Hummasti S, Tontonoz P. The peroxisome proliferator-activated receptor N-terminal domain controls isotype-selective gene expression and adipo-genesis. Mol Endocrinol 2006;20:1261-1275.

[13] Gearing KL, Gottlicher M, Teboul M, Widmark E, Gustafsson JA. Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor. Proc Natl Acad Sci U S A 1993;90:1440-1444.

[14] Juge-Aubry C, Pernin A, Favez T, Burger AG, Wahli W, Meier CA, et al. DNA binding properties of peroxisome proliferator-activated receptor subtypes

on various natural peroxisome proliferator response elements. Importance of the 5'-flanking region. J Biol Chem 1997;272:25252-25259.

[15] Dowell P, Ishmael JE, Avram D, Peterson VJ, Nevrivy DJ, Leid M. Identification of nuclear receptor corepressor as a peroxisome proliferator-activated receptor alpha interacting protein. J Biol Chem 1999;274:15901-15907.

[16] Blanquart C, Mansouri R, Paumelle R, Fruchart JC, Staels B, Glineur C. The protein kinase C signaling pathway regulates a molecular switch between transactivation and transrepression activity of the peroxisome proliferator-activated receptor alpha. Mol Endocrinol 2004;18:1906-1918.

[17] Pourcet B, Pineda-Torra I, Derudas B, Staels B, Glineur C. SUMOylation of human peroxisome proliferator-activated receptor alpha inhibits its trans-activity through the recruitment of the nuclear corepressor NCoR. J Biol Chem 2010;285:5983-5992.

[18] Xu HE, Lambert MH, Montana VG, Plunket KD, Moore LB, Collins JL, et al. Structural determinants of ligand binding selectivity between the perox-isome proliferator-activated receptors. Proc Natl Acad Sci U S A 2001;98:13919-13924.

[19] Gampe Jr RT, Montana VG, Lambert MH, Miller AB, Bledsoe RK, Milburn MV, et al. Asymmetry in the PPARgamma/RXRalpha crystal structure reveals the molecular basis of heterodimerization among nuclear receptors. Mol Cell 2000;5:545-555.

[20] Batista FA, Trivella DB, Bernardes A, Gratieri J, Oliveira PS, Figueira AC, et al. Structural insights into human peroxisome proliferator activated receptor delta (PPAR-delta) selective ligand binding. PLoS One 2012;7:e33643.

[21] Cronet P, Petersen JF, Folmer R, Blomberg N, Sjoblom K, Karlsson U, et al. Structure of the PPARalpha and -gamma ligand binding domain in complex with AZ 242; ligand selectivity and agonist activation in the PPAR family. Structure 2001;9:699-706.

[22] Leuenberger N, Pradervand S, Wahli W. Sumoylated PPARalpha mediates sex-specific gene repression and protects the liver from estrogen-induced toxicity in mice. J Clin Invest 2009;119:3138-3148.

[23] Fan CY, Pan J, Chu R, Lee D, Kluckman KD, Usuda N, et al. Hepatocellular and hepatic peroxisomal alterations in mice with a disrupted peroxisomal fatty acyl-coenzyme A oxidase gene. J Biol Chem 1996;271:24698-24710.

[24] Fan CY, Pan J, Usuda N, Yeldandi AV, Rao MS, Reddy JK. Steatohepatitis, spontaneous peroxisome proliferation and liver tumors in mice lacking peroxisomal fatty acyl-CoA oxidase. Implications for peroxisome prolifer-ator-activated receptor alpha natural ligand metabolism. J Biol Chem 1998;273:15639-15645.

[25] Yu K, Bayona W, Kallen CB, Harding HP, Ravera CP, McMahon G, et al. Differential activation of peroxisome proliferator-activated receptors by eicosanoids. J Biol Chem 1995;270:23975-23983.

[26] Delerive P, Furman C, Teissier E, Fruchart J, Duriez P, Staels B. Oxidized phospholipids activate PPARalpha in a phospholipase A2-dependent manner. FEBS Lett 2000;471:34-38.

[27] Chakravarthy MV, Pan Z, Zhu Y, Tordjman K, Schneider JG, Coleman T, et al. "New" hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis. Cell Metab 2005;1:309-322.

[28] Chakravarthy MV, Lodhi IJ, Yin L, Malapaka RR, Xu HE, Turk J, et al. Identification of a physiologically relevant endogenous ligand for PPARalpha in liver. Cell 2009;138:476-488.

