• 75 Views
  • 42 Downloads
nurr: Vol. 4
Research Article
Nuclear Receptor Research
Vol. 4 (2017), Article ID 101310, 52 pages
doi:10.11131/2017/101310

Therapeutic Potential of Peroxisome Proliferator-Activated Receptor Modulation in Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis

Leonie Gellrich and Daniel Merk

Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany

Received 13 September 2017; Accepted 26 October 2017

Editor: Li Wang

Copyright © 2017 Leonie Gellrich and Daniel Merk. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

A long neglected hepatic manifestation of the metabolic syndrome, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) arise as serious health burden with alarming global prevalence. The disease complex is currently attracting considerable interest of drug discovery and many experimental approaches are studied in all stages of clinical development. Peroxisome proliferator-activated receptors (PPARs) have a successful history as pharmaceutical targets in the treatment of several aspects of the metabolic syndrome and, therefore, a putative therapeutic value of PPAR modulators in NAFLD/NASH is obvious. However, so far only the PPARα/δ agonist elafibranor has revealed clear efficacy and reached an advanced stage of development while the far more established PPAR subtypes PPARα and PPARγ have disappointed. Still, clinical trial design and population might have obscured beneficial activities and, in addition, synergistic multi-target approaches as well as selective PPAR modulators could generate safer approaches with higher therapeutic efficacy.

1. Introduction

Non-alcoholic steatohepatitis (NASH) is the long-term consequence of non-alcoholic fatty liver disease (NAFLD). NAFLD is defined as hepatic lipid accumulation (steatosis) in the absence of pathologies such as viral hepatitis or alcohol abuse [1] with triglyceride levels above 55 mg/g measured by magnetic resonance imaging (MRI) [2] or determined histologically by a grading system [3]. When hepatic inflammation, hepatocellular injury [4] and hepatocyte ballooning occur additionally, the disease progresses to NASH that may finally lead to fibrosis, cirrhosis, or hepatocellular carcinoma (HCC) [5,6].

Drug discovery for novel NASH therapies is extensive and there are promising approaches in all stages of clinical development. Nuclear receptors have high significance amongst the experimental targets studied for NASH and as well-established targets in the treatment of metabolic diseases. PPARs have been evaluated for the therapeutic potential as well. The available data of PPAR modulation in the disease complex NAFLD and NASH will be focus of this review.

2. Pathophysiology of Non-Alcoholic Steatohepatitis

Related to the pathologies obesity, insulin resistance, hypertension and hyperlipidemia, NAFLD is considered as the hepatic manifestation of the metabolic syndrome [7]. It has an alarming estimated global prevalence of 25% [8]. Furthermore, 75% of patients with obesity-associated type 2 diabetes have some form of fatty liver [9]. According to recent investigations, 7%-30% of NAFLD patients have NASH making the overall prevalence of NASH range between 1.5% and 6.45% [8]. Without countermeasures, patients with NASH will develop cirrhosis within 10 years at 29% [10] and have a 10-fold higher risk of liver-related mortality [11]. During the next decade NASH is estimated to overtake hepatitis C virus infection as the leading cause of liver transplantation in the US [12].

Most NAFLD patients are asymptomatic [13] or have unspecific symptoms such as fatigue, malaise and right upper quadrant discomfort [14]. Thus, an early diagnosis of the disease is difficult. Additionally, there are diagnostic complexities since only few obesity-independent circulating markers for NAFLD and NASH have been reported [15]. Elevated serum transaminase levels are often used in clinical trials for NASH as surrogate marker, especially alanine aminotransferase (ALT) and aspartate transaminase (AST), because they can indicate hepatocellular damage. However, these surrogate parameters have several limitations, because on one hand drugs can influence transaminase levels and on the other hand, fatty liver and fibrosis can occur without affecting ALT and AST levels [14,16,17,18]. Studies relying only on transaminase levels as readout should therefore carefully evaluated. Therefore, new surrogate parameters are required, that have better correlation with the histological outcome. One such potential marker could be adiponectin, which will be discussed later.

Imaging techniques such as ultrasound and MRI are often used to diagnose lipid accumulation in the liver but also have their limitations. With these methods steatosis cannot be distinguished from steatohepatitis and the severity of inflammation, the degree of fibrosis and the stage of the disease cannot be measured [14].

Hence, the gold standard for the diagnosis of NASH is an invasive method, namely liver biopsy followed by histological analysis [19]. However, invasive methods cannot be repeated multiple times and the location, where the liver biopsy is taken is crucial, because parenchymal injury and fibrosis vary in different regions of the liver [20,21]. In a review of Day [22], which recapitulates the current knowledge about NASH, it is postulated, that a liver biopsy is indicated, when some of the following points apply: ALT is greater than twice normal, AST level are higher than ALT, at least moderate central obesity occurs, non-insulin dependent diabetes mellitus (NIDDM) or impaired glucose tolerance arises, hypertension and hypertriglyceridemia develops [23].

To avoid liver biopsy, a NAFLD fibrosis activity score (NAS) was established with 90% prediction accuracy. It predicts the appearance of fibrosis in NAFLD patients considering age, hypertriglyceridemia, body mass index, platelet count, albumin, and AST/ALT ratio [24].

To understand the pathogenesis of the disease a two-hit hypothesis was suggested in 1998 postulating that the first hit is steatosis caused by insulin resistance, which is the reason for enhanced lipolysis and delivery of free fatty acids to the liver. The first hit determines a sensitizing factor for a second hit through metabolic injuries that cause inflammation in the liver. These metabolic injuries include oxidative stress, abnormal cytokine production, lipid peroxidation, endoplasmatic reticulum stress, elevated intrahepatic ceramide levels, elevated plasma fatty acids, elevated intestine derived endotoxins and pathogens or high plasma cholesterol levels. After this second hit, hepatic inflammation, necrosis and fibrosis develop [25].

Steatosis under physiological conditions is a common reversible state in homoeostasis to maintain energy balance. In the fasting state the liver accumulates triglycerides and fatty acid flux from adipose tissue is increased [26,27]. Thus, hepatic steatosis is not necessarily pathological.

Approximately 60% of triglycerides localized in the liver derive from plasma albumin-bound fatty acids and 15% from nutrition. About 25% are result from de novo synthesis [28]. This indicates potential causes and targets for steatosis and that the main source of hepatic lipids is not derived from nutrition or de novo synthesis in the liver but steatosis basically arises because of accumulation of free fatty acids from plasma. One reason for enhanced hepatic fatty acid accumulation is indeed insulin resistance and accordingly type 2 diabetes [29,30]. When carbohydrate levels are increased, insulin levels are enhanced and thus the transcription of sterol regulatory element-binding protein 1c (SREBP-1c) in adipose tissue and liver X receptor (LXR) in hepatic tissue is upregulated leading to induction of lipogenic genes. SREBP-1c and LXR induce L-type pyruvate kinase (L-PK), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), which are key glycolytic enzymes converting carbohydrates into triglycerides [31,32]. SREBP-1c is regulated by peroxisome proliferator-activator receptor (PPAR)α and farnesoid X receptor (FXR). FXR decreases SREBP-1c mRNA expression [33] while PPARα enhances the activity of the SREBP-1c promoter [34]. SREBP-1c is a transcription factor, which regulates multiple genes involved in fatty acid and triglyceride synthesis by binding to a promotor sequence of different genes, called sterol regulatory element-1 (SRE1). SRE1 regulated genes include ATP citrate lyase, which generates acetyl-CoA as well as ACC and FAS, which is involved in the conversion of pyruvate into fatty acids. Both produce palmitate. Another SREBP-1c target gene is stearoyl-CoA desaturase (SCD), which catalyses a rate-limiting step in the synthesis of unsaturated fatty acids and plays a key role in hepatic synthesis of triglycerides and very-low-density lipoproteins (VLDL) [35]. Furthermore, SREBP-1c causes massive production of malonyl-Co-A, which inhibits carnitine palmitoyl transferase-1 (CPT-1), a pivotal enzyme involved in the transport of fatty acids through the inner mitochondrial membrane to the mitochondrial matrix, where their metabolism by β-oxidation takes place. Therefore inhibition of CPT-1 decreases fatty acid transport into the mitochondrial matrix, which in turn lowers β-oxidation [36,37]. Recent studies have reported, that PPARγ is also responsible for the upregulation of lipogenic genes beyond SREBP-1c.

Both transcription factors are crucial for the lipogenic effects detected in NAFLD patients and their expression is upregulated in these patients [38,39]. Due to elevated plasma glucose levels caused by insulin resistance, the transcription factor carbohydrate response element binding protein (ChREBP) is increased [36]. In the liver, ChREBP is responsible for transactivation of L-PK, ACC and FAS, the above mentioned key glycolytic enzymes, converting carbohydrates to triglycerides [40]. Thus ChREBP initiates triglyceride synthesis from carbohydrates in response to glucose. The transcription factors SREBP-1c and LXR in contrast, are activated by insulin signalling and induce the same pathway of carbohydrate conversion to triglycerides. Forkhead box protein O1 (FOXO1) is a transcription factor that is related to lipolysis and insulin resistance and considered as important player in NAFLD and NASH. It induces phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Both enzymes are also induced by glucagon during fasting and in the postprandial state, whereas insulin inhibits their expression. Both are involved in hepatic gluconeogenesis, and therefore, insulin resistance causes a loss of gluconeogenesis inhibition, thus elevating plasma glucose levels. FOXO1 additionally suppresses glucose oxidation, recruits the fatty acid translocase (FAT) also referred to as cluster of differentiation (CD)36, induces lipoprotein lipase (LPL) and stimulates pyruvate dehydrogenase kinase 4 (PDK4), an inhibitor of the oxidation of glucose to acetyl-CoA leading to further glucose accumulation [41,42].

NAFLD patients display low plasma adiponectin levels and the expression of adiponectin receptors in the liver is decreased [43,45,46]. Adiponectin is a peptide hormone produced by adipocytes and is involved in fatty acid oxidation, which could improve hepatic insulin sensitivity and reduce steatosis. Furthermore, the hormone has anti-inflammatory properties. It is secreted from adipose tissue into the bloodstream and responsible for decreased gluconeogenesis and enhanced glucose uptake through improved insulin sensitivity. Furthermore, adiponectin promotes β-oxidation, upregulates uncoupling proteins and reduces tumor necrosis factor-α (TNF-α) [47,48,49].

Besides insulin resistance, obesity plays a major role in the development of steatosis, because high caloric intake and obesity-driven lipolysis of triglycerides dramatically enhance the levels of free fatty acids. Additionally, excessive intake of carbohydrates induces de novo lipogenesis (DNL) in the liver and from a non-lipid source further free fatty acids are synthesised exacerbating steatosis [50]. Finally, dysregulated gut physiology and microbiome impairment may also increase dietary fat uptake [51,52].

Extraordinarily high free fatty acid levels promote the rate of mitochondrial β-oxidation but can overburden this metabolic pathway [53,54] and the lack of (alternative) fatty acid metabolism routes can contribute to steatosis. Imbalance of fatty acid metabolism can arise from mitochondrial dysfunction leading to increased levels of malonyl-CoA [55]. Moreover, mitochondrial dysfunction upregulates compensatory pathways to decrease pathologically high fatty acid levels such as peroxisomal β-oxidation and microsomal omega-oxidation, which cause oxidative stress [56,57].

Also decreased fatty acid efflux from the liver can significantly contribute to steatosis via impaired VLDL synthesis or transport by apolipoprotein (APO) B expression, which is essential for triglyceride accumulation into VLDL [14,58]. In this context, insulin resistance plays a role as well, because in postprandial state insulin targets APO B and leads to VLDL degradation and decreased trigyceride efflux [30].

The progression from NAFLD to NASH is mainly induced through hepatic inflammation, caused by excessive cytokine production, e.g. TNF-α, interleukin (IL)-1, IL-6, macrophage disorder, oxidative stress, direct release of TNF-α by adipose tissue and bacterial overgrowth in small intestine [3].

Arising from hepatic steatosis and alterations in gut microbiota, the liver is sensitized for noxious agents such as endotoxins, a component of the lipopolysaccarid cell wall of gram negative bacteria [59] and other proinflammatory stimuli whereby cytokines are released and macrophages as well as Kupffer cells are activated. Thus inflammation and excessive cytokine production, especially of TNF-α, and nuclear factor-κB (NF-κB) signalling are triggered [60,62,63].

Oxidative stress develops from reactive oxygen species (ROS) released by mitochondria. ROS are produced dependent on the amount of free fatty acids, which are highly increased in hepatocytes from NAFLD patients [64]. ROS lead to lipid peroxidation which in turn alters mitochondrial DNA. Additionally, lipid peroxidation products bind to mitochondrial proteins inhibiting the transfer of electrons along the respiratory chain. In consequence, further ROS are produced leading to a vicious cycle. These findings are confirmed by NASH patients displaying mitochondrial lesions and reduced activity of respiratory chain complexes [54].

Another source of oxidative stress could be the cytochrome P450 enzymes (CYP)2E1 and CYP3A4 [65]. CYP2E1 plays a key role in alcoholic liver disease and is also important in the pathogenesis of oxidative stress in NAFLD patients, because of its ability to stimulate lipid peroxidation. During phase I of xenobiotic metabolism CYPs bind substrates via the formation of an oxy complex which is subsequently reduced to a peroxy-complex. Thus ROS is generated during the intermediate stages of CYP-mediated biotransformation [66]. Elevated hepatic iron concentrations might also play a role in the development of ROS and NAFLD patients display elevated serum iron in form of serum ferritin. Furthermore, increased hepatic iron storage is correlated to hepatic damage and fibrosis by inducing oxidative stress, understood as an increase in the steady state concentration of oxygen radical intermediates [67,68].

Hepatic inflammation leads to activation of hepatic stellate cells (HSCs) which occurs in all cases of liver damage. In resting state, HSCs contain vitamin A lipid droplets and are important for fat storage [69]. Activated HSC, so-called myofibroblast-like cells, are the major cell type involved in liver fibrosis, where fibrotic scar tissue replaces liver cells when chronic liver injury occurs [70,71]. Upon activation by inflammatory stimuli, HSCs enter proliferation and induce contractility. Moreover, activated HSCs influence microcirculation and portal hypertension caused by vasoactive substances and induce chemotaxis of granulocytes and macrophages. Additionally, their vitamin A content decreases and they start secreting collagen, which is responsible for the development of scar tissue leading to fibrotic remodelling and, terminally, cirrhosis [3].

Although NAFLD is one of the most common liver diseases there is currently no FDA-approved pharmacological therapy available but pharmacological interventions are urgently required since lifestyle changes are rarely effective to treat NASH [12]. Drug discovery should especially focus on decreasing hepatic inflammation and hepatocyte damage, no worsening of fibrosis and a decrease in the NAFLD fibrosis activity score.

So far, fibrates as PPARα agonists, thiazolidinediones (TZDs) as PPARγ agonists and different dual agonists of PPAR, e.g. the dual PPARα/δ agonist elafibranor [72], have been studied in clinical trials. Furthermore, vitamin E [12], the FXR agonist obeticholic acid [73] and succeeding compounds as well as insulin-sensitizing agents, particularly metformin are investigated as therapeutic option for the treatment of NAFLD or NASH. In addition, inhibition of stearoyl-CoA desaturase 1 (SCD1) to enhance fatty acid degradation is evaluated. Earlier stages of clinical development also study the potential of apoptosis signal regulating kinase 1 (ASK1) inhibitors and caspase inhibitors as direct anti-inflammatory and anti-fibrotic approaches.

PPARs play a crucial role in drug development for NAFLD and NASH and it is important to consider this group of nuclear receptors more in detail to assess its opportunities and limitations.