[29] Sapiro JM, Mashek MT, Greenberg AS, Mashek DG. Hepatic triacylglycerol hydrolysis regulates peroxisome proliferator-activated receptor alpha activity. J Lipid Res 2009;50:1621-1629.

[30] Reid BN, Ables GP, Otlivanchik OA, Schoiswohl G, Zechner R, Blaner WS, et al. Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation, stimulates direct release of free fatty acids, and ameliorates steatosis. J Biol Chem 2008;283:13087-13099.

[31] Fruchart JC. Selective peroxisome proliferator-activated receptor alpha modulators (SPPARMalpha): the next generation of peroxisome prolifera-tor-activated receptor alpha-agonists. Cardiovasc Diabetol 2013;12:82.

[32] Willson TM, Brown PJ, Sternbach DD, Henke BR. The PPARs: from orphan receptors to drug discovery. J Med Chem 2000;43:527-550.

[33] Cariou B, Staels B. GFT505 for the treatment of nonalcoholic steatohepatitis and type 2 diabetes. Expert Opin Investig Drugs 2014;23:1441-1448.

[34] Surapureddi S, Yu S, Bu H, Hashimoto T, Yeldandi AV, Kashireddy P, et al. Identification of a transcriptionally active peroxisome proliferator-acti-vated receptor alpha -interacting cofactor complex in rat liver and characterization of PRIC285 as a coactivator. Proc Natl Acad Sci U S A 2002;99:11836-11841.

[35] Pawlak M, Bauge E, Bourguet W, De Bosscher K, Lalloyer F, Tailleux A, et al. The transrepressive activity of Pparalpha is necessary and sufficient to prevent liver fibrosis. Hepatology 2014;60:1593-1606.

[36] Jia Y, Qi C, Kashireddi P, Surapureddi S, Zhu YJ, Rao MS, et al. Transcription coactivator PBP, the peroxisome proliferator-activated receptor (PPAR)-binding protein, is required for PPARalpha-regulated gene expression in liver. J Biol Chem 2004;279:24427-24434.

[37] IJpenberg A, Tan NS, Gelman L, Kersten S, Seydoux J, Xu J, et al. In vivo activation of PPAR target genes by RXR homodimers. EMBO J 2004;23:2083-2091.

[38] Boergesen M, Pedersen TA, Gross B, van Heeringen SJ, Hagenbeek D, Bindesboll C, et al. Genome-wide profiling of liver X receptor, retinoid X receptor, and peroxisome proliferator-activated receptor alpha in mouse liver reveals extensive sharing of binding sites. Mol Cell Biol 2012;32:852-867.

[39] van der Meer DL, Degenhardt T, Vaisanen S, de Groot PJ, Heinaniemi M, de Vries SC, et al. Profiling of promoter occupancy by PPARalpha in human hepatoma cells via ChIP-chip analysis. Nucleic Acids Res 2010;38:2839-2850.

[40] McMullen PD, Bhattacharya S, Woods CG, Sun B, Yarborough K, Ross SM, et al. A map of the PPARalpha transcription regulatory network for primary human hepatocytes. Chem Biol Interact 2014;209:14-24.

[41] Rakhshandehroo M, Hooiveld G, Muller M, Kersten S. Comparative analysis of gene regulation by the transcription factor PPARalpha between mouse and human. PLoS One 2009;4:e6796.

[42] Rakhshandehroo M, Sanderson LM, Matilainen M, Stienstra R, Carlberg C, de Groot PJ, et al. Comprehensive analysis of PPARalpha-dependent regulation of hepatic lipid metabolism by expression profiling. PPAR Res 2007;2007:26839.

[43] Delerive P, De Bosscher K, Besnard S, Vanden Berghe W, Peters JM, Gonzalez FJ, et al. Peroxisome proliferator-activated receptor alpha negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1. J Biol Chem 1999;274:32048-32054.

[44] Bougarne N, Paumelle R, Caron S, Hennuyer N, Mansouri R, Gervois P, et al. PPARalpha blocks glucocorticoid receptor alpha-mediated transactivation but cooperates with the activated glucocorticoid receptor alpha for transrepression on NF-kappaB. Proc Natl Acad Sci U S A 2009;106:7397-7402.

[45] Xu X, Otsuki M, Saito H, Sumitani S, Yamamoto H, Asanuma N, et al. PPARalpha and GR differentially down-regulate the expression of nuclear factor-kappaB-responsive genes in vascular endothelial cells. Endocrinology 2001;142:3332-3339.