3. Role of PPARs in Metabolic Syndrome and NASH

3.1. Peroxisome proliferator-activator receptors (PPARs)

In 1969 for the first time, peroxisome-like particles were detected and in 1990 the first PPAR was cloned from mouse liver [74,75]. Since then, research on their function and potential use as drug target was very intensive. PPARs are members of the family of nuclear receptors and exist in the subtypes PPARα, PPARβ/δ (from now on named PPARδ) and PPARγ [76]. All subtypes act as ligand-activated transcription factors and usually form a heterodimer with a retinoid X receptor (RXR). PPARs have five or six structural regions (A–F) in four functional domains. The structural region C represents the DNA-binding domain (DBD), which binds to PPAR response elements (PPRE) that constitute direct repeats interspaced by a single residue (DR-1). The E/F domain mediates ligand-binding. In a comparative study of mammalian and amphibian samples, the amino acid sequence discrepancy in the E domain between both lineages was investigated. In contrast to the thyroid hormone receptor (THR) and retinoic acid receptor (RAR) the E domain of PPARs has evolved three times faster which could be the reason for the increasing specialisation of PPAR subtypes [77]. In addition, PPARs have large ligand binding sites and accommodate a variety of ligands [78].

When an agonist binds to the ligand binding domain (LBD), a conformational change in the transactivation domain (activation function 2, AF2) is induced leading to release of corepressors and recruitment of coactivators [79]. Typical corepressors for PPAR which are bound to the LBD in inactive state include nuclear corepressor (NCoR) and the silencing mediator of retinoid and thyroid hormone receptors (SMRT). Characteristic coactivators that are recruited upon activation are cAMP response element-binding protein (CREB) and p300, which acetylate histones, PPAR binding protein (PBP), which bridges PPAR and the transcription initiation machinery, and the PPARγ coactivator 1-alpha (PGC-1α), whose role as coactivator is not fully clarified on molecular level [76,80,81]. The DBD of PPAR binds to the PPRE in the promoter region of the corresponding target gene and the transcription of PPAR target genes is upregulated [80,82,83]. PPARs can also repress target genes through mechanisms that are less well investigated. This “trans-repression” could be the result of stabilization of corepressor recruitment after posttranslational PPAR modification by for example sumoylation [76,84].

Despite large differences in signalling pathways and tissue distribution, PPARs mainly regulate metabolic pathways and inflammatory processes [77]. In rodents, PPARs also promote peroxisome proliferation and before it was discovered that this activity lacks in humans, PPAR modulating agents were expected to have carcinogenic potential especially in liver, and therefore the research of PPAR as drug target was discontinued for a while. PPAR expression and function differs between rodents and humans, which makes it difficult to evaluate the function and involvement in diseases of PPARs by using animal models. Table 1 gives an overview over significant species differences concerning PPARs in human and rodents [82].

Known available drugs targeting PPARs are the insulin-sensitizing TZDs e.g. pioglitazone and rosiglitazone which activate PPARγ and were used to treat diabetes mellitus [85]. PPARα agonists, named fibrates, play a role in the treatment of dyslipidemia by their triglyceride lowering and high-density lipoprotein (HDL) raising properties but their therapeutic relevance has decreased [86,87].

PPARs are still in the focus of current drug discovery mainly as targets for inflammatory liver diseases but also in chronic inflammatory disorders such as multiple sclerosis [88]. This review gathers available data on the role of PPARs in NAFLD and NASH and evaluates their therapeutic potential. Each PPAR subtype has individual effects on metabolism in different tissues but all affect the various aspects of the metabolic syndrome including NAFLD and NASH (Figure 1).

T1

Table 1: Comparison of PPAR expression and function between rodents and humans.

3.1.1. PPARα

PPARα is mainly expressed in the liver and other tissues where metabolic processes, especially lipid metabolism, and gluconeogenesis occur, such as adipose tissue, heart, skeletal muscle, intestine, renal cortex and kidney. PPARα transcriptionally regulates multiple metabolic processes including β-oxidation, lipid transport and gluconeogenesis and is also involved in inflammatory processes [76,82].

Activation of PPARα improves plasma lipids. Plasma triglyceride levels are decreased since the balance between hepatic fatty-acid oxidation and glycerolipid esterification is switched towards the catabolic route and, therefore, less triglycerides are available for VLDL synthesis. The metabolism of triglycerides is triggered by the transport of fatty acids through the inner mitochondrial membrane to the mitochondrial matrix, where their metabolism takes place, catalysed by the pivotal enzyme CPT-1 [112] whose expression is regulated by PPARα. Furthermore, expression of other enzymes, which are important for fatty acid uptake, intracellular transport and β-oxidation are under control of PPARα. Besides CPT-1 the most important enzymes are fatty acid transport protein (FATP), CD36 and long-chain fatty acid acetyl-coenzyme A synthase (LC-FACS) [113].

Transporter of long-chain fatty-acids (LCFAs) can be graduated in three groups, namely FATP 1-6, CD36 and plasma membrane-associated fatty-acid binding protein (FABP)pm. Two of three groups count to PPARα target genes. FATP is a transporter for LCFAs with Acyl-CoA synthetase (ACS) activity, which leads to an esterification of the fatty acids and therefore an activation for β-oxidation and a prevention of their efflux. Besides its own ACS activity, FATP forms a complex with a long-chain-fatty-acid-CoA ligase (ACSL) to acetylate fatty acids [114,115,116]. CD36 belongs to the scavenger receptors which recognize oxidized or acetylated low-density lipoprotein (LDL). Except LDL, CD36 recognizes LCFAs and facilitates their transport through membrane barriers into cell compartments [117]. Acetylation of LCFAs via LC-FACS leads to enhanced β-oxidation and decreased efflux of LCFAs as explained above. By the increased β-oxidation and the reduction of DNL, PPARα also improves hepatic insulin resistance [118].

PPARα not only promotes degradation of triglycerides, but also induces the hydrolysis of lipoproteins, since LPL expression is induced by PPARα and the inhibitor of LPL, APO CIII, is repressed by PPARα [119,120]. In addition, the amount of HDL cholesterol is increased, because PPARα activation induces APO AI [121] and APO AII [122] expression in hepatocytes, which are the major HDL APOs. In contrast, in rats PPARα activation suppresses APO AI expression and lowers HDL.

Besides its role in lipid homeostasis, PPARα activation inhibits inflammatory genes induced by NF-κB and directly inhibits NF-κB by increasing the transcription of the Inhibitor of κB (IκB) [123,124]. Furthermore, it decreases the expression of acute-phase response genes, IL-6 and C-reactive protein (CRP) [125,126]. In the endothelial tissue, vascular cell adhesion molecule-1 (VCAM-1) expression is reduced lowering the formation of atherosclerotic plaques [127,128,129]. Another anti-atherosclerotic effect results from PPARα activation in macrophages, where the cholesterol efflux protein ATP-binding cassette transporter (ABCA1) is induced, causing an increased cholesterol efflux [130]. Furthermore PPARα reduces induction of COX-2 [82] and stimulates peroxisomal β-oxidation of leukotriene B4 (LTB4), which is important for the negative feedback control of inflammatory lipid mediators [131].

3.1.2. PPARγ

PPARγ is predominantly expressed in adipose tissue and immune cells and has a major impact on adipocyte differentiation and lipid metabolism [82]. Furthermore, glucose transporters are induced by PPARγ [132], which rendered PPARγ an attractive drug target for the therapy of diabetes with insulin sensitizers in form of TZDs. In addition, PPARγ interferes with inflammatory processes and several proteins, such as uncoupling protein 1 (UCP-1), that have an important role in thermogenesis, are upregulated by PPARγ [133].

PPARγ is expressed in white and brown adipose tissue at similar levels and is essential for their growth, because PPARγ upregulates genes, involved in adipocyte functions, such as fatty acid uptake, transport and esterification, lipogenesis, lipolysis and thermogenesis as well as adipokine synthesis and secretion [134]. Preadipocyte cell lines were used to investigate the molecular background underlying PPARγ induced adipocyte differentiation. It turned out, that PPARγ is especially required in the early stage of adipogenesis where it induces the transcription factors CCAAT-enhancer-binding proteins (C/EBP)-α C/EBPβ and C/EBPδ [98,135,136]. C/EBP proteins control cellular proliferation, growth and differentiation. C/EBPα has a positive feedback on PPARγ, because its activation induces PPARγ expression [137].

Adipogenesis is an essential process in lipid homoeostasis, because adipocytes are important energy storages. When energy intake exceeds the current need, adipocyte differentiation is upregulated by insulin. Energy mobilization from adipose tissue can be triggered by epinephrine, glucagon and adrenocorticotropic hormone (ACTH) [138].

PPARγ activation can effectuate the conversion of white adipose tissue into brown adipose tissue. Brown-like adipocytes are called brite or beige adipocytes, which develop from their white counterparts or from adipocyte precursor cells [139,140]. White adipose tissue is the storage of energy in form of fat while brown adipose tissue is mainly responsible for thermogenesis and negatively associated with adiposity, insulin resistance and aging [141].

Besides adipogenesis, PPARγ regulates genes involved in lipid metabolism. For instance, LPL and FABP4 also referred as adipocyte Protein 2 (aP2), a carrier protein for fatty acids that facilitates the transport of fatty acids into the adipocytes, are upregulated via PPARγ [85,135,142,143]. FATP [144] and oxidized LDL receptor 1 (OLR1) [145] are supplemental target genes of PPARγ, which arrange fatty acid influx into adipocytes leading to internalization and degradation of oxidatively modified low density lipoprotein (oxLDL) by vascular endothelial cells [145]. Thus, PPARγ plays a role in adipocyte differentiation and transport of fatty acids from periphery to adipocytes and other tissues, but, furthermore, PPARγ is important for fatty acid biosynthesis, where the PPARγ target genes fatty-acyl-CoA synthase (FACS) [142], glycerol kinase [145] and the glycerol transporter aquaporin 7 (AQP7) [146] are involved.

Moreover PPARγ induces the transcription of a class B scavenger receptor, called CD36, that leads to binding and internalisation of oxLDL, but as well recognizes LCFAs and supports their transport through membrane barriers into cell compartments, which is important for following β-oxidation in mitochondria [147]. Therefore CD36 is important, when elevated fatty acid plasma level occur such as in NAFLD/NASH.

So far, the only approved therapeutic use of PPARγ agonists is their insulin sensitizing effect. Insulin resistance occurs often in combination with obesity, in particular visceral obesity. High glucose levels, caused by insulin resistance, induce DNL and, therefore, arrange further adipose tissue and enhance free fatty acid concentrations in plasma leading to accumulation of fat in the liver. In addition, when insulin resistance occurs, insulin levels in plasma are elevated as compensatory effect. Under physiological conditions, insulin has anti-lipolytic effects and prevents hydrolysis of triglycerides into glycerol and free fatty acids. Since free fatty acids play a crucial role in progression of NAFLD to NASH, insulin has positive effects on fatty liver, which could be utilised as therapeutic option by the insulin sensitizing effect of PPARγ agonists.

NAFLD patients are frequently obese. In obesity, dysfunctional fat cells develop that release cytokines, such as TNF-α, IL-6 and resistin supporting the development of insulin resistance and decreasing insulin sensitizing cytokines like adiponectin [148,149]. PPARγ activation on one hand leads to elevated adiponectin levels and on the other hand can resolve insulin resistance by increasing the expression and translocation of the glucose transporters 1 (GLUT)-1 and -4, which improves glucose uptake into liver and skeletal muscle cells [150]. Furthermore PPARγ agonists decrease IL-1β, IL-6 and TNF-α levels [151,152].

Additionally, it was found out that PPARγ increases nitric oxide bioavailability in cultured endothelial cells potentially due to a repression of the NADPH oxidase enzyme complex. Therefore, a decreased superoxide anion production and less oxidative stress occur, which is another beneficial effect preventing steatohepatitis in NAFLD patients [153,154].

PPARγ activation has positive and negative impact on NAFLD an NASH. Adipogenesis and differentiation of adipocytes is not desirable for the treatment, but the weight gain induced by PPARγ agonists mainly tended to be peripheral fat rather than central, thus no exacerbation of NAFLD and NASH is expected by PPARγ activation. The positive aspect of PPARγ as drug target are the insulin sensitizing and anti-inflammatory effects as well as its adiponectin increasing property and its beneficial influence on oxidative stress [155].

3.1.3. PPARδ

PPARδ is ubiquitously expressed throughout the body, but the highest expression rates are found in small intestine, colon, heart, adipose tissue, inflammatory cells, skin and brain [113,156]. Its main function is regulation of fatty acid β-oxidation in skeletal muscle. In contrast to PPARα, PPARδ has little influence on β-oxidation in the liver. The receptor can influence lipid metabolism and reduce inflammation and insulin resistance [157,158,159,160] but, so far, PPARδ is the least investigated receptor amongst PPARs. The PPARδ agonist GW501516 was studied as promising drug candidate for the treatment of dyslipidaemia but it was withdrawn due to safety concerns in clinical trials [161]. One reason was its potential carcinogenicity. It was detected that in colon cancer cells PPARδ is upregulated by the adenomatous polyposis coli (APC) tumour suppressor pathway [162]. Potentially PPARδ could then be activated by products of cyclooxygenase-2 (COX-2). This could also explain the beneficial effects of non-steroidal anti-inflammatory drugs against colorectal tumorigenesis. The carcinogenic potential of GW501516 was the main reason for its failure during development. Of note, the dual PPARα and PPARδ ligand elafibranor showed none of these effects [162].

Apart from GW501516 and elafibranor, which will be discussed later, most experiments to study PPARδ were conducted in cell culture models and some animal studies. Since there are major differences between animal and human PPAR functions, their results must be evaluated carefully.

To identify target genes of PPARδ subcutaneous white adipose tissue isolated from obese patients was incubated with selective agonists of all three PPARs (GW7647 for PPARα, GW0742 for PPARδ and BRL49653 (rosiglitazone) for PPARγ). All subtypes decreased leptin and IL-6 secretion, whereas only PPARα and PPARδ agonists increased hepatocyte growth factor secretion. The PPARδ agonist down-regulated angiogenin and induced TIMP metallopeptidase inhibitor 1(TIMP-1) release [163].

Other studies have reported that PPARδ activation induces SCD1. Although SCD1 has lipogenic properties by stimulating hepatic synthesis of triglycerides and VLDL, it has beneficial effects on oxidative stress. Endoplasmic reticulum stress is induced by saturated fatty acids and SCD1 catalyses the formation of monounsaturated fatty acids from unsaturated fatty acids [164,165].

In brown fat, Twist-related protein 1 (Twist-1) is expressed and inhibits PGC-1α. PGC-1α mediates mitochondrial oxidative metabolism and uncoupling. PPARδ seems to act downstream of Twist-1 signal transduction but also increases Twist-1 expression indicating that Twist-1 serves as a negative-feedback to modulate the PGC-1α/PPARβ/δ-regulated brown fat metabolism [166].

In a mouse myoblast cell line (C2C12), PPARδ induced the expression of FOXO1, which suppresses glucose oxidation, recruits CD36, induces LPL and stimulates PDK4, an inhibitor of the oxidation of glucose to acetyl-CoA [167]. Furthermore, many genes involved in lipid utilisation, cholesterol efflux, energy uncoupling, and β-oxidation were upregulated [168] including UCP-1, UCP-2, UCP-3, FABP3, LPL, M-CPT-1 and adipose differentiation-related protein (ADRP). UCP carrier proteins are expressed in the mitochondria of brown fat and involved in thermogenesis [169]. FABP3 supports the transport of LCFAs from cytoplasm to the nucleus and M-CPT-1, the isoform of carnitine palmitoyl transferase 1 in muscles, catalyses the transport of LCFAs from cytosol into mitochondria by the transfer of an acyl group [168]. ADRP is involved in the formation of lipid droplets [170].