[46] Gervois P, Vu-Dac N, Kleemann R, Kockx M, Dubois G, Laine B, et al. Negative regulation of human fibrinogen gene expression by peroxi-some proliferator-activated receptor alpha agonists via inhibition of CCAAT box/enhancer-binding protein beta. J Biol Chem 2001;276: 33471-33477.

[47] Oka S, Alcendor R, Zhai P, Park JY, Shao D, Cho J, et al. PPARalpha-Sirt1 complex mediates cardiac hypertrophy and failure through suppression of the ERR transcriptional pathway. Cell Metab 2011;14:598-611.

[48] Oka S, Zhai P, Alcendor R, Park JY, Tian B, Sadoshima J. Suppression of ERR targets by a PPARalpha/Sirt1 complex in the failing heart. Cell Cycle 2012;11:856-864.

[49] Mogilenko DA, Kudriavtsev IV, Shavva VS, Dizhe EB, Vilenskaya EG, Efremov AM, et al. Peroxisome proliferator-activated receptor alpha positively regulates complement C3 expression but inhibits tumor necrosis factor alpha-mediated activation of C3 gene in mammalian hepatic-derived cells. J Biol Chem 2013;288:1726-1738.

[50] Schaffer JE, Lodish HF. Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein. Cell 1994;79: 427-436.

[51] Martin G, Schoonjans K, Lefebvre AM, Staels B, Auwerx J. Coordinate regulation of the expression of the fatty acid transport protein and acyl-CoA synthetase genes by PPARalpha and PPARgamma activators. J Biol Chem 1997;272:28210-28217.

[52] Frohnert BI, Hui TY, Bernlohr DA. Identification of a functional peroxisome proliferator-responsive element in the murine fatty acid transport protein gene. J Biol Chem 1999;274:3970-3977.

[53] Motojima K, Passilly P, Peters JM, Gonzalez FJ, Latruffe N. Expression of putative fatty acid transporter genes are regulated by peroxisome prolif-erator-activated receptor alpha and gamma activators in a tissue- and inducer-specific manner. J Biol Chem 1998;273:16710-16714.

[54] Helledie T, Grontved L, Jensen SS, Kiilerich P, Rietveld L, Albrektsen T, et al. The gene encoding the Acyl-CoA-binding protein is activated by peroxi-some proliferator-activated receptor gamma through an intronic response element functionally conserved between humans and rodents. J Biol Chem 2002;277:26821-26830.

[55 [56 [57

[58 [59 [60

[63 [64

[67 [68 [69 [70

[71 [72

Hostetler HA, Mcintosh AL, Atshaves BP, Storey SM, Payne HR, Kier AB, et al.

L-FABP directly interacts with PPARalpha in cultured primary hepatocytes. J

Lipid Res 2009;50:1663-1675. [76

Velkov T. Interactions between human liver fatty acid binding protein and

peroxisome proliferator activated receptor selective drugs. PPAR Res

2013;2013:938401. [77

Wolfrum C, Borrmann CM, Borchers T, Spener F. Fatty acids and hypolip-

idemic drugs regulate peroxisome proliferator-activated receptors alpha -

and gamma-mediated gene expression via liver fatty acid binding protein:

a signaling path to the nucleus. Proc Natl Acad Sci U S A 2001;98: [78

2323-2328.

LouetJF, Chatelain F, Decaux JF, Park EA, Kohl C, Pineau T, et al. Long-chain

fatty acids regulate liver carnitine palmitoyltransferase I gene (L-CPT I) [79

expression through a peroxisome-proliferator-activated receptor alpha

(PPARalpha)-independent pathway. Biochem J 2001;354:189-197.

Mascaro C, Acosta E, Ortiz JA, Marrero PF, Hegardt FG, Haro D. Control of [80

human muscle-type carnitine palmitoyltransferase I gene transcription by

peroxisome proliferator-activated receptor. J Biol Chem 1998;273:

8560-8563.