In C2C12 cells and in human myotubes angiopoietin-like protein 4 (ANGPTL4) was identified as target gene of PPARβ/δ [171]. ANGPTL4 inhibits LPL and increases lipolysis and fatty acid oxidation and decreases blood glucose levels. Furthermore, it is an inhibitor of angiogenesis and apoptose [172]. However, PPARδ has as well pro-angiogenic properties in heart via calcineurin and in skeletal muscle via vascular endothelial growth factor A (VEGF-A) and platelet endothelial cell adhesion molecule (PECAM-1) [173,174].

PPARδ selective agonists elevated HDL levels in diabetic mice [175] and GW501516 increased HDL while decreasing triglyceride and insulin levels in obese rhesus monkeys [176]. A first study in healthy volunteers displayed increased circulating HDL levels [177]. In contrast, in overweight volunteers, GW501516 treatment did not affect HDL levels. In the same trial, circulating triglycerides, APO B, LDL, insulin levels, liver fat content and urinary isoprostanes were decreased [161]. Further mouse studies with GW501516 also showed attenuated weight gain and insulin resistance by increasing β-oxidation of fatty acids in skeletal muscle [178]. Additionally, PPARδ exhibits anti-inflammatory activity by preventing the activation of macrophages and Kupffer cells [179]. Accordingly, GW501516 reduced NF-κB activity and stimulated IκBα expression [180]. In adipocytes, PPARδ activation decreased IL-6 levels [181].

F1
Figure 1: Contributing factors and molecular targets in NAFLD & NASH. Insulin resistance is an important factor in the pathology of non-alcoholic fatty liver disease (NAFLD) and elevated insulin levels play a crucial role in disease manifestation and progression. Insulin enhances the transcription of sterol regulatory element-binding protein 1c (SREBP-1c) and liver X receptor (LXR), which upregulate the three key glycolytic enzymes L-type pyruvate kinase (L-PK), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). These enzymes catalyse conversion of carbohydrates to triglycerides. SREBP-1c additionally causes malonyl-Co-A production, which inhibits carnitine palmitoyl transferase-1 (CPT-1), the pivotal enzyme for the transport of fatty acids through the inner mitochondrial membrane to the mitochondrial matrix, where their metabolism by β-oxidation takes place. When high glucose levels occur, carbohydrate response element binding protein (ChREBP) is upregulated, which further promotes conversion of carbohydrates to triglycerides. The transcription factor forkhead box protein O1 (FOXO1) induces phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) which are usually induced by glucagon and repressed by insulin. Both enzymes are involved in hepatic gluconeogenesis, and therefore, insulin resistance leads to a loss of gluconeogenesis inhibition, thus elevating plasma glucose levels. FOXO 1 additionally induces cluster of differentiation (CD)-36, which recognizes long-chain fatty-acid (LCFAs) and mediates their transport through membrane barriers into cellular compartments. Lipoprotein lipase (LPL) which hydrolyses triglycerides to free fatty acids and glycerol is also upregulated by FOXO1 further increasing the amount of free fatty acids. Another FOXO1 target gene is pyruvate dehydrogenase kinase 4 (PDK4), an inhibitor of glucose oxidation to acetyl-CoA leading to further glucose accumulation. The peptide hormone adiponectin also seems to be crucially involved in the pathogenesis of NAFLD and is downregulated in affected patients. It improves insulin sensitivity and promotes β-oxidation, reduces gluconeogenesis and has anti-inflammatory properties by inhibiting tumor necrosis factor-α (TNF-α). When free fatty acids are elevated, mitochondrial β-oxidation can be overburdened leading to further hepatic accumulation and steatosis. As compensatory effect peroxisomal β-oxidation and microsomal omega-oxidation are activated and cause oxidative stress. Impaired very low density lipoprotein (VLDL) synthesis and transport by apolipoprotein (APO)-B expression, which is essential for triglyceride accumulation into VLDL, may also contribute to hepatic fat accumulation. Insulin is also involved in this context, because in postprandial state insulin targets APO-B and leads to VLDL degradation. The progression of NAFLD to non-alcoholic steatohepatitis (NASH) is mainly caused by hepatic inflammation. Inflammation is triggered by alterations in gut microbiota leading to release of endotoxins by gram negative bacteria and reactive oxygen species (ROS). ROS are produced as a result of dysfunctional mitochondria, by CYP enzymes in lipid peroxidation, or by iron, which increases the steady state concentration of oxygen radical intermediates. Hepatic inflammation activates hepatic stellate cells (HSCs) leading to fibrotic remodelling and, terminally, cirrhosis. PPARα can counteract the disease by inducing CPT-1 and CD36, thus improving the transport of fatty acids into mitochondrial matrix. Furthermore, PPARα upregulates enzymes that mediate β-oxidation, decreases de novo lipogenesis and inhibits NF-κB signalling by increasing the transcription of the Inhibitor of κB (IκB). However, PPARα also promotes hydrolysis of lipoproteins by inducing LPL expression and LPL inhibitor APO CIII repression which increases free fatty acid levels. PPARγ also induces CD36 promoting fatty acid uptake into mitochondria and increases adiponectin levels that improve insulin sensitivity. Furthermore, PPARγ can reduce ROS formation and oxidative stress by repression of the NADPH oxidase enzyme and TNF-α. On the other hand, PPARγ activation promotes adipocyte differentiation, fatty acid biosynthesis and LPL expression, which enhance fatty acid formation. PPARδ can influence lipid metabolism, but mainly in skeletal muscle (not depicted). The receptor induces the expression of FOXO1, which has negative and positive effects on NAFLD. On one hand, flux of fatty acids into mitochondria is improved by CD36, but on the other hand glucose levels are enhanced by PDK4, PEPCK and G6Pase. In addition, LPL is induced by FOXO1 and directly by PPARδ. APO B levels are decreased when PPARδ is activated. A positive impact of PPARδ is the reduction of NF-κB activity and stimulation of IκBα expression.

4. textitIn Vivo Effects of PPAR Modulation in NASH

4.1. PPARα

PPARα agonists have been studied to evaluate their potential efficacy in the treatment of NAFLD and NASH in animal models and in clinical trials. Most results are derived from rodent models using a variety of high fat diets to induce fatty liver disease. However, several animal studies were conducted with Wy-14,643, which is defined as selective for PPARα by many authors, but has considerable potency on PPARγ, as well [182,183]. Thus, interpretation of these studies should consider this concern and evaluate the results as arising from activation of PPARα and PPARγ.

4.1.1. PPARα animal studies

There are different methods to induce NASH in animal models. Mainly different diets were conducted, that result in liver injury similar to NASH. One method is to apply a methionine and choline deficient (MCD) diet to rodents which in consequence develop progressive fibrosing steatohepatitis. In one study, after 5 weeks MCD diet-fed wild-type and PPARα knockout mice displayed moderate steatohepatitis and elevated ALT levels. Afterwards the mice were treated with Wy-14,643 which prevented intrahepatic triglyceride accumulation and resolved steatohepatitis in wild-type mice. Wy-14,643 increased FABP and enzymes essential for β-oxidation. The knockout mice were unaffected by Wy-14,643 [184]. In another trial, MCD diet-fed mice were treated with Wy-14,643 and examined after five and twelve days to detect, whether Wy-14,643 has additional antifibrotic effects, beyond the already observed effect on steatohepatitis. The study revealed significantly reduced ALT levels and hepatic lipoperoxides and less severe steatohepatitis. After twelve days, hepatic triglycerides and histology were normal. To identify the fibrosis reducing mode of action, expression of relevant genes including matrix metalloproteinases (MMPs), its inhibitors TIMP-1 and TIMP-2 and transforming growth factor beta 1 (TGF-β1) were studied but only TIMP-2 and MMP-13 were decreased [185]. Thus, beyond the benefit on steatohepatitis no additional effect on fibrosis could be confirmed.

Fibrates which are less potent PPAR agonists compared to Wy-14,643 were also studied in NASH animal models. The single fibrates have a different pattern of selectivity. Clofibric and fenofibric acid activating both PPARα and PPARγ, with a 10-fold selectivity for PPARα. Bezafibrate has no specificity for any of the three PPAR subtypes [186]. Again this lack of selectivity must be considered for the interpretation of the following studies.

APO E2 knockout mice who were fed a western-type high fat diet under fenofibrate treatment revealed a decrease in hepatic macrophage accumulation and reduced steatosis. Expression of inflammatory genes was diminished and genes involved in β-oxidation were increased [187]. Another mouse model, using a high-caloric and high-cholesterol diet (HCD) to induce NAFLD, reported promising data for fenofibrate which caused a significant improvement of NAFLD symptoms, reduced apoptosis and repressed genes related to oxidative stress in endoplasmic reticulum such as inositol-requiring enzyme 1α (IRE1α) and X-box binding protein 1 (XBP1). Moreover, phosphorylation of c-Jun N-terminal kinase (JNK) was decreased [188]. In a very recent animal study with mice on fructose-enriched diet (FED) to induce hepatic dyslipidemia and insulin resistance, fenofibrate positively affected the expression of several genes involved in hepatic metabolism, inflammation and fibrosis such as suppressor of cytokine signaling 3 (SOCS-3), apoptosis antigen 1 (CD95), mast cell degranulating peptide, TGF-β1 and leptin. Additionally, reductions in body weight, insulin resistance estimated by Homeostatic Model Assessment (HOMA), liver triglycerides, AST/ALT ratio and TNF-α were observed [189].

Overall, PPARα agonists showed promising effects on steatosis and steatohepatitis in the few animal models. Their impact on fibrosis is not consistent. Wy-14,643 revealed no effect on fibrosis, whereas fenofibrate might possess some therapeutic potential. But it is possible, that its activity on PPARγ was responsible for this outcome. While the trials in rodents indicate promising outcomes, studies in humans showed more variation (Table 2).

4.1.2. PPARα clinical trials

In 1996 a pilot study with clofibrate in 16 patients with liver biopsy proven NASH and hypertriglyceridemia was conducted. Besides 2 g per day clofibrate treatment, 24 patients were treated with 13-15 mg/kg/d ursodeoxycholic acid and both treatments were compared to each other, but no placebo control group was recruited. No changes in ALT, AST, γ-glutamyltransferase (GGT), bilirubin, triglycerides or cholesterol levels were observed. Only alkaline phosphatase (ALP) levels were significantly reduced under clofibrate. Furthermore, no improvement in the histological grade of steatosis, inflammation or fibrosis could be confirmed after 12 months of treatment. In contrast, ursodeoxycholic acid improved values of ALP, ALT, GGT and the histological grade of steatosis [190]. Another trial enrolled 46 patients with histologically proven NASH that received 600 mg gemfibrozil per day or placebo. After 4 weeks, mean serum ALT, AST and GGT levels were significantly decreased contradicting the former trial, but the higher number of patients might have enabled a more robust result [191]. Collectively 74% of treated patients in comparison to 30% in the placebo group had a significant reduction of ALT levels. No changes in triglyceride values or mean body weight could be detected. Neither a biopsy to examine a histological effect nor an analysis of side effects were conducted. A third pilot trial investigated not only surrogate markers, but also analysed histological parameters. It enrolled 16 patients with biopsy-confirmed NAFLD that received 200 mg fenofibrate per day for 48 weeks. Unfortunately, a placebo control was missing. At the end of treatment, histological outcomes were determined from liver biopsy revealing a reduced grade of hepatocellular ballooning and degeneration, but no effects on steatosis, lobular inflammation, fibrosis or NAFLD activity score could be observed. Surrogate parameters showed a significant decrease in triglyceride, glucose, ALP, GGT, ALT and AST levels as well as increased APO A1 levels. Furthermore, an improvement of insulin sensitivity and decreased insulin levels were observed [192]. A rather short trial reported by Riserus et al. for only 2 weeks with 12 healthy moderately overweight participants receiving 20 µg of the selective PPARα agonist GW590735 or placebo per day, revealed triglyceride lowering activity but no effects on APO B, LDL, insulin or HDL were observed. A histological examination to determine the efficacy of GW590735 was not conducted [161].

In summary, animal models suggest significant beneficial effects of PPARα or PPARα/γ activation in NAFLD and NASH. The few and rather small clinical trials on humans fail to confirm the promising in vivo activity. Their outcomes are inconsistent and not very promising. This might in part be due to different functions and distribution patterns of PPARα or PPARs in general in human and rodents. Additionally, the lack of clinical efficacy could also arise from underpowered studies.

4.2. PPARδ

Similar to PPARα, most data for PPARδ modulation in NAFLD/NASH has been reported from animal studies while there are only few clinical trials (Table 2).

4.2.1. PPARδ animal studies

The PPARδ agonist GW501516 was evaluated in rat L6 myotubes and a mouse model with high fat diet to induce NAFLD/NASH. In vitro data from a microarray indicated that enzymes of mitochondrial fatty acid-oxidation, electron transport, and ketogenesis were induced. Especially PDK4, which is involved in glucose oxidation, and genes important for fatty acid transport and activation, e.g. FATP, LC-FACS and CPT-1 were upregulated. Accordingly, GW501516 ameliorated diet-induced obesity and insulin resistance in mice. Furthermore, PPARδ activation led to an improved metabolic rate by enhanced β-oxidation. In addition, the proliferation of mitochondria was improved and lipid droplets were reduced in the skeletal muscles of GW501516-treated mice [178].

Another animal study compared GW501516 as selective PPARδ agonist and bezafibrate, a PPAR pan-agonist, activating PPARα and PPARδ and with lower potency PPARγ [193], in a MCD diet model to induce NASH in mice. Both agents, administered over 5 weeks, normalized hepatic triglyceride levels and the amount of fat droplets within hepatocytes. In addition, inflammation and the number of activated HSC, that cause fibrosis, were decreased. Specific target genes were monitored in hepatic tissue after isolation of the liver, indicating increased fatty acid β-oxidation after bezafibrate and GW501516 treatment by enhanced acyl-CoA oxidase (ACO), CPT-1, FABP and peroxisomal ketothiolase gene expression. Furthermore, inflammatory cytokines, such as TGF-β1, IL-6, IL-1β, monocyte chemoattractant protein (MCP)-1, TNF-α and NF-κB were decreased. Bezafibrate but not GW501516 reduced plasma ALT levels and enhanced plasma adiponectin. This could be due to its dual agonism on PPARα and PPARδ [194].

An alternative PPARδ agonist that has been used in NAFLD animal models is GW0742. It was studied in Otsuka Long Evans Tokushima Fatty (OLETF) rats which are often used as model of type II diabetes with obesity or to evaluate fatty liver and hepatic inflammation. To induce NASH, mice were fed a MCD diet. Upon treatment with GW0742 over 5 weeks, intrahepatic triglyceride content, expression of inflammatory cytokines such as TNF-α and MCP-1 as well as PGC-1α gene expression were significantly decreased [180].

4.2.2. PPARδ clinical trials

Beyond these preclinical animal data, there are just two small clinical trials examining potential effects on fatty liver of selective PPARδ agonists. A small study reported by Risérus et al. investigated the effect of 10 mg GW501516 in 18 healthy but moderately overweight participants over 2 weeks versus placebo and 20 µg of the PPARα agonist GW590735. In comparison to the PPARα agonist, GW501516 showed much more promising results with significant reductions in fasting plasma triglycerides, APO B, LDL, insulin, liver fat content and urinary isoprostanes. Urinary isoprostanes are prostaglandin-like substances, which develop when fatty acids react with free radicals in a peroxidation. They serve as inflammatory mediators and markers for lipid peroxidation, respectively oxidative stress. No effect was found on HDL levels. Furthermore, an improvement of β-oxidation was indicated by a higher skeletal muscle expression of CPT-1b [161]. The second PPARδ agonist used in a clinical trial was MBX-8025 which was evaluated in 181 dyslipidaemic and overweight patients. 50 or 100 mg MBX-8025 were compared to 20 mg atorvastatin or combined with atorvastatin for 8 weeks. Both, MBX-8025 alone and the combination with atorvastatin showed promising metabolic effects. APO B, non-HDL-cholesterol, especially LDL, triglycerides, free fatty acids, high-sensitivity CRP and the liver enzymes GGT and ALP were reduced while HDL levels were increased. Unfortunately, specific parameters concerning NAFLD or NASH or biopsies were not studied [196].