Barrero MJ, Camarero N, Marrero PF, Haro D. Control of human carnitine [81

palmitoyltransferase II gene transcription by peroxisome proliferator-activated receptor through a partially conserved peroxisome proliferator-responsive element. BiochemJ 2003;369:721-729. [82

Gulick T, Cresci S, Caira T, Moore DD, Kelly DP. The peroxisome proliferator-activated receptor regulates mitochondrial fatty acid oxidative enzyme gene expression. Proc Natl Acad Sci U S A 1994;91:11012-11016. [83

Aoyama T, Peters JM, Iritani N, Nakajima T, Furihata K, Hashimoto T, et al. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem 1998;273:5678-5684. [84

Reddy JK, Azarnoff DL, Hignite CE. Hypolipidaemic hepatic peroxisome proliferators form a novel class of chemical carcinogens. Nature 1980;283:397-398. [85

Yu S, Cao WQ, Kashireddy P, Meyer K, Jia Y, Hughes DE, et al. Human peroxisome proliferator-activated receptor alpha (PPARalpha) supports the induction of peroxisome proliferation in PPARalpha-deficient mouse liver. J Biol Chem 2001;276:42485-42491.

Morimura K, Cheung C, Ward JM, Reddy JK, Gonzalez FJ. Differential [86

susceptibility of mice humanized for peroxisome proliferator-activated receptor alpha to Wy-14,643-induced liver tumorigenesis. Carcinogenesis 2006;27:1074-1080.

Shah YM, Morimura K, Yang Q, Tanabe T, Takagi M, Gonzalez FJ. Peroxisome [87

proliferator-activated receptor alpha regulates a microRNA-mediated signaling cascade responsible for hepatocellular proliferation. Mol Cell Biol 2007;27:4238-4247. [88

De La Iglesia FA, Lewis JE, Buchanan RA, Marcus EL, McMahon G. Light and electron microscopy of liver in hyperlipoproteinemic patients under long-term gemfibrozil treatment. Atherosclerosis 1982;43:19-37. Blumcke S, Schwartzkopff W, Lobeck H, Edmondson NA, Prentice DE, Blane GF. Influence of fenofibrate on cellular and subcellular liver structure in [89

hyperlipidemic patients. Atherosclerosis 1983;46:105-116. Bonovas S, Nikolopoulos GK, Bagos PG. Use of fibrates and cancer risk: a systematic review and meta-analysis of 17 long-term randomized placebo-controlled trials. PLoS One 2012;7:e45259. [90

Rodriguez JC, Gil-Gomez G, Hegardt FG, Haro D. Peroxisome proliferator-activated receptor mediates induction of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene by fatty acids. J Biol Chem 1994;269:18767-18772. [91

Kersten S, Seydoux J, Peters JM, Gonzalez FJ, Desvergne B, Wahli W. Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting. J Clin Invest 1999;103:1489-1498.

Djouadi F, Weinheimer CJ, Saffitz JE, Pitchford C, Bastin J, Gonzalez FJ, et al. [92

A gender-related defect in lipid metabolism and glucose homeostasis in peroxisome proliferator-activated receptor alpha-deficient mice. J Clin Invest 1998;102:1083-1091.

Badman MK, Pissios P, Kennedy AR, Koukos G, Flier JS, Maratos-Flier E. [93

Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states. Cell Metab 2007;5:426-437.

Galman C, Lundasen T, Kharitonenkov A, Bina HA, Eriksson M, Hafstrom I, [94

et al. The circulating metabolic regulator FGF21 is induced by prolonged fasting and PPARalpha activation in man. Cell Metab 2008;8: [95

169-174.

Schoonjans K, Peinado-Onsurbe J, Lefebvre AM, Heyman RA, Briggs M, Deeb S, et al. PPARalpha and PPARgamma activators direct a distinct

tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene. EMBO J 1996;15:5336-5348.

Hertz R, Bishara-Shieban J, Bar-Tana J. Mode of action of peroxisome proliferators as hypolipidemic drugs. Suppression of apolipoprotein C-III. J Biol Chem 1995;270:13470-13475.

Caron S, Verrijken A, Mertens I, Samanez CH, Mautino G, Haas JT, et al. Transcriptional activation of apolipoprotein CIII expression by glucose may contribute to diabetic dyslipidemia. Arterioscler Thromb Vasc Biol 2011;31:513-519.

Altomonte J, Cong L, Harbaran S, Richter A, Xu J, Meseck M, et al. Foxol mediates insulin action on apoC-III and triglyceride metabolism. J Clin Invest 2004;114:1493-1503.

Qu S, Su D, Altomonte J, Kamagate A, He J, Perdomo G, et al. PPAR(alpha) mediates the hypolipidemic action of fibrates by antagonizing FoxO1. Am J Physiol Endocrinol Metab 2007;292:E421-E434.

Berthou L, Duverger N, Emmanuel F, Langouet S, Auwerx J, Guillouzo A, et al. Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice. J Clin Invest 1996;97:2408-2416.

Staels B, van Tol A, Andreu T, Auwerx J. Fibrates influence the expression of genes involved in lipoprotein metabolism in a tissue-selective manner in the rat. Arterioscler Thromb 1992;12:286-294.

Peters JM, Hennuyer N, Staels B, Fruchart JC, Fievet C, Gonzalez FJ, et al. Alterations in lipoprotein metabolism in peroxisome proliferator-activated receptor alpha-deficient mice. J Biol Chem 1997;272:27307-27312. Vu-Dac N, Schoonjans K, Kosykh V, Dallongeville J, Fruchart JC, Staels B, et al. Fibrates increase human apolipoprotein A-II expression through activation of the peroxisome proliferator-activated receptor. J Clin Invest 1995;96:741-750.

Fruchart-Najib J, Bauge E, Niculescu LS, Pham T, Thomas B, Rommens C, et al. Mechanism of triglyceride lowering in mice expressing human apolipoprotein A5. Biochem Biophys Res Commun 2004;319:397-404. Schaap FG, Rensen PC, Voshol PJ, Vrins C, van der Vliet HN, Chamuleau RA, et al. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis. J Biol Chem 2004;279: 27941-27947.

Merkel M, Loeffler B, Kluger M, Fabig N, Geppert G, Pennacchio LA, et al. Apolipoprotein AV accelerates plasma hydrolysis of triglyceride-rich lipo-proteins by interaction with proteoglycan-bound lipoprotein lipase. J Biol Chem 2005;280:21553-21560.

Pennacchio LA, Olivier M, Hubacek JA, Cohen JC, Cox DR, Fruchart JC, et al. An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing. Science 2001;294:169-173. Prieur X, Lesnik P, Moreau M, Rodriguez JC, Doucet C, Chapman MJ, et al. Differential regulation of the human versus the mouse apolipoprotein AV gene by PPARalpha. Implications for the study of pharmaceutical modifiers of hypertriglyceridemia in mice. Biochim Biophys Acta 2009;1791: 764-771.

Vu-Dac N, Gervois P, Jakel H, Nowak M, Bauge E, Dehondt H, et al. Apolipoprotein A5, a crucial determinant of plasma triglyceride levels, is highly responsive to peroxisome proliferator-activated receptor alpha activators. J Biol Chem 2003;278:17982-17985.

Brautbar A, Barbalic M, Chen F, Belmont J, Virani SS, Scherer S, et al. Rare APOA5 promoter variants associated with paradoxical HDL cholesterol decrease in response to fenofibric acid therapy. J Lipid Res 2013;54: 1980-1987.

Brautbar A, Covarrubias D, Belmont J, Lara-Garduno F, Virani SS, Jones PH, et al. Variants in the APOA5 gene region and the response to combination therapy with statins and fenofibric acid in a randomized clinical trial of individuals with mixed dyslipidemia. Atherosclerosis 2011;219:737-742. Lai CQ, Arnett DK, Corella D, Straka RJ, Tsai MY, Peacock JM, et al. Fenofibrate effect on triglyceride and postprandial response of apolipopro-tein A5 variants: the GOLDN study. Arterioscler Thromb Vasc Biol 2007;27:1417-1425.

Cardona F, Guardiola M, Queipo-Ortuno MI, Murri M, Ribalta J, Tinahones FJ. The -1131T>C SNP of the APOA5 gene modulates response to fenofibrate treatment in patients with the metabolic syndrome: a postprandial study. Atherosclerosis 2009;206:148-152. Ferre P, Foufelle F. Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c. Diabetes Obes Metab 2010;12:83-92. Fernandez-Alvarez A, Alvarez MS, Gonzalez R, Cucarella C, Muntane J, Casado M. Human SREBP1c expression in liver is directly regulated by peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem 2011;286:21466-21477.

[96 [97 [98 [99

[104 [105 [106 [107 [108 [109

[113 [114

[115 [116

Patel DD, Knight BL, Wiggins D, Humphreys SM, Gibbons GF. Disturbances in the normal regulation of SREBP-sensitive genes in PPAR alpha-deficient mice. J Lipid Res 2001;42:328-337.

Miller CW, Ntambi JM. Peroxisome proliferators induce mouse liver stearoyl-CoA desaturase 1 gene expression. Proc Natl Acad Sci U S A 1996;93:9443-9448.