In summary, available data on PPARδ in NAFLD/NASH is exiguous, similar to PPARα. Based on data from rodent models, PPARδ seems very promising to treat NASH and clinical data is less disappointing. Still, more experiments with selective PPARδ agonists are required to reveal the receptor's exact molecular signalling pathways and to confirm the therapeutic value of PPARδ modulators in clinical trials.

4.3. PPARγ

PPARγ has long been successfully used as therapeutic target for the treatment of type 2 diabetes since its activation has insulin sensitizing activity. Additionally, anti-inflammatory effects have been reported, that could be very beneficial in the therapy of NAFLD and NASH as well [85]. Since TZDs were approved drugs, many clinical data from large patient collectives are available (Table 2). Most of them have been withdrawn because of safety concerns. Pioglitazone was associated with bladder tumors [197], rosiglitazone revealed an increased risk of cardiac events (although the FDA in 2013 revised the assessment) [198] and troglitazone, an early member of the TZDs, caused drug-induced hepatitis and was already withdrawn in 2000. Moreover, there are numerous animal experiments for TZDs as experimental NAFLD/NASH therapeutics.

4.3.1. PPARγ animal studies

Pioglitazone was studied in a rat model of liver fibrosis induced by a choline-deficient L-amino acid-defined (CDAA) diet. First assessment after 2 weeks indicated that pioglitazone improved hepatic steatosis, because liver weight was reduced and triacylglycerol content was decreased. Furthermore, pioglitazone prevented liver fibrosis, and reduced pre-neoplastic lesions in the liver. After another 10 weeks pioglitazone prevented the activation of HSCs resulting in reduced expression of type I procollagen, MMP-2, that is increased when HSCs are activated and may be involved in liver carcinogenesis, TIMP-1 and TIMP-2. MMP-13 was not affected, which mainly hydrolyses extracellular matrix and can degrade fibrous collagen, e.g. types I and III collagens, two major factors in the formation of hepatic fibrosis. In addition, pioglitazone reduced serum ALP and bile acid levels in a dose dependent manner, but had no influence on ALT levels. It was observed that activated HSC also referred to as myofibroblast-like cells, who express alpha smooth muscle actin (aSMA), showed proliferation in the livers of rats fed CDAA diet for 10 weeks and pioglitazone was able to reduce the area of aSMA-positive cells. Moreover the area of placental glutathione S-transferase (GST-P), a marker for hepatocarcinogenesis, was reduced by pioglitazone [199].

Another animal study using various methods (carbon tetrachloride (CCl4) injection, choline-deficient diet, bile duct ligation(BDL)) of inducing fibrosis showed less beneficial effects. Mice were analysed for severity of hepatic fibrosis in pioglitazone treated animals versus untreated controls. In the CCl4 and in choline-deficient diet induced fibrosis, pioglitazone revealed beneficial effects in reducing hepatic fibrosis, hydroxyproline content, a major component of the protein collagen which plays a key role for collagen stability, hepatic mRNA expression of collagen type I, and profibrotic genes, as well as the amount of aSMA. However, antifibrotic effects were lost when pioglitazone was administered after 5 weeks instead of 2 weeks after fibrosis induction. Moreover, pioglitazone failed to reduce fibrosis in the BDL model, independent from the time of administration. Thus, the study indicates a time and severity dependent effect of pioglitazone on fibrosis [200].

Pioglitazone was also tested in a murine MSD diet model. Besides steatohepatitis, effects on systemic insulin sensitivity were studied after pioglitazone treatment. However, no effect on insulin sensitivity was observed, although pioglitazone is known to have insulin sensitizing effects [85]. Still, pioglitazone was able to reduce hepatic fat accumulation and steatohepatitis. Adiponectin levels were enhanced, but ACO and ACC, which are important for β-oxidation, were unchanged [201].

Hsiao et al. evaluated the hepatoprotective effect of pioglitazone in male C57BL/6 mice treated with a 30% fat diet and 100 mg/kg/day pioglitazone for 8 weeks. Tissue oxidative stress was measured by malondialdehyde concentration, which arises when ROS degrades polyunsaturated lipids. Pioglitazone normalized malondialdehyde levels. Additionally, oxidative DNA damage was studied by immunohistochemical 8-oxoguanine (8-oxoG) staining. 8-oxoG develops as most common DNA lesions resulting from ROS. In this experiment, pioglitazone reduced the number of DNA lesions. Furthermore, the expression of antioxidative genes and the potential to repair oxidative DNA damage was evaluated. Under high fat diet, the expression of superoxide dismutase (Sod)1, Sod2, 8-Oxoguanine glycosylase (Ogg1) repairing DNA by base excision and MutY, another DNA glycosylase involved in oxidative DNA damage repair, were significantly decreased but this reduction could be reversed by pioglitazone treatment [202].

4.3.2. PPARγ clinical trials

In 2001, the first clinical trial investigating the efficacy of a TZD in the treatment of NASH was reported. 10 female patients with histologically proven NASH were treated with 400 mg troglitazone per day for 6 months. During the study, seven out of ten patients were responder and only this group of patients was included in data evaluation. ALT and AST levels fell significantly, but biopsy comparisons before and after therapy showed persistent steatohepatitis in all cases. Four of these seven patients experienced a one-point improvement in necroinflammatory grade. Via electron microscopy, an elongation of the mitochondria after therapy was detected, but the number of mitochondria did not change significantly. Additionally, no data concerning hepatic dysfunction has been reported [203]. It was a rather short and small study, so it is difficult to assess its outcome. Furthermore, using ALT and AST levels to evaluate the efficacy on steatohepatitis is rather unspecific and histological examinations revealed no efficacy. Still, a longer trial might be able to confirm the positive outcome of the surrogate parameters [203].

Neuschwander-Tetri et al. studied rosiglitazone during a longer and larger trial. Within the class of TZDs, rosiglitazone is the most potent derivative [204]. 30 patients, including both men and women underwent liver biopsies before and after treatment. All displayed biopsy proven NASH, obesity and elevated ALT levels. The participants received 4 mg rosiglitazone twice daily for 48 weeks, which was the highest recommended dose for the treatment of diabetes. No placebo group was recruited, so the outcome cannot be evaluated without concerns. It was observed that the global necroinflammatory score was significantly improved and biopsies of 10 patients proved that they did not meet the criteria for NASH anymore. Furthermore, hepatocellular ballooning and zone 3 perisinusoidal fibrosis were improved, but the global fibrosis score remained unchanged. Serum levels of ALT, AST, ALP and GGT were decreased, which implicates less cholestatic injury [205]. However, no correlation between baseline liver fat and baseline GGT level or changes in liver fat and changes in GGT level could be demonstrated. Furthermore, no significant correlation was found between changes in ALT level and changes in ballooning or changes in the global necroinflammatory score. Cholesterol and triglyceride levels remained stable throughout the trial. Histological examinations showed a shift in parenchymal localization of steatosis and changes in the relative grade of portal inflammation. Moreover, a shift of inflammation from predominantly lobular to more portal based, was observed. After 48 weeks the treatment with rosiglitazone was discontinued but patients were observed for another 6 months. After this follow-up, enzyme levels had increased to near pre-treatment levels, and the insulin-sensitizing effects of the therapy was not sustained. As side effects, haemoglobin levels were decreased and typically for TZDs patients gained weight. This weight gain mainly tended to be peripheral rather than central fat [155] and therefore it might not increase risks associated with the metabolic syndrome since central or abdominal obesity is strongly correlated with cardiovascular diseases, diabetes and dyslipidaemia. The risk of the appearance of heart diseases, hypertension, insulin resistance and type 2 diabetes is much higher in patients with central obesity than peripheral obesity [206]. In conclusion, there was no correlation between changes in ALT level and changes in ballooning or changes in the global necroinflammatory score. The authors state this could be due to the relatively small cohort size [149] but eventually liver enzyme levels are not perfect surrogate parameters to estimate the severity of the disease. Nonetheless rosiglitazone was able to resolve NASH in nearly half of the patients without improving fibrosis. Additionally, all benefits lasted only as long as the drug intervention.

Promrat et al. reported a trial for NASH that excluded diabetic patients. Only adult non-diabetic patients with biopsy-proven NASH and elevated serum ALT or AST were enrolled. 3 months before treatment, participants were instructed to lose weight, follow a healthy diet and avoid over-the-counter vitamin, mineral, or herbal supplements. After this, 18 out of 20 included patients entered 48-week therapy with 30 mg pioglitazone per day. According to liver biopsy, two thirds of the patients achieved improvements in histological features including steatosis, cellular injury, parenchymal inflammation, mallory bodies, which are damaged intermediate filaments in hepatocytes and fibrosis. The NASH activity index was reduced by at least one point in all patients. For the defined histological response, a reduction in the NASH activity index by at least 3 points with improvements of at least 1 point each in steatosis, parenchymal inflammation, and hepatocellular injury was required which was matched by 67% of patients. Liver fat and volume examination by MRI revealed significant reductions in both parameters. Secondary outcomes included a reduction of serum ALT and AST levels. ALT values were normalized in 72% of patients. This decrease was gradual with an onset after 4 weeks of treatment and lowest levels between weeks 40 and 48. In addition, AST and ALP levels were reduced, insulin sensitivity was improved while serum glucose levels remained constant, hepatic fat content and size decreased and free fatty acid levels were decreased to some extent. There were no significant changes in total cholesterol, triglycerides, LDL and HDL cholesterol levels. Again, the degree of histological improvements was not reliably correlated with changes in serum aminotransferase levels. 83% of patients with a histological response had normal ALT levels, but 50% of patients who did not achieve a histological response had normal ALT values as well. As major side effect, weight gain of 3.5 kg in average occurred in 72% of patients. Unfortunately, the study enrolled no placebo treated control group. Moreover, no follow-up evaluating long-term effects was conducted [207].

Another clinical trial (NCT00227110) in 2006 enrolled patients with impaired glucose tolerance or type 2 diabetes and liver biopsy–confirmed NASH. They were treated with 45 mg pioglitazone per day in combination with a hypocaloric diet over 6 months. This study included a control group, treated with a hypocaloric diet plus placebo. During the trial, participants were assessed by MRI to diagnose hepatic histologic features and hepatic fat content. Additionally, oral glucose tolerance tests were conducted. Diet in combination with pioglitazone improved glycaemic control and glucose tolerance. Furthermore, hepatic insulin sensitivity and glucose clearance were improved, which resulted in reduced free fatty acids, glucose and insulin in plasma. ALT and AST values were reduced, as well as hepatic fat content. Histological examination showed an improvement in steatosis, ballooning, necrosis and inflammation. TNF-α and TGF-β levels were reduced while adiponectin levels were significantly increased. However, also this trial failed to confirm a significant reduction in fibrosis. As side effects fatigue and mild lower-extremity edema occurred and potentially due to dietary intervention. Pioglitazone intake caused only a modest weight gain (2.5±0.5 kg) and increase in body fat of 1.5±0.5% [208].

Lutchman et al. conducted a small study in 18 patients with NASH treated with 30 mg pioglitazone daily for 48 weeks to investigate the serum levels of selected adipokines and proinflammatory cytokines. The aim was to correlate changes to improvements in liver histology and identify a better surrogate parameter to estimate the efficacy of TZD treatment. As result, they only detected increased adiponectin levels while the levels of other studied cytokines, including leptin, IL-1a, IL-6, and TNF-α, remained unchanged. Hepatic steatosis and the NASH activity index score could be correlated to the adiponectin level indicating on one hand, that adiponectin might be a major target of TZDs, rather than proinflammatory cytokines. On the other hand, it could be a better surrogate parameter for estimation of histological outcomes in NAFLD/NASH treatment [209].

The long-term efficacy of 30 mg pioglitazone per day was assessed in another trial lasting 12 months. Only non-diabetic patients with histologically proven NASH were included. Overall 74 patients were recruited and received 30 mg pioglitazone daily or placebo. Both groups were instructed to reduce their calorie intake by 500 Kcal/day, and to perform modest exercise, starting 3-months before drug intervention. 61 patients of 74 had a liver biopsy both at the beginning and the end of the study. After 12 months a reduction in glucose levels (−0.1 mmol/L vs. +0.4 mmol/L), haemoglobin A1c (HbA1c; −0.18% vs. +0.16%), insulin C peptide level (−78 pmol/L vs. +42 pmol/L), ALT (−36.2 U/L vs. −10.9 U/L), GGT (−41.2 U/L vs. −9.4 U/L) and ferritin (−11.3 µg/L vs. −90.53 µg/L), a storage for iron, were observed. Histological examination detected reduced degree of steatosis, hepatocellular injury, lobular inflammation, mallory bodies and fibrosis. In addition, there was a modest increase in serum adiponectin upon pioglitazone treatment. Hepatic steatosis was improved by dietary intervention and physical exercise alone, too. Improvements in fibrosis could be observed as well but failed to reach statistical significance upon treatment with pioglitazone. Only hepatocellular injury and the amount of Mallory bodies were significantly improved by pioglitazone [210].

Ratziu et al. studied the efficacy of rosiglitazone in two trials whereby FLIRT2 was an enlargement of the former FLIRT1 trial. 63 Patients with a histological diagnosis of NASH and elevated ALT levels were enrolled in the placebo-controlled trial FLIRT1. The verum group was treated with 4 mg/day rosiglitazone for the first month and 8 mg/day thereafter for 1 year in total. 4 months after the end of treatment, patients were examined again. Both groups, verum and placebo, were instructed to lose weight if they were obese or overweight. In addition, they had to follow a healthy diet and to exercise at least twice a week. Until then, this was the longest placebo-controlled study of a TZD derivative in NASH. Primary end points for the assessment of the trial were improvements in histologic score of steatosis, normalization of serum transaminase levels and improvements in necroinflammation and fibrosis. Steatosis was reduced in 47% verum patients vs 16% in the placebo group. 38% vs 7% had a reduction in transaminase levels. This effect occurred rapidly during the first 4 months and was stable during the whole treatment period. No significant improvement in liver necroinflammation or liver fibrosis could be detected. Some secondary end points were also not matched, as there was no improvement in other histologic lesions, including hepatocyte ballooning. Still, progression of hepatocyte ballooning, portal inflammation, perisinusoidal fibrosis and overall fibrosis was significantly lower with rosiglitazone. Furthermore, significant reductions in fasting glucose level, HbA1c, and surrogate serum markers of insulin resistance (hyperinsulinemia, HOMA index) were observed. Another aspect of the study was the detection of responder and non-responder. It turned out, that only half of the patients responded and the authors assume that a predictor for response is the absence of diabetes and in the presence of strong steatosis. Decreases in insulin levels, serum glucose and HOMA levels were significantly more pronounced in responders. The loss of liver fat was associated with an improvement of these parameters. In addition, it was observed that serum adiponectin levels correlated negatively with reduction in steatosis and responders had higher baseline adiponectin levels. Moreover, responders had lower median-GGT values, less frequently diabetes and a higher grade of steatosis than non-responders. An improvement of steatosis correlated with reduction of transaminase levels, improvement in insulin sensitivity and increase in adiponectin levels but not with weight changes. 4 months after end of treatment, the long-term efficacy of rosiglitazone was evaluated. ALT, glucose, HbA1c, cholesterol, LDL and haemoglobin levels were normalized to a healthy level and there was still an improvement in insulin levels and HOMA values. Therefore, the authors assume that long term use of TZDs is required and a safety evaluation of long-term TZD intake is required. Side effects detected in this trial beyond weight gain were painfully swollen legs, muscular cramps and reduced serum haemoglobin levels. No hepatotoxicity or cardiovascular events occurred [211].

To study, whether a longer treatment had beneficial effects on the prognosis of the disease, the extensional trial FLIRT2 was conducted. 40 patients that completed FLIRT1 including the final examination after 4 months without treatment were enrolled. 22 received placebo during FLIRT1 and 18 were treated with rosiglitazone. In FLIRT2, all 40 were treated with 8 mg rosiglitazone daily for another 2 years. Both groups demonstrated a reduction in serum insulin by 26%, in HOMA by 30%, and ALT level by 24% but no significant change in the mean NAS, ballooning score, fibrosis stage, or area of fibrosis, was achieved. In the group receiving placebo in FLIRT1, steatosis significantly decreased after 2 years but in the group, that had already received verum previously, no additional improvement could be detected. Thus, the promising first trial, indicating a longer treatment might improve the symptoms of NAFLD an NASH, was not confirmed [212].

In 2010, a phase 3 trial (NCT00063622, PIVENS) was reported that lasted 96 weeks and enrolled 247 adults without diabetes who had biopsy-confirmed NASH. Three groups received either 30 mg pioglitazone daily, 800 IU vitamin E daily or placebo. 90% of all patients underwent a liver biopsy after the end of treatment. The primary outcome was resolution of steatohepatitis, which was only achieved in the vitamin E group. Pioglitazone displayed no benefit compared to placebo. As secondary outcomes, histological features were evaluated and both vitamin E and pioglitazone showed an improvement. Liver biopsies indicated that both treatments lead to a significant reduction in steatosis, lobular inflammation and the activity score for NAFLD but fibrosis scores were not significantly reduced. Vitamin E improved scores for hepatocellular ballooning while pioglitazone was not effective in this scoring system. The serum levels of ALT and AST were reduced by both interventions. Additionally, pioglitazone reduced insulin resistance. Pioglitazone treated patients gained more weight than those who received vitamin E or placebo. The mean increase was about 4.7 kg at week 96. Also this long study enrolling nearly 250 participants failed to demonstrate beneficial effects of a PPARγ agonist on fibrosis in NASH. Treatment with vitamin E seemed superior to pioglitazone, although patients with diabetes or with cirrhosis were excluded from the trial and thus the results cannot be generalized [213].

Another trial evaluated, whether a drug combination could be more beneficial for NAFLD and NASH treatment and ameliorate weight-gain of TZDs. Therefore, 4 mg rosiglitazone and 500 mg metformin twice daily were combined and compared to 4 mg rosiglitazone twice daily and 50 mg losartan once daily and both treatments were matched with 4 mg rosiglitazone twice daily treatment alone. The trial lasted 48 weeks, enrolled 137 patients with biopsy-proven NASH and assessed differences in the improvement of steatosis, hepatocellular inflammation, and fibrosis. Histological examinations revealed no significant difference between the 3 groups but in all groups, an improvement in steatosis, hepatocellular inflammation, ballooning degeneration and especially fibrosis was detectable. Serum aminotransferase levels were reduced in all three groups, as well. No reduced weight gain was achieved by the combination of rosiglitazone with metformin or losartan. Thus, use of combination therapy with metformin or losartan had no additional beneficial effects in this study. Still, this trial was able to detect a significant fibrosis improvement in contrast to all former studies with glitazones in NASH. Unfortunately there was no comparison to a placebo group, which lowers the impact of this study [214].

An Indian study compared 1200 mg pentoxifylline divided in 3 doses per day and 30 mg pioglitazone per day in 60 biopsy proven NASH patients with elevated ALT levels for 6 months. In addition to drug intake, patients were instructed to reduce their caloric intake by 500 kcal/day and perform exercise. Pioglitazone was superior to pentoxifylline but both agents were reported to improve transaminase levels, insulin resistance (HOMA), adiponectin levels and steatosis. TNF-α was not significantly reduced with either intervention. Pioglitazone improved lobular inflammation, portal inflammation and Brunts grade which is a staging score developed to reflect localization and extent of fibrosis [215].

Another study (NCT00994682) was conducted to assess the efficacy and safety of long-term pioglitazone treatment in patients with NASH and prediabetes or diabetes. 101 biopsy proven NASH patients received 45 mg pioglitazone per day or placebo over 18 months and were instructed to follow a hypocaloric diet. A reduction of at least 2 points in the NAFLD disease activity score in 2 histologic categories without worsening of fibrosis was defined as primary outcome and occurred in 58% of pioglitazone treated patients. Additionally, 51% had resolution of NASH. As secondary outcomes, histological improvements, hepatic triglyceride content and metabolic parameters were evaluated. Some individual histologic scores, including the fibrosis score could be improved. Although the fibrosis score was reduced in the verum group, the effect on fibrosis was rather modest with a reduction by -0.5 points. Placebo treatment had no effect on fibrosis. Furthermore, hepatic triglyceride content was reduced and insulin sensitivity was improved. Consistent with previous trials pioglitazone caused weight gain by about 2.5 kg compared to placebo. Although the study aimed at evaluating the efficacy of pioglitazone treatment in patients with NASH and diabetes, a subgroup analysis of the population which is actually suffering from diabetes and NASH is missing [216].

Since the outcome of fibrosis improvement by TZD treatment is not obvious from the above discussed studies, a meta-analysis evaluated the available four high quality randomized, placebo-controlled trials (NCT00063622 [213], Aithal et al. [210], NCT00227110 [208], FLIRT1 [211]). Three of the trial used pioglitazone and one study rosiglitazone. The meta-analysis concludes, that TZDs are able to improve ballooning degeneration, lobular inflammation and steatosis. Additionally, necroinflammation could be significantly reduced, calculated by statistical analysis over the 4 studies. However, including all 4 studies no significance regarding fibrosis improvement could be detected. Without involving the FLIRT1 trial, the improvement in fibrosis was statistically significant. However, evaluating each of the single trials revealed no significant fibrosis improvement. Thus, it is possible, that pioglitazone only has moderate beneficial effects on fibrosis but more clinical data would be required to confirm this activity [217].

Another systematic review supports the thesis that pioglitazone is superior to rosiglitazone. But the key aim of this systematic analysis was to evaluate, whether adiponectin is a main target of TZD treatment. Again 4 studies were analysed, including the study of Belfort et al. [208], Lutchman et al. [209], Ratziu et al. (FLIRT1) [211] and Sharma et al. [215]. In total, 187 patients with liver biopsy proven NASH were investigated, which were treated 6-12 months with a TZD. In all studies, a significant elevation of adiponectin was observed, as well as a significant improvement of steatosis. Lobular inflammation was only significantly improved during pioglitazone treatment, not with rosiglitazone, supporting the former thesis that pioglitazone might be superior to the more potent rosiglitazone. The best efficacy in ballooning and fibrosis was found in trials with pioglitazone in the highest dose or the longest duration of therapy. Insulin resistance and liver function were improved as well. Moreover, it could be verified, that an increase in circulating adiponectin levels correlates with histological improvement. These findings might support the development of selective PPARγ modulators (SPPARMs), which are discussed below [218].

In summary, the variety of trials with TZD in NAFLD/NASH fail to confirm the promising data from animal models. Still, significant improvements in histological parameters, especially hepatic steatosis and inflammation could be observed during TZD treatment. Whether the agents can reduce or reverse fibrosis remains to be confirmed. Potentially, a less efficacious and potent PPARγ agonist is superior since the more potent agents might promote adipocyte differentiation and fat storage. This assumption and the problem of significant weight gain during TZD treatment further highlight the promising role SPPARMs could have in NASH to bypass side effects and improve for example adiponectin levels.

4.4. Dual PPAR agonists
4.4.1. Dual PPAR agonists animal studies

As mentioned above, bezafibrate constitutes a pan agonist of PPARα, PPARδ with lower potency on PPARγ [193]. A few rodent models respective NAFLD and NASH demonstrated potential efficacy of bezafibrate. In addition to the examinations of Nagasawa et al. who compared bezafibrate with the PPARδ agonist GW501516 [194], Nakano et al. studied bezafibrate and pioglitazone in male KK-Ay/TaJcl mice fed a MCD diet for 7 weeks as type 2 diabetes mouse model. Moreover, an in vitro HSC model with an immortalized rat HSC (RI-T) was conducted. The cells were stimulated with TGF-β1 to induce fibrosis. In vivo, bezafibrate was able to reduce hepatic triglyceride content and the accumulation of fat droplets within hepatocytes. Pioglitazone in comparison did not affect triglyceride content of the liver. Furthermore, in contrast to pioglitazone, bezafibrate decreased ALT levels and the concentration of thiobarbituric acid-reactive substances (TBARS). TBARS develop during lipid peroxidation as side-products and can be used to estimate the extend of ROS creation. Both, bezafibrate and pioglitazone increased adiponectin levels. The effect of bezafibrate on inflammation was determined by the number of foamy macrophage clusters and neutrophil infiltration, which were significant reduced. Moreover, bezafibrate and pioglitazone prevented HSC activation, measured by the number of αSMA-positive HSC. Via Masson trichrome stain, a three-colour staining to distinguish keratin or muscle fibres from collagen or bone, and to distinguish cytoplasm from cell nuclei was conducted. Thereby, collagen can be detected as fibrosis marker. Bezafibrate, but not pioglitazone, seemed to prevent hepatic fibrosis in the mouse model. Moreover, bezafibrate increased the mRNA levels of ACO and CPT-1, indicating an enhanced β-oxidation. Also TNF-α, IL-1β, IL-6 and MCP-1 were reduced during bezafibrate treatment and anti-oxidative enzymes such as SOD-1 were elevated. Pioglitazone only affected Il-6 and SOD-1. In the in vitro HSC model, the fibrogenic response of several different PPAR modulators was studied by investigating αSMA, α1(I)-collagen and fibronectin-1 levels. All were decreased with bezafibrate superior to pioglitazone. In addition, fenofibrate as specific PPARα agonist had the strongest decreasing effect on fibrogenic target genes. The PPARδ agonist GW501516 had no significant effect. The authors assume that an anti-fibrotic effect could be result of mainly PPARα and additionally PPARγ activation but to verify this hypothesis, more target genes need to be considered [219].

Although these animal models indicate a superior efficacy of the pan agonist bezafibrate, no clinical studies for the treatment of NASH with humans have been reported that would be required to confirm the promising in vivo data.

With the glitazars acting as dual agonists of PPARα and PPARγ there is another class of PPAR modulators. Glitazars were developed with the aim to treat diabetic patients, which develop dyslipidemia as comorbidity resulting in atherosclerotic coronary heart disease [220]. However, as consequence of several cardiovascular [221,222] and renal [223] side effects, most glitazars have been withdrawn. Only saroglitazar is marketed in India.

Ji-Ming et al. reported promising in vivo data from rats on high fat diet treated with ragaglitazar, Wy-14,643 or rosiglitazone. Insulin sensitivity and lipid metabolism were assessed and indeed, hepatic triglyceride accumulation as well as visceral adiposity were stronger improved upon glitazar treatment than with rosiglitazone or Wy-14,643. Furthermore, in comparison with rosiglitazone and Wy-14,643 insulin sensitivity improvement was more pronounced and adiponectin values were elevated, significantly correlating with lipid content and insulin activity in liver. Hence, the dually active glitazars might be superior to selective PPAR modulators, but due to safety concerns their future is unknown. In India, saroglitazar is currently investigated in a phase 3 study (NCT02265276, GLAZED), evaluating its efficacy in NAFLD in comparison with pioglitazone. Though, according to the clinical trial database of United States National Library of Medicine, the completion date has passed and the status has not been verified in more than two years [224].

Another dual PPAR agonist, in particular the dual PPARα and PPARδ agonist elafibranor (GFT505), is currently amongst the farthest developed pharmacological NASH treatments. It has proven therapeutically effective during several animal studies and a clinical trial (GOLDEN-505). Elafibranor is currently the most promising PPAR targeting approach in NASH treatment (Table 2).

Staels et al. have extensively studied elafibranor in NASH models with human APO E2 transgenic mice fed with a western diet as well as MCD fed mice and rats, where fibrosis was induced by CCl4. All the rodent models showed that elafibranor decreased steatosis and inflammation with lower levels of IL-1β, TNF-α and EGF-like module-containing mucin-like hormone receptor-like 1 (F4/80), a member of adhesion G protein-coupled receptors and a specific marker for eosinophils. Furthermore, profibrotic genes such as TGF-β, TIMP-2 and collagens were downregulated. Human APO E2 transgenic mice where used for PPARα knockout experiments to assess the effects of elafibranor in absence PPARα revealing that western diet induced steatosis and inflammation were resolved, despite of PPARα knockdown, but plasma triglyceride levels and the concentration of free fatty acids were not affected, suggesting involvement of PPARα. In rats, GFT505 underwent extensive enterohepatic cycling.

4.4.2. Dual PPAR agonists clinical trials

Two clinical studies of elafibranor reported by Cariou et al. evaluated effects and safety of elafibranor in abdominally obese patients with either combined dyslipidemia or prediabetes. The trials were not designed to study the treatment of fatty liver, but already indicate a potential use regarding fatty liver diseases. The first trial enrolled 94 patients with obesity and dyslipidemia while the second study was conducted in 47 obese patients with prediabetes. Patients were treated with 80 mg elafibranor per day for 28 days in trial one and 35 days in trial two. Elafibranor was able to significantly reduce fasting plasma triglycerides and increased HDL cholesterol in both studies. However, LDL only decreased in obese prediabetic patients. Additionally, a significant decrease of HOMA-index and fasting plasma glucose was detected in the same population. A reduction of GGT levels was observed in both studies. No verum specific side effects could be detected. These two trials both indicate a beneficial effect of the dual PPARα and PPARδ agonist for the treatment of NAFLD and NASH, because insulin sensitivity is a major contributor of fatty liver disease and decreases in triglycerides as well as circulating non-HDL-cholesterol levels seem very beneficial for a therapy of NAFLD and NASH [225].

Elafibranor was then specifically studied as potential therapy of NAFLD and NASH in the GOLDEN-505 trial (NCT01694849). Selection criterion to be included in this phase IIb trial was a histologic diagnosis of non-cirrhotic NASH. Patients were excluded if they consumed alcohol daily, if steatohepatitis had a secondary cause, or if any other chronic liver disease was detected. In total, 276 patients were enrolled and separated in three treatment groups receiving 80 mg or 120 mg elafibranor once daily or placebo over 52 weeks. The primary outcome was the resolution of NASH without fibrosis worsening. Resolution of NASH was defined as the absence of at least one of the following symptoms: steatosis, ballooning or inflammation. Worsening of fibrosis was defined as no progression to bridging fibrosis or when bridging fibrosis was the initial diagnosis, no progression to cirrhosis. The endpoint was not matched, because there was no statistically significant difference between elafibranor and placebo but NASH resolution occurred in a higher number of patients in the 120-mg elafibranor group. Using a post-hoc analysis with a modified definition, the results of the response rate changed. The modified definition describes resolution of NASH as disappearance of ballooning in combination with either disappearance of lobular inflammation or the persistence of mild lobular inflammation only. Furthermore, any increase in fibrosis is regarded as fibrosis worsening. With this definition, the response rate was significantly higher for the 120-mg arm than for placebo while the 80-mg group revealed no superiority to placebo for both definitions of response. As secondary outcomes, changes in NASH activity score from baseline biopsy at the end of treatment as well as changes and improvements in individual histologic scores of steatosis, ballooning, inflammation, and fibrosis were defined. Again no significant difference between elafibranor and placebo could be detected. The efficacy of the 120-mg dose to reduce the NASH activity score by 2 points and to improve steatosis, ballooning and lobular inflammation was slightly improved, but did not reach a level of statistical significance. Patients receiving elafibranor had improved ALT, GGT and ALP levels. Furthermore, lipid parameters, including triglycerides, LDL and HDL were improved and in diabetic patients, elafibranor improved fasting serum glucose and HbA1c, as well as markers of insulin resistance such as fasting insulin, HOMA-index and circulating free fatty acids. Additionally, there was a reduction in systemic inflammatory markers, mainly high-sensitivity CRP and fibrinogen at both doses. Histologic changes were estimated by a panel of serum biomarkers of steatosis and fibrosis (SteatoTest, Fatty Liver Index, Fibrotest/FibroSure, and the NAFLD Fibrosis score) and showed significant reductions in patients treated with elafibranor 120 mg only. Overall, high placebo induced NASH improvements affected the outcome of the study. Patients with mild steatohepatitis, had a high placebo response rate leading to a lack of significance in the verum groups. Therefore, the authors suggest that elafibranor might have higher efficacy in more severe disease. Further studies to confirm this assumption are required, however. Concerning side effects, elafibranor was well tolerated and did not cause weight gain or cardiac events. Only a mild, reversible increase in serum creatinine was observed, indicating renal side effects [72].