Knight BL, Hebbachi A, Hauton D, Brown AM, Wiggins D, Patel DD, et al. A role for PPARalpha in the control of SREBP activity and lipid synthesis in the liver. Biochem J 2005;389:413-421.

Chen G, Liang G, Ou J, Goldstein JL, Brown MS. Central role for liver X receptor in insulin-mediated activation of Srebp-1c transcription and stimulation of fatty acid synthesis in liver. Proc Natl Acad Sci U S A 2004;101:11245-11250.

Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, Shimomura I, et al. Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta. Genes Dev 2000;14:2819-2830.

Hebbachi AM, Knight BL, Wiggins D, Patel DD, Gibbons GF. Peroxisome proliferator-activated receptor alpha deficiency abolishes the response of lipogenic gene expression to re-feeding: restoration of the normal response by activation of liver X receptor alpha. J Biol Chem 2008;283:4866-4876. Juge-Aubry CE, Hammar E, Siegrist-Kaiser C, Pernin A, Takeshita A, Chin WW, et al. Regulation of the transcriptional activity of the peroxisome proliferator-activated receptor alpha by phosphorylation of a ligand-independent trans-activating domain. J Biol Chem 1999;274:10505-10510. Jiang Y, Cypess AM, Muse ED, Wu CR, Unson CG, Merrifield RB, et al. Glucagon receptor activates extracellular signal-regulated protein kinase 1/ 2 via cAMP-dependent protein kinase. Proc Natl Acad Sci U S A 2001;98:10102-10107.

Lazennec G, Canaple L, Saugy D, Wahli W. Activation of peroxisome

proliferator-activated receptors (PPARs) by their ligands and protein kinase

A activators. Mol Endocrinol 2000;14:1962-1975.

Sengupta S, Peterson TR, Laplante M, Oh S, Sabatini DM. MTORC1 controls

fasting-induced ketogenesis and its modulation by ageing. Nature

2010;468:1100-1104.

Kim K, Pyo S, Um SH. S6 kinase 2 deficiency enhances ketone body production and increases peroxisome proliferator-activated receptor alpha activity in the liver. Hepatology 2012;55:1727-1737. Jensen-Urstad AP, Song H, Lodhi IJ, Funai K, Yin L, Coleman T, et al. Nutrient-dependent phosphorylation channels lipid synthesis to regulate PPARalpha. J Lipid Res 2013;54:1848-1859.

Bronner M, Hertz R, Bar-Tana J. Kinase-independent transcriptional co-activation of peroxisome proliferator-activated receptor alpha by AMP-activated protein kinase. Biochem J 2004;384:295-305. Joly E, Roduit R, Peyot ML, Habinowski SA, Ruderman NB, Witters LA, et al. Glucose represses PPARalpha gene expression via AMP-activated protein kinase but not via p38 mitogen-activated protein kinase in the pancreatic beta-cell. J Diabetes 2009;1:263-272.

Ravnskjaer K, Boergesen M, Dalgaard LT, Mandrup S. Glucose-induced repression of PPARalpha gene expression in pancreatic beta-cells involves PP2A activation and AMPK inactivation. J Mol Endocrinol 2006;36: 289-299.

Yoon MJ, Lee GY, Chung JJ, Ahn YH, Hong SH, Kim JB. Adiponectin increases fatty acid oxidation in skeletal muscle cells by sequential activation of AMP-activated protein kinase, p38 mitogen-activated protein kinase, and peroxisome proliferator-activated receptor alpha. Diabetes 2006;55: 2562-2570.

Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y, et al. Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol 2000;20: 1595-1599.

Mansouri RM, Bauge E, Staels B, Gervois P. Systemic and distal repercussions of liver-specific peroxisome proliferator-activated receptor-alpha control of the acute-phase response. Endocrinology 2008;149:3215-3223. Stienstra R, Saudale F, Duval C, Keshtkar S, Groener JE, van Rooijen N, et al. Kupffer cells promote hepatic steatosis via interleukin-1beta-dependent suppression of peroxisome proliferator-activated receptor alpha activity. Hepatology 2010;51:511-522.

Stienstra R, Mandard S, Tan NS, Wahli W, Trautwein C, Richardson TA, et al. The Interleukin-1 receptor antagonist is a direct target gene of PPARalpha in liver. J Hepatol 2007;46:869-877.