Currently the phase 3 study RESOLVE-IT (NCT02704403) to evaluate the efficacy and safety of elafibranor in NASH patients is ongoing. Inclusion criteria for participants are liver biopsy confirmed diagnosis of NASH, and in contrast to GOLDEN-505, at least 1 point in each component of the NAS score (steatosis scored 0-3, ballooning degeneration scored 0-2, and lobular inflammation scored 0-3) with a total score of at least 4. In addition, a fibrosis stage between 1 and 4 according to the NASH CRN fibrosis staging system is required [226].

T2

Table 2: Clinical trials of PPAR modulators for NAFLD/NASH.

5. Synergistic Multi-Target Modulation as Superior NASH Treatment?

As discussed above, NASH is a very multi-factorial disease that arises from a variety of causes and results in several pathological conditions leading to liver damage and promoting further disease progression. It might, therefore, be rather effective to address more than one molecular target in order to generate an effective pharmacological response in NASH treatment. In theory, modulation of each PPAR subtype provides numerous beneficial effects for NASH treatment but so far, only the dual PPARα/PPARδ agonist elafibranor could translate PPAR modulation into clinical efficacy supporting the assumption that modulation of a single target might not be sufficient to treat NASH. Combined activation of PPARα and PPARδ by elafibranor might generate a synergistic effect by producing beneficial effects in different tissues. While PPARα activation amongst others promotes hepatic degradation of lipids by β-oxidation, PPARδ agonism predominantly exhibits extrahepatic activity improving metabolic balance which in turn improves liver health. Similar potential synergies with PPAR modulation are also plausible for several other targets that have revealed beneficial effects in NASH treatment.

Although anti-inflammatory effects of PPAR activation have repeatedly been reported and elafibranor caused improvement of hepatitis in clinical trials, PPARs predominantly regulate metabolic pathways and many of their anti-inflammatory effects in liver are a consequence of improved metabolic balance than a direct activity. PPARs promote β-oxidation and lipolysis and enhance clearance of lipids from the liver leading to reduced steatosis. To support these metabolic improvements with anti-inflammatory, liver cell protective or anti-fibrotic activity, combining PPAR activation with an anti-inflammatory approach holds much promise as potentially synergistic therapeutic strategy. In NASH, metabolic stress in the liver leads to oxidative stress and a permanent inflammatory process causing cell damage and ultimately cell death through apoptosis. Several anti-inflammatory targets have already proven efficacy against this aspect of NASH in animal models or even clinical trials. Except for the anti-oxidative activity of vitamin E, two anti-inflammatory strategies evolve as experimental approaches in NASH treatment [12,228,229]. The chemokine receptor antagonist cenicriviroc blocking the chemokine receptors (CCR)2 and CCR5 is currently the most advanced anti-inflammatory agent in the NASH pipeline. Chemokine ligand (CCL)2 and CCL5 amongst others have been found upregulated in NASH patient biopsies [230] and might significantly contribute to hepatic inflammation. Cenicriviroc currently undergoes a prospective phase IIb trial [231] to assess its anti-inflammatory and anti-fibrotic potential in NASH. Inhibitors of the ASK1 such as selonsertib (GS-4997) also have great anti-inflammatory and cell-protective potential and three studies for the ASK1 inhibitor selonsertib as NASH treatment are recruiting (NCT02781584, NCT03053050, NCT03053063). These anti-inflammatory approaches could support the beneficial metabolic effects of PPAR activation by directly reducing hepatic inflammation and thereby providing liver cell protective activity. Under reduced inflammatory conditions, liver cell apoptosis and fibrosis would also be expected to decrease.

A direct reversal of fibrosis is very desirable in NASH treatment but promising anti-fibrotic effects from animal models have not translated to a comparable activity in clinical trials so far. Two strategies directly aiming at preventing and reducing fibrosis in NASH are in clinical development with the caspase inhibitor emricasan in phase IIa and galectin 3 inhibitors in phase I [228,229,232,233,234]. Although there is also some limited evidence that PPARγ activation itself might have anti-fibrotic activity [235], combination of anti-fibrotic approaches with PPAR agonists holds therapeutic promise. By preventing apoptosis of hepatocytes and reducing the formation of fibrotic tissue, caspase inhibitors or galectin 3 inhibitors could protect the liver architecture and retain hepatic function which might also result in synergistic efficacy against NASH when combined with PPAR activation that predominantly ameliorates hepatic metabolism and reverses steatosis.

Beyond combining agents to improve metabolic balance with direct anti-inflammatory or anti-fibrotic mechanisms also targeting metabolic anti-NASH effects in different tissues holds promise for synergistic activity. In addition to the dual activity of elafibranor, this might also be achievable by simultaneously activating FXR and PPARδ. FXR exhibits its effects mainly in liver and intestine. Via PPARα and the transcription factor sterol regulatory element binding protein 1c (SREBP1c), the nuclear receptor promotes β-oxidation and lipid clearance in liver. Additionally, FXR enhances cholesterol excretion from liver into bile and prevents biosynthesis of hepatotoxic bile acids by repressing cholesterol 7α hydroxylase (CYP7A1). Finally, FXR-mediated repression of NF-κB signaling in hepatocytes and hepatic macrophages/Kupffer cells can reduce hepatic inflammation and fibrogenesis [229]. PPARδ activation in contrast, only moderately affects hepatic β-oxidation and lipid metabolism but improves metabolic balance by promoting utilization of lipids for energy generation in muscles. Additionally, PPARδ activation has beneficial effects on cholesterol levels and the ratio of HDL/LDL which might ameliorate the cholesterol accumulation caused by FXR activation. Finally, dual modulation of FXR and PPARα might provide synergistic effects by activating hepatic lipid clearance via two essential regulators. As discussed above, both receptors promote anti-steatotic effects in the liver, PPARα mainly by enhancing β-oxidation and FXR by inducing the expression of PPARα as well as by SREBP1c mediated effects on hepatic lipid and glucose metabolism. Clinical trials and practice will have to reveal the most favorable synergistic combinations of pharmacological approaches in NASH treatment but the multifactorial nature of the disease especially in later stages will most likely require more than one pharmacodynamic mechanism for sufficient therapeutic efficacy.

6. Selective PPARγ Modulators

Selective modulation of nuclear receptors is getting increasingly into focus of drug discovery as an improved strategy to exploit their pharmacological potential as targets with reduced side effects. Such selective strategies include tissue selective modulation, gene selective modulation and partial activation with reduced transactivation efficacy.

Also some SPPARMs have been reported (Table 3) that might have value in NASH treatment. Amongst SPPARMs, partial agonists gained most attention. Several potent examples have been developed and studied in vivo. PAM-1616 [236], KR62980 [237], SPPARγM5 [238], INT131 [239,240], L312 [241], MK-0533 [242] and MBX-102 [243] constitute partial PPARγ agonists that have revealed anti-diabetic activities with lower typical PPARγ mediated side-effects such as weight gain, cardiac hypertrophy and edema. Clinical trials of INT131 [244] have already confirmed that such partial agonism can translate into robust effects on glycemic control in human since INT131 was not inferior to pioglitazone but did not cause weight gain and fluid retention in phase 2.

T3

Table 3: Selective PPARγ modulators (SPPARγMs).

Structural data from an INT131-PPARγ co-crystal structure indicates that partial activation of the receptor is explained by incomplete stabilization of the transactivation helix (H12). INT131 forms only lipophilic contacts to this helix whereas full agonistic TZDs participate in several H-bonds with H12 [240]. Partial PPARγ activation seems superior to full agonists concerning side effect profiles in vivo but retain beneficial metabolic effects. However, the effects on metabolism did not exceed the activity of classical TZDs. As discussed above, pioglitazone or rosiglitazone alone were not effective in NASH treatment and, therefore, it is unlikely that partial PPARγ agonists alone will perform significantly better.

Gene-selective PPARγ modulation might hold even more therapeutic potential. But in contrast to the large number of partial PPARγ agonists, only few compounds have been found to selectively modulate PPARγ activation in a gene-selective manner which is much more difficult to achieve. So far, the most specific modulation of PPARγ has been achieved with SPPARMs displaying significantly distinct co-activator recruitment profiles.

S26948 is a PPARγ ligand with comparable affinity and transactivation efficacy as rosiglitazone in reporter gene assays. However, its co-activator recruitment profile was significantly different from rosiglitazone as it only recruited glutamate receptor interacting protein 1 (GRIP1) while rosiglitazone also recruited DRIP205 and PGC-1α. The compound did not cause adipocyte differentiation or lipid accumulation in vitro but in vivo exhibited comparable antidiabetic and lipid-lowering activity as rosiglitazone. In contrast to rosiglitazone, S26948 caused no weight gain in vivo. Additionally, the PPARγ modulator reduced liver weight and liver fat content while promoting hepatic lipid oxidation. In a mouse model of atherosclerosis, S26948 reduced plasma cholesterol and lipid levels to a less atherogenic profile. These results indicate that specific PPARγ modulation might also be effective in improving liver health in NASH [245].

The SPPARM FK614 [246,247] revealed a clearly different pharmacodynamic profile than S26948. It robustly dissociated co-repressors SMRT and NCor from PPARγ and recruited PGC-1α with comparable efficacy as thiazolidindiones while C/EBP and steroid receptor coactivator-1 (SRC-1) were significantly less recruited upon FK614 binding. In contrast to S26948, FK614 promoted adipocyte differentiation and lipid accumulation with similar efficacy as thiazolidindiones. However, the long-term effect of FK614 on mature adipocytes differed from thiazolidindiones as treatment with the SPPARM resulted in a more favorable expression profile of genes involved in insulin resistance. This activity might as well improve liver health in NASH by redistributing fat from the liver to the periphery but clinical trials with thiazolidindiones in NASH have revealed that this effect alone is not sufficient and the resulting promotion of adipose tissue formation and weight gain is hardly desirable.

Beyond its partial PPARγ transactivation potency, MBX-102 has been discussed as more effective in PPARγ transrepression activity. While it only moderately induced PPARγ target gene expression, repression of pro-inflammatory genes was markedly more pronounced. In vitro, MBX-102 displayed nearly full efficacy in inducing the release of co-repressors NCOR and SMRT from PPARγ but only moderately recruited the co-activators C/EBP, SRC-1, PGC-1α, DRIP205 and transcriptional mediators/intermediary factor 2 (TIF2) with efficacies below 30% which gives a hint for the mechanism of the compound's interesting pharmacological profile [243]. According to the limited available data, the partial PPARγ agonist PAM-1616 might also have gene-selective modulatory properties as it induced the glucose transporter GLUT-4 with higher efficacy compared to rosiglitazone than other PPARγ target genes [236].

LG100641 was identified as PPARγ antagonist with specific modulatory profile. It inhibited co-activator recruitment to PPARγ induced by rosiglitazone, prevented adipocyte differentiation upon rosiglitazone treatment and blocked TZD induced PPARγ target gene expression. However, LG100641 did not reduce basal glucose uptake by adipocytes but a clear molecular mechanism to explain the compound's activity has not been demonstrated and other targets might be involved [248].

Recently, inhibition of cyclin-dependent kinase 5 (CdK-5) mediated phosphorylation of PPARγ has been proposed as another way of modulating the nuclear receptor [249,250,251]. Under high fat diet, PPARγ phosphorylation at Ser-273 was found significantly upregulated in adipose tissue changing the expression of adiponectin and other genes involved in insulin sensitivity. However, the phosphorylation only affected a subset of PPARγ target genes which might be due to differential recruitment of co-regulatory proteins. PPARγ phosphorylation is blocked by PPARγ agonists such as rosiglitazone but independent from their activating properties since PPARγ ligands lacking agonistic potency could also be identified. Such non-agonist ligand SR1664 revealed robust antidiabetic activity in vivo suggesting that specific modulation of PPARγ that prevents phosphorylation at Ser-273 might be sufficient to retain anti-diabetic activity. The expression profile of PPARγ target genes under treatment with the non-agonist ligand thereby significantly differed from a typical PPARγ agonist expression profile. As major advantage, the non-agonist modulator did not induce fluid retention in the animals or promote body fat mass whereas TZDs increased body fat and caused weight gain. Since insulin resistance and type 2 diabetes are closely linked to NAFLD and NASH, these preliminary observations indicate that blocking PPARγ phosphorylation at Ser-273 might also be effective in NASH treatment. Several studies have developed small-molecule blockers of PPARγ phosphorylation lacking agonistic potency and have shown their anti-diabetic activity [241,252,253]. However, their effect on hepatic fat content and liver health has not been studied, so far. PPARγ agonists tend to reduce liver fat by redistributing and disposing fat into peripheral adipose tissue causing weight gain. Since increased body fat mass and weight gain have not been observed with the non-agonist PPARγ modulators that block phosphorylation at Ser-273, their effect on liver steatosis and hepatic lipid metabolism might be significantly different and the value of this mode of PPARγ modulation in NASH needs to be further studied.

7. Conclusion & Outlook

PPARs have a changeful history as drug targets. PPARγ agonistic thiazolidinediones markedly improved the therapy of type 2 diabetes mellitus as insulin sensitizers but few years ago their therapeutic significance ended with market withdrawals and warnings due to severe side effects. PPARα agonistic fibrates are still marketed and can be used to treat hyperlipidemia but have also significantly lost in significance since alternative therapeutic approaches turned out superior. And finally, PPARδ was considered as very promising target to treat metabolic disorders until the first selective PPARδ agonist GW501516 failed in clinical trials due to unresolved safety concerns. However, the growing interest in therapeutic options for non-alcoholic steatohepatitis might help PPARs to a new rise as drug targets.

As discussed above, all three PPAR subtypes are crucially involved in the various aspects of the metabolic syndrome. PPARα regulates transport and distribution as well as synthesis and degradation of lipids. Its activation promotes the utilization of lipids as energy source in β-oxidation leading to decreased lipid concentrations especially in liver. Thus, PPARα activation seems a very reasonable strategy to treat NASH which is highly linked to increased hepatic lipid levels. Accordingly, promising efficacy was observed in rodent models but most clinical trials failed to translate this into robust therapeutic benefit. A significant issue of almost all studies on PPARα in NASH might be the used PPARα agonists since hardly any selective compound was studied. Therefore, the results of isolated PPARα activation in NASH are hardly known and further studies with potent and selective agents will be necessary to clearly analyze its therapeutic value.