Kleemann R, Gervois PP, Verschuren L, Staels B, Princen HM, Kooistra T. Fibrates down-regulate IL-1-stimulated C-reactive protein gene expression in hepatocytes by reducing nuclear p50-NFkappa B-C/EBP-beta complex formation. Blood 2003;101:545-551.

[117] Allard JP, Aghdassi E, Mohammed S, Raman M, Avand G, Arendt BM, et al. Nutritional assessment and hepatic fatty acid composition in non-alcoholic fatty liver disease (NAFLD): a cross-sectional study. J Hepatol 2008;48: 300-307.

118] Tailleux A, Wouters K, Staels B. Roles of PPARs in NAFLD: potential therapeutic targets. Biochim Biophys Acta 2012;1821:809-818.

119] Ip E, Farrell G, Hall P, Robertson G, Leclercq I. Administration of the potent PPARalpha agonist, Wy-14,643, reverses nutritional fibrosis and steato-hepatitis in mice. Hepatology 2004;39:1286-1296.

120] Peters JM, Rusyn I, Rose ML, Gonzalez FJ, Thurman RG. Peroxisome proliferator-activated receptor alpha is restricted to hepatic parenchymal cells, not Kupffer cells: implications for the mechanism of action of peroxisome proliferators in hepatocarcinogenesis. Carcinogenesis 2000;21:823-826.

121] Rommelaere S, Millet V, Gensollen T, Bourges C, Eeckhoute J, Hennuyer N, et al. PPARalpha regulates the production of serum Vanin-1 by liver. FEBS Lett 2013;587:3742-3748.

122] van Diepen JA, Jansen PA, Ballak DB, Hijmans A, Hooiveld GJ, Rommelaere S, et al. PPAR-alpha dependent regulation of vanin-1 mediates hepatic lipid metabolism. J Hepatol 2014;61:366-372.

123] Jha P, Claudel T, Baghdasaryan A, Mueller M, Halilbasic E, Das SK, et al. Role of adipose triglyceride lipase (PNPLA2) in protection from hepatic inflammation in mouse models of steatohepatitis and endotoxemia. Hepatology 2014;59:858-869.

124] Arsov T, Silva DG, O'Bryan MK, Sainsbury A, Lee NJ, Kennedy C, et al. Fat aussie-a new Alstrom syndrome mouse showing a critical role for ALMS1 in obesity, diabetes, and spermatogenesis. Mol Endocrinol 2006;20: 1610-1622.

125] Larter CZ, Yeh MM, Van Rooyen DM, Teoh NC, Brooling J, Hou JY, et al. Roles of adipose restriction and metabolic factors in progression of steatosis to steatohepatitis in obese, diabetic mice. J Gastroenterol Hepatol 2009;24: 1658-1668.

126] Collin GB, Cyr E, Bronson R, Marshall JD, Gifford EJ, Hicks W, et al. Alms1-disrupted mice recapitulate human Alstrom syndrome. Hum Mol Genet 2005;14:2323-2333.

127] Larter CZ, Yeh MM, Van Rooyen DM, Brooling J, Ghatora K, Farrell GC. Peroxisome proliferator-activated receptor-alpha agonist, Wy 14,643, improves metabolic indices, steatosis and ballooning in diabetic mice with non-alcoholic steatohepatitis. J Gastroenterol Hepatol 2011;27: 341-350.

128] Abdelmegeed MA, Yoo SH, Henderson LE, Gonzalez FJ, Woodcroft KJ, Song BJ. PPARalpha expression protects male mice from high fat-induced nonalcoholic fatty liver. J Nutr 2011;141:603-610.

129] Guerre-Millo M, Gervois P, Raspe E, Madsen L, Poulain P, Derudas B, et al. Peroxisome proliferator-activated receptor alpha activators improve insulin sensitivity and reduce adiposity. J Biol Chem 2000;275:16638-16642.

130] Tordjman K, Bernal-Mizrachi C, Zemany L, Weng S, Feng C, Zhang F, et al. PPARalpha deficiency reduces insulin resistance and atherosclerosis in apoE-null mice. J Clin Invest 2001;107:1025-1034.

131] Haluzik M, Gavrilova O, LeRoith D. Peroxisome proliferator-activated receptor-alpha deficiency does not alter insulin sensitivity in mice maintained on regular or high-fat diet: hyperinsulinemic-euglycemic clamp studies. Endocrinology 2004;145:1662-1667.

132] Haluzik MM, Haluzik M. PPAR-alpha and insulin sensitivity. Physiol Res 2006;55:115-122.