PPARγ mainly modulates glucose homeostasis and promotes insulin sensitivity. Thus, its relation to NASH becomes clear only at second glance because insulin resistance, diabetes and NAFLD/NASH are connected as manifestations of the metabolic syndrome. Notably, type 2 diabetes is amongst the strongest risk factors of NAFLD/NASH and some trials with thiazolidinediones have indeed revealed some efficacy in NASH treatment. Still, PPARγ activation is well-known for causing weight gain which constitutes a major issue in NAFLD/NASH patients that often already are obese. Although PPARγ activation was shown to decrease hepatic fat content, this effect was mainly due to redistribution of fat from liver to visceral fat. PPARγ modulators have been most extensively studied in clinical trials for NASH but with merely disappointing results. However, PPARγ drug discovery remains very intensive and there are many ligands of newer generations that either partially activate the nuclear receptor, cause a specific interaction profile with different co-activators and repressors or modulate the receptor's phosphorylation state. These new approaches have shown retained anti-diabetic activity in vivo but caused significantly less side effects such as weight gain. Thus, selective PPARγ modulators also hold considerable potential in NASH but much more research will be necessary to confirm this hypothesis.

PPARδ remains the least studied PPAR subtype also with the great success of elafibranor in clinical trials. It holds a lot of promise in NASH treatment by improving lipid utilization in peripheral organs, especially in skeletal muscle, combined with anti-inflammatory activity. Elafibranor is very likely to become the first approved PPAR targeting agent to treat NASH and revealed efficacy in reducing steatosis, hepatitis and fibrosis in clinical trials, at least in severe cases. However, the great efficacy of elafibranor might arise from its dual activity profile since it also activates PPARα and thereby combines peripheral and hepatic anti-NASH activity. On one hand, this makes the interpretation of PPARδ's isolated role in NASH treatment rather difficult but on the other hand indicates that dual or multi-target agents might possess superior efficacy in this multifactorial disease complex.

Multi-target modulation as innovative, additive and potentially synergistic strategy holds promise especially in a multifactorial context as it is present in the metabolic syndrome in general and in NASH in specific. In this context, PPARs seem very promising targets that can be combined with supportive modulation of other signaling pathways or enzymatic cascades. Since PPARs mainly cause metabolic improvements that positively affect liver health and can reduce hepatic steatosis by promoting metabolic lipid elimination, combination of PPAR activation with anti-inflammatory and anti-apoptotic strategies has obvious potential. Thereby, the beneficial metabolic consequences of PPAR modulation could be markedly supported potentially leading to superior efficacy.

Despite of intensive research, several putative therapeutic effects of PPAR modulation in NASH have not been successfully translated into clinical efficacy, yet. Potential reasons are the considerable species differences in PPAR functions and expression pattern. Therefore, animal models and trials in human are not comparable in various aspects (Table 1). Additionally this might also be due to the design, population and biomarkers of the respective clinical studies. Improved NASH research requires better biomarkers with more predictive potential as well as more innovative study design. In addition, a more individualized therapeutic approach might be required in NASH. The great number of patients suffering from the disease, of course, have individual genetic background and individually suffer from different co-morbidities. Accordingly, several of the above discussed trials have identified specific populations of responders and non-responders that must be considered in future drug development for NASH. Then, not only elafibranor but also other PPAR targeting drugs might reveal great therapeutic efficacy in NASH treatment.

8. Abbreviations Used

8-oxoG: 8-oxoguanine

ABCA1: ATP-binding cassette transporter

ACC: acetyl-CoA carboxylase

ACO: acyl-CoA oxidase

ACS: acyl-CoA synthetase

ACSL: long-chain-fatty-acid-CoA ligase

ACTH: adrenocorticotropic hormone

ADRP: adipose differentiation-related protein

AF2: activation function 2

ALP: alkaline phosphatase

ALT: alanine aminotransferase

ANGPTL4: angiopoietin-like protein 4

aP2: adipocyte Protein 2

APC: adenomatous polyposis coli

APO: apolipoprotein

AQP7: aquaporin 7

ASK1: apoptosis signal-regulating kinase 1

aSMA: alpha smooth muscle actin

AST: aspartate transaminase

BDL: bile duct ligation

C/EBP: CCAAT-enhancer-binding proteins

C2C12: mouse myoblast cell line

CCL: chemokine ligand

CCl4: carbon tetrachloride

CCR: chemokine receptors

CD: cluster of differentiation

CD95: apoptosis antigen 1

CDAA: choline-deficient L-amino acid-defined

CdK-5: cyclin-dependent kinase 5

ChREBP: carbohydrate response element binding protein

COX-2: cyclooxygenase-2

CPT-1: carnitine palmitoyl transferase-1

CREB: cAMP response element-binding protein

CRP: C-reactive protein

CYP: cytochrome P450 enzyme

CYP7A1: cholesterol 7α hydroxylase

DBD: DNA-binding domain

DNL: de novo lipogenesis

DR-1: direct repeat 1

DRIP205: vitamin D-interacting protein 205

F4/80: EGF-like module-containing mucin-like hormone receptor-like 1

FABP: fatty acid binding protein

FACS: fatty-acyl-CoA synthase

FAS: fatty acid synthase

FAT/CD36: fatty acid translocase

FATP: fatty acid transport protein

FDA: Food and Drug Administration

FED: fructose-enriched diet

FOXO1: forkhead box protein O1

FXR: farnesoid X receptor

G6Pase: glucose-6-phosphatase

GGT: γ-glutamyl transpeptidase

GLUT: glucose transporter

GRIP1: glutamate receptor interacting protein 1

GST-P: glutathione S-transferase

HbA1c: haemoglobin A1c

HCC: hepatocellular carcinoma

HCD: high-caloric and high-cholesterol diet

HDL: high-density lipoprotein

HOMA: Homeostatic Model Assessment

HSC: hepatic stellate cell

IL: interleukin

IRE1α: inositol-requiring enzyme 1α

IκB: Inhibitor of κB

JNK: c-Jun N-terminal kinase

LBD: ligand-binding domain

LCFA: long-chain fatty acid

LC-FACS: long-chain fatty acid acetyl-coenzyme A synthase

LDL: low-density lipoprotein

L-PK: L-type pyruvate kinase

LPL: lipoprotein lipase

LTB4: leukotriene B4

LXR: liver X receptor

MCD: methionine and choline deficient diet

MCP: monocyte chemoattractant protein

MMP: matrix metalloproteinase

MRI: magnetic resonance imaging

NAFLD: non-alcoholic fatty liver disease

NAS: NAFLD fibrosis activity score

NASH: non-alcoholic steatohepatitis

NCoR: nuclear corepressor

NF-κB: nuclear factor-κB

NIDDM: non insulin dependent diabetes mellitus

Ogg1: 8-Oxoguanine glycosylase

OLETF: Otsuka Long Evans Tokushima Fatty

OLR1: oxidized LDL receptor 1

oxLDL: oxidatively modified low density lipoprotein

PBP: PPAR binding protein

PDK4: pyruvate dehydrogenase kinase 4

PECAM-1: platelet endothelial cell adhesion molecule

PEPCK: phosphoenolpyruvate carboxykinase

PGC-1α: PPARγ coactivator 1-alpha

PPAR: peroxisome proliferator-activator receptor

PPRE: PPAR response element

RAR: retinoic acid receptor

ROS: reactive oxygen species

RXR: retinoid X receptor

SCD1: stearoyl-CoA desaturase 1

SMRT: silencing mediator of retinoid and thyroid hormone receptors

SOCS-3: suppressor of cytokine signaling 3

Sod: superoxide dismutase

SPPARM: selective PPARγ modulators

SRC-1: steroid receptor coactivator-1

SRE1: sterol regulatory element-1

SREBP-1c: sterol regulatory element-binding protein 1c

TBARS: thiobarbituric acid-reactive substance

TGF-β1: transforming growth factor beta 1

THR: thyroid hormone receptor

TIF2: transcriptional mediators/intermediary factor 2

TIMP: tissue inhibitor of metalloproteinase

TNF-α: tumor necrosis factor-α

Twist-1: Twist-related protein 1

TZD: thiazolidinedione

UCP: uncoupling protein

VCAM-1: vascular cell adhesion molecule-1

VEGF-A: vascular endothelial growth factor A

VLDL: very low density lipoprotein

XBP1: X-box binding protein 1

Competing Interests

The authors declare no competing interests.