133] Shiri-Sverdlov R, Wouters K, van Gorp PJ, Gijbels MJ, Noel B, Buffat L, et al. Early diet-induced non-alcoholic steatohepatitis in APOE2 knock-in mice and its prevention by fibrates. J Hepatol 2006;44:732-741.

134] Lalloyer F, Wouters K, Baron M, Caron S, Vallez E, Vanhoutte J, et al. Peroxisome proliferator-activated receptor-alpha gene level differently affects lipid metabolism and inflammation in apolipoprotein E2 knock-in mice. Arterioscler Thromb Vasc Biol 2011;31:1573-1579.

135] Jun HJ, Kim J, Hoang MH, Lee SJ. Hepatic lipid accumulation alters global histone h3 lysine 9 and 4 trimethylation in the peroxisome proliferator-activated receptor alpha network. PLoS One 2012;7:e44345.

136] Svegliati Baroni G, D'Ambrosio L, Ferretti G, Casini A, Di Sario A, Salzano R, et al. Fibrogenic effect of oxidative stress on rat hepatic stellate cells. Hepatology 1998;27:720-726.

137] De Bleser PJ, Xu G, Rombouts K, Rogiers V, Geerts A. Glutathione levels discriminate between oxidative stress and transforming growth factor-beta signaling in activated rat hepatic stellate cells. J Biol Chem 1999;274: 33881-33887.

138] Toyama T, Nakamura H, Harano Y, Yamauchi N, Morita A, Kirishima T, et al. PPARalpha ligands activate antioxidant enzymes and suppress hepatic fibrosis in rats. Biochem Biophys Res Commun 2004;324:697-704.

[139] Rodriguez-Vilarrupla A, Lavina B, Garcia-Caldero H, Russo L, Rosado E, Roglans N, et al. PPARalpha activation improves endothelial dysfunction and reduces fibrosis and portal pressure in cirrhotic rats. J Hepatol 2012;56:1033-1039.

[140] Fernandez-Miranda C, Perez-Carreras M, Colina F, Lopez-Alonso G, Vargas C, Solis-Herruzo JA. A pilot trial of fenofibrate for the treatment of nonalcoholic fatty liver disease. Dig Liver Dis 2008;40:200-205.

[141] Nakamuta M, Morizono S, Soejima Y, Yoshizumi T, Aishima S, Takasugi S, et al. Short-term intensive treatment for donors with hepatic steatosis in living-donor liver transplantation. Transplantation 2005;80:608-612.

[142] Basaranoglu M, Acbay O, Sonsuz A. A controlled trial of gemfibrozil in the treatment of patients with nonalcoholic steatohepatitis. J Hepatol 1999;31:384.

[143] Laurin J, Lindor KD, Crippin JS, Gossard A, Gores GJ, Ludwig J, et al. Ursodeoxycholic acid or clofibrate in the treatment ofnon-alcohol-induced steatohepatitis: a pilot study. Hepatology 1996;23:1464-1467.

[144] Holden PR, Tugwood JD. Peroxisome proliferator-activated receptor alpha: role in rodent liver cancer and species differences. J Mol Endocrinol 1999;22:1-8.

[145] Palmer CN, Hsu MH, Griffin KJ, Raucy JL, Johnson EF. Peroxisome proliferator activated receptor-alpha expression in human liver. Mol Pharmacol 1998;53:14-22.

[146] Cariou B, Zair Y, Staels B, Bruckert E. Effects of the new dual PPAR alpha/ delta agonist GFT505 on lipid and glucose homeostasis in abdominally obese patients with combined dyslipidemia or impaired glucose metabolism. Diabetes Care 2011;34:2008-2014.

[147] Cariou B, Hanf R, Lambert-Porcheron S, Zair Y, Sauvinet V, Noel B, et al. Dual peroxisome proliferator-activated receptor alpha/delta agonist GFT505 improves hepatic and peripheral insulin sensitivity in abdominally obese subjects. Diabetes Care 2013;36:2923-2930.

[148] Parker HM, Johnson NA, Burdon CA, Cohn JS, O'Connor HT, George J. Omega-3 supplementation and non-alcoholic fatty liver disease: a systematic review and meta-analysis. J Hepatol 2012;56:944-951.

[149] Lu Y, Boekschoten MV, Wopereis S, Muller M, Kersten S. Comparative transcriptomic and metabolomic analysis of fenofibrate and fish oil treatments in mice. Physiol Genomics 2011;43:1307-1318.