References

  1. P. Angulo and K. D. Lindor, “Non-alcoholic fatty liver disease,” Journal of Gastroenterology and Hepatology, vol. 17, supplement s1, pp. S186–S190, 2002. Publisher Full Text | Google Scholar
  2. L. S. Szczepaniak, P. Nurenberg, D. Leonard et al., “Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population,” American Journal of Physiology-Renal Physiology, vol. 288, no. 2, pp. E462–E468, 2005. Publisher Full Text | Google Scholar
  3. E. M. Brunt, “Grading and staging the histopathological lesions of chronic hepatitis: the Knodell histology activity index and beyond,” Hepatology, vol. 31, no. 1, pp. 241–246, 2000. Publisher Full Text | Google Scholar
  4. G. C. Farrell and C. Z. Larter, “Nonalcoholic fatty liver disease: from steatosis to cirrhosis,” Hepatology, vol. 43, no. 2, pp. 99–112, 2006. Publisher Full Text | Google Scholar
  5. T. Karlas, J. Wiegand, and T. Berg, “Gastrointestinal complications of obesity: non-alcoholic fatty liver disease (NAFLD) and its sequelae,” Best Practice & Research Clinical Endocrinology & Metabolism, vol. 27, no. 2, pp. 195–208, 2013. Publisher Full Text | Google Scholar
  6. V. Ratziua, S. Bellentanib, and H. Cortez-Pintoc, “A position statement on NAFLD/NASH based on the EASL 2009 special conference,” Journal of Hepatology, vol. 53, no. 2, pp. 372–384, 2010. Publisher Full Text | Google Scholar
  7. M. Charlton, “Nonalcoholic fatty liver disease: a review of current understanding and future impact,” Clinical Gastroenterology and Hepatology, vol. 2, no. 12, pp. 1048–1058, 2004. Publisher Full Text | Google Scholar
  8. Z. M. Younossi, A. B. Koenig, D. Abdelatif, Y. Fazel, L. Henry, and M. Wymer, “Global epidemiology of nonalcoholic fatty liver disease—meta-analytic assessment of prevalence, incidence, and outcomes,” Hepatology, 2015. Publisher Full Text | Google Scholar
  9. G. Marchesini, M. Brizi, A. M. Morselli-Labate et al., “Association of nonalcoholic fatty liver disease with insulin resistance,” American Journal of Medicine, vol. 107, no. 5, pp. 450–455, 1999. Publisher Full Text | Google Scholar
  10. S. Caldwell and C. Argo, “The natural history of non-alcoholic fatty liver disease,” Digestive Diseases, vol. 28, no. 1, pp. 162–168, 2010. Publisher Full Text | Google Scholar
  11. M. Ekstedt, L. E. Franzén, U. L. Mathiesen et al., “Long-term follow-up of patients with NAFLD and elevated liver enzymes,” Hepatology, vol. 44, no. 4, pp. 865–873, 2006. Publisher Full Text | Google Scholar
  12. A. M. Oseini and A. J. Sanyal, “Therapies in non-alcoholic steatohepatitis (NASH),” Liver International, vol. 37, pp. 97–103, 2017. Publisher Full Text | Google Scholar
  13. Y. Falck-Ytter, Z. M. Younossi, G. Marchesini, and A. J. McCullough, “Clinical features and natural history of nonalcoholic steatosis syndromes,” Seminars in Liver Disease, vol. 21, no. 1, pp. 17–26, 2001. Publisher Full Text | Google Scholar
  14. S. Parekh and F. A. Anania, “Abnormal lipid and glucose metabolism in obesity: implications for nonalcoholic fatty liver disease,” Gastroenterology, vol. 132, no. 6, pp. 2191–2207, 2007. Publisher Full Text | Google Scholar
  15. A. Kotronen and H. Yki-Järvinen, “Fatty liver: a novel component of the metabolic syndrome,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 28, no. 1, pp. 27–38, 2008. Publisher Full Text | Google Scholar
  16. P. Mofrad, M. J. Contos, M. Haque et al., “Clinical and histologic spectrum of nonalcoholic fatty liver disease associated with normal ALT values,” Hepatology, vol. 37, no. 6, pp. 1286–1292, 2003. Publisher Full Text | Google Scholar
  17. L. A. Adams, S. Sanderson, K. D. Lindor, and P. Angulo, “The histological course of nonalcoholic fatty liver disease: a longitudinal study of 103 patients with sequential liver biopsies,” Journal of Hepatology, vol. 42, no. 1, pp. 132–138, 2005. Publisher Full Text | Google Scholar
  18. G. Donati, B. Stagni, F. Piscaglia et al., “Increased prevalence of fatty liver in arterial hypertensive patients with normal liver enzymes: Role of insulin resistance,” Gut, vol. 53, no. 7, pp. 1020–1023, 2004. Publisher Full Text | Google Scholar
  19. B. A. Neuschwander-Tetri and S. H. Caldwell, “Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference,” Hepatology, vol. 37, no. 5, pp. 1202–1219, 2003. Publisher Full Text | Google Scholar
  20. N. S. Goldstein, F. Hastah, M. V. Galan, and S. C. Gordon, “Fibrosis heterogeneity in nonalcoholic steatohepatitis and hepatitis C virus needle core biopsy specimens,” American Journal of Clinical Pathology, vol. 123, no. 3, pp. 382–387, 2005. Publisher Full Text | Google Scholar
  21. M. M. Yeh and E. M. Brunt, “Pathology of nonalcoholic fatty liver disease,” American Journal of Clinical Pathology, vol. 128, no. 5, pp. 837–847, 2007. Publisher Full Text | Google Scholar
  22. C. P. Day, “Non-alcoholic steatohepatitis (NASH): Where are we now and where are we going?” Gut, vol. 50, no. 5, pp. 585–588, 2002. Publisher Full Text | Google Scholar
  23. E. M. Brunt, C. G. Janney, A. M. Di Bisceglie, B. A. Neuschwander-Tetri, and B. R. Bacon, “Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions,” American Journal of Gastroenterology, vol. 94, no. 9, pp. 2467–2474, 1999. Publisher Full Text | Google Scholar
  24. P. Angulo, J. M. Hui, G. Marchesini et al., “The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD,” Hepatology, vol. 45, no. 4, pp. 846–854, 2007. Publisher Full Text | Google Scholar
  25. C. P. Day and O. F. W. James, “Steatohepatitis: a tale of two “Hits”?” Gastroenterology, vol. 114, no. 4, pp. 842–845, 1998. Publisher Full Text | Google Scholar
  26. H. J. Seitz, M. J. Müller, W. Krone, and W. Tarnowski, “Coordinate control of intermediary metabolism in rat liver by the insulin/glucagon ratio during starvation and after glucose refeeding. Regulatory significance of long-chain acyl-CoA and cyclic AMP,” Archives of Biochemistry and Biophysics, vol. 183, no. 2, pp. 647–663, 1977. Publisher Full Text | Google Scholar
  27. T. Hashimoto, W. S. Cook, C. Qi, A. V. Yeldandi, J. K. Reddy, and M. S. Rao, “Defect in peroxisome proliferator-activated receptor α-inducible fatty acid oxidation determines the severity of hepatic steatosis in response to fasting,” The Journal of Biological Chemistry, vol. 275, no. 37, pp. 28918–28928, 2000. Publisher Full Text | Google Scholar
  28. K. L. Donnelly, C. I. Smith, S. J. Schwarzenberg, J. Jessurun, M. D. Boldt, and E. J. Parks, “Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease,” The Journal of Clinical Investigation, vol. 115, no. 5, pp. 1343–1351, 2005. Publisher Full Text | Google Scholar
  29. S. K. Fried, C. D. Russell, N. L. Grauso, and R. E. Brolin, “Lipoprotein lipase regulation by insulin and glucocorticoid in subcutaneous and omental adipose tissues of obese women and men,” The Journal of Clinical Investigation, vol. 92, no. 5, pp. 2191–2198, 1993. Publisher Full Text | Google Scholar
  30. H. N. Ginsberg, “Is the slippery slope from steatosis to steatohepatitis paved with triglyceride or cholesterol?” Cell Metabolism, vol. 4, no. 3, pp. 179–181, 2006. Publisher Full Text | Google Scholar
  31. J. B. Kim, P. Sarraf, M. Wright et al., “Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1,” The Journal of Clinical Investigation, vol. 101, no. 1, pp. 1–9, 1998. Publisher Full Text | Google Scholar
  32. C. Biosynthesis, K. A. R. Tobin, S. M. Ulven et al., “Liver X receptors as insulin-mediating factors in fatty acid and cholesterol biosynthesis,” The Journal of Biological Chemistry, vol. 277, no. 12, pp. 10691–10697, 2002. Publisher Full Text | Google Scholar
  33. Y. Zhang, L. W. Castellani, C. J. Sinal, F. J. Gonzalez, and P. A. Edwards, “Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) regulates triglyceride metabolism by activation of the nuclear receptor FXR,” Genes & Development, vol. 18, no. 2, pp. 157–169, 2004. Publisher Full Text | Google Scholar
  34. A. Fernández-Alvarez, M. Soledad Alvarez, R. Gonzalez, C. Cucarella, J. Muntané, and M. Casado, “Human SREBF1c expression in liver is directly regulated by peroxisome proliferator-activated receptor α (PPARα),” The Journal of Biological Chemistry, vol. 286, no. 24, pp. 21466–21477, 2011. Publisher Full Text | Google Scholar
  35. J. D. Horton, J. L. Goldstein, and M. S. Brown, “SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver,” The Journal of Clinical Investigation, vol. 109, no. 9, pp. 1125–1131, 2002. Publisher Full Text | Google Scholar
  36. R. Dentin, J. Girard, and C. Postic, “Carbohydrate responsive element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c): Two key regulators of glucose metabolism and lipid synthesis in liver,” Biochimie, vol. 87, no. 1, pp. 81–86, 2005. Publisher Full Text | Google Scholar
  37. D. Chien, D. Dean, A. K. Saha, J. P. Flatt, and N. B. Ruderman, “Malonyl-CoA Content and Fatty Acid Oxidation in Rat Muscle and Liver in Vivo,” Am. J. Physiol. Endocrinol. Metab, vol. 279, pp. 259–265, 2000.
  38. P. Pettinelli and L. A. Videla, “Up-regulation of PPAR-γ mRNA expression in the liver of obese patients: an additional reinforcing lipogenic mechanism to SREBP-1c induction,” The Journal of Clinical Endocrinology & Metabolism, vol. 96, no. 5, pp. 1424–1430, 2011. Publisher Full Text | Google Scholar
  39. P. Pettinelli, T. del Pozo, J. Araya et al., “Enhancement in liver SREBP-1c/PPAR-α ratio and steatosis in obese patients: Correlations with insulin resistance and n-3 long-chain polyunsaturated fatty acid depletion,” Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, vol. 1792, no. 11, pp. 1080–1086, 2009. Publisher Full Text | Google Scholar
  40. K. Iizuka and Y. Horikawa, “ChREBP: a glucose-activated transcription factor involved in the development of metabolic syndrome,” Endocrine Journal, vol. 55, no. 4, pp. 617–624, 2008. Publisher Full Text | Google Scholar
  41. V. T. Samuel, C. S. Choi, T. G. Phillips et al., “Targeting Foxo1 in mice using antisense oligonucleotide improves hepatic and peripheral insulin action,” Diabetes, vol. 55, no. 7, pp. 2042–2050, 2006. Publisher Full Text | Google Scholar
  42. L. Valenti, R. Rametta, P. Dongiovanni et al., “Increased expression and activity of the transcription factor FOXO1 in nonalcoholic steatohepatitis,” Diabetes, vol. 57, no. 5, pp. 1355–1362, 2008. Publisher Full Text | Google Scholar
  43. F. Abbasi, S.-A. Chang, J. W. Chu et al., “Improvements in insulin resistance with weight loss, in contrast to rosiglitazone, are not associated with changes in plasma adiponectin or adiponectin multimeric complexes,” American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, vol. 290, no. 1, pp. R139–R144, 2006. Publisher Full Text | Google Scholar
  44. M. Tiikkainen, A.-M. Häkkinen, E. Korsheninnikova, T. Nyman, S. Mäkimattila, and H. Yki-Järvinen, “Effects of rosiglitazone and metformin on liver fat content, hepatic insulin resistance, insulin clearance, and gene expression in adipose tissue in patients with type 2 diabetes,” Diabetes, vol. 53, no. 8, pp. 2169–2176, 2004. Publisher Full Text | Google Scholar
  45. S. Kaser, A. Maschen, A. Cayon et al., “Adiponectin and its receptors in non-alcoholic steatohepatitis,” Gut, vol. 54, no. 1, pp. 117–121, 2005. Publisher Full Text | Google Scholar
  46. J. Fruebis, T. S. Tsao, S. Javorschi et al., “Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 98, no. 4, pp. 2005–2010, 2001. Publisher Full Text | Google Scholar
  47. N. Ouchi, S. Kihara, Y. Arita et al., “Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-κB signaling through a cAMP-dependent pathway,” Circulation, vol. 102, no. 11, pp. 1296–1301, 2000. Publisher Full Text | Google Scholar
  48. A. H. Berg, T. P. Combs, X. Du, M. Brownlee, and P. E. Scherer, “The adipocyte-secreted protein Acrp30 enhances hepatic insulin action,” Nature Medicine, vol. 7, no. 8, pp. 947–953, 2001. Publisher Full Text | Google Scholar
  49. J. B. Moore, P. J. Gunn, and B. A. Fielding, “The role of dietary sugars and de novo lipogenesis in non-alcoholic fatty liver disease,” Nutrients, vol. 6, no. 12, pp. 5679–5703, 2014. Publisher Full Text | Google Scholar
  50. M. Asrih and F. R. Jornayvaz, “Diets and nonalcoholic fatty liver disease: the good and the bad,” Clinical Nutrition, vol. 33, no. 2, pp. 186–190, 2014. Publisher Full Text | Google Scholar
  51. J. E. Lambert, M. A. Ramos-Roman, J. D. Browning, and E. J. Parks, “Increased de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease,” Gastroenterology, vol. 146, no. 3, pp. 726–735, 2014. Publisher Full Text | Google Scholar
  52. A. J. Sanyal, C. Campbell-Sargent, F. Mirshahi et al., “Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities,” Gastroenterology, vol. 120, no. 5, pp. 1183–1192, 2001. Publisher Full Text | Google Scholar
  53. D. Pessayre, A. Berson, B. Fromenty, and A. Mansouri, “Mitochondria in steatohepatitis,” Seminars in Liver Disease, vol. 21, no. 1, pp. 57–69, 2001. Publisher Full Text | Google Scholar
  54. J. D. McGarry and D. W. Foster, “Regulation of hepatic fatty acid oxidation and ketone body production.,” Annual Review of Biochemistry, vol. 49, pp. 395–420, 1980. Publisher Full Text | Google Scholar
  55. J. K. Reddy and T. Hashimoto, “Peroxisomal β-oxidation and peroxisome proliferator—activated receptor α: an adaptive metabolic system,” Annual Review of Nutrition, vol. 21, pp. 193–230, 2001. Publisher Full Text | Google Scholar
  56. G. A. MacDonald and J. B. Prins, “Peroxisomal fatty acid metabolism, peroxisomal proliferator-activated receptors and non-alcoholic fatty liver disease,” Journal of Gastroenterology and Hepatology, vol. 19, no. 12, pp. 1335–1337, 2004. Publisher Full Text | Google Scholar
  57. M. den Boer, P. J. Voshol, F. Kuipers, L. M. Havekes, and J. A. Romijn, “Hepatic steatosis: a mediator of the metabolic syndrome. Lessons from animal models,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 24, no. 4, pp. 644–649, 2004. Publisher Full Text | Google Scholar
  58. S. Q. Yang, H. Z. Lin, M. D. Lane, M. Clemens, and A. M. Diehl, “Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 94, no. 6, pp. 2557–2562, 1997. Publisher Full Text | Google Scholar
  59. H. Z. Lin, S. Q. Yang, C. Chuckaree, F. Kuhajda, G. Ronnet, and A. M. Diehl, “Metformin reverses fatty liver disease in obese, leptin-deficient mice,” Nature Medicine, vol. 6, no. 9, pp. 998–1003, 2000. Publisher Full Text | Google Scholar
  60. M. C. Arkan, A. L. Hevener, F. R. Greten et al., “IKK-β links inflammation to obesity-induced insulin resistance,” Nature Medicine, vol. 11, no. 2, pp. 191–198, 2005. Publisher Full Text | Google Scholar
  61. H. Tilg, P. D. Cani, and E. A. Mayer, “Gut microbiome and liver diseases,” Gut, vol. 65, no. 12, pp. 2035–2044, 2016. Publisher Full Text | Google Scholar
  62. C.-Y. Fan, J. Pan, N. Usuda, A. V. Yeldandi, M. S. Rao, and J. K. Reddy, “Steatohepatitis, spontaneous peroxisome proliferation and liver tumors in mice lacking peroxisomal fatty acyl-Coa oxidase: Implications for peroxisome proliferator-activated receptor α natural ligand metabolism,” The Journal of Biological Chemistry, vol. 273, no. 25, pp. 15639–15645, 1998. Publisher Full Text | Google Scholar
  63. I. A. Leclercq, G. C. Farrell, J. Field, D. R. Bell, F. J. Gonzalez, and G. R. Robertson, “CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis,” The Journal of Clinical Investigation, vol. 105, no. 8, pp. 1067–1075, 2000. Publisher Full Text | Google Scholar
  64. S. Banerjee and J. Ghosh, “Drug Metabolism and Oxidative Stress: Cellular Mechanism and New Therapeutic Insights,” Biochemistry & Analytical Biochemistry, vol. s3, 2016. Publisher Full Text | Google Scholar
  65. D. K. George, S. Goldwurm, G. A. Macdonald et al., “Increased hepatic iron concentration in nonalcoholic steatohepatitis is associated with increased fibrosis,” Gastroenterology, vol. 114, no. 2, pp. 311–318, 1998. Publisher Full Text | Google Scholar
  66. S. Puntarulo, “Iron, oxidative stress and human health,” Molecular Aspects of Medicine, vol. 26, no. 4-5, pp. 299–312, 2005. Publisher Full Text | Google Scholar
  67. W. S. Blaner, S. M. O'Byrne, N. Wongsiriroj et al., “Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage,” Biochimica et Biophysica Acta, vol. 1791, no. 6, pp. 467–473, 2009. Publisher Full Text | Google Scholar
  68. S. L. Friedman, “Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver,” Physiological Reviews, vol. 88, no. 1, pp. 125–172, 2008. Publisher Full Text | Google Scholar
  69. U. E. Lee and S. L. Friedman, “Mechanisms of hepatic fibrogenesis,” Best Practice & Research: Clinical Gastroenterology, vol. 25, no. 2, pp. 195–206, 2011. Publisher Full Text | Google Scholar
  70. V. Ratziu, S. A. Harrison, S. Francque et al., “Elafibranor, an agonist of the peroxisome proliferator-activated receptor-α and -δ, induces resolution of nonalcoholic steatohepatitis without fibrosis worsening,” Gastroenterology, vol. 150, no. 5, pp. 1147.e5–1159.e5, 2016. Publisher Full Text | Google Scholar
  71. B. A. Neuschwander-Tetri, R. Loomba, and A. J. Sanyal, “Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial,” The Lancet, 2014. Publisher Full Text | Google Scholar
  72. C. de Duve, “Evolution of the Peroxisome,” Annals of the New York Academy of Sciences, vol. 168, no. 2, pp. 369–381, 1969. Publisher Full Text | Google Scholar
  73. I. Issemann and S. Green, “Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators,” Nature, vol. 347, no. 6294, pp. 645–650, 1990. Publisher Full Text | Google Scholar
  74. J. D. Brown and J. Plutzky, “Peroxisome proliferator-activated receptors as transcriptional nodal points and therapeutic targets,” Circulation, vol. 115, no. 4, pp. 518–533, 2007. Publisher Full Text | Google Scholar
  75. C. Dreyer, H. Keller, A. Mahfoudi, V. Laudet, G. Krey, and W. Wahli, “Positive regulation of the peroxisomal β-oxidation pathway by fatty acids through activation of peroxisome proliferator-activated receptors (PPAR),” Biology of the Cell, vol. 77, no. C, pp. 67–76, 1993. Publisher Full Text | Google Scholar
  76. T. M. Willson, P. J. Brown, D. D. Sternbach, and B. R. Henke, “The PPARs: from orphan receptors to drug discovery,” Journal of Medicinal Chemistry, vol. 43, no. 4, pp. 527–550, 2000. Publisher Full Text | Google Scholar
  77. C. K. Glass and S. Ogawa, “Combinatorial roles of nuclear receptors in inflammation and immunity,” Nature Reviews Immunology, vol. 6, no. 1, pp. 44–55, 2006. Publisher Full Text | Google Scholar
  78. J. Berger and D. E. Moller, “The mechanisms of action of PPARs,” Annual Review of Medicine, vol. 53, pp. 409–435, 2002. Publisher Full Text | Google Scholar
  79. P. Puigserver, Z. Wu, C. W. Park, R. Graves, M. Wright, and B. M. Spiegelman, “A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis,” Cell, vol. 92, no. 6, pp. 829–839, 1998. Publisher Full Text | Google Scholar
  80. J. Vamecq and N. Latruffe, “Medical significance of peroxisome proliferator-activated receptors,” The Lancet, vol. 354, no. 9173, pp. 141–148, 1999. Publisher Full Text | Google Scholar
  81. W. W. Harrington, C. S. Britt, J. G. Wilson et al., “The effect of PPARα, PPARδ, PPARγ, and PPARpan agonists on body weight, body mass, and serum lipid profiles in diet-induced obese AKR/J Mice,” PPAR Research, Article ID 97125, 2007. Publisher Full Text | Google Scholar
  82. G. Pascual, A. L. Fong, S. Ogawa et al., “A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-γ,” Nature, vol. 437, no. 7059, pp. 759–763, 2005. Publisher Full Text | Google Scholar
  83. M. Lehrke and M. A. Lazar, “The many faces of PPARγ,” Cell, vol. 123, no. 6, pp. 993–999, 2005. Publisher Full Text | Google Scholar
  84. B. Staels and J.-C. Fruchart, “Therapeutic roles of peroxisome proliferator-activated receptor agonists,” Diabetes, vol. 54, no. 8, pp. 2460–2470, 2005. Publisher Full Text | Google Scholar
  85. B. M. Forman, J. Chen, and R. M. Evans, “Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors α and δ,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 94, no. 9, pp. 4312–4317, 1997. Publisher Full Text | Google Scholar
  86. T. Hanke, D. Merk, D. Steinhilber, G. Geisslinger, and M. Schubert-Zsilavecz, “Small molecules with anti-inflammatory properties in clinical development,” Pharmacology & Therapeutics, vol. 157, pp. 163–187, 2016. Publisher Full Text | Google Scholar
  87. H. H. Steineger, H. N. Sørensen, J. D. Tugwood, S. Skrede, Ø. Spydevold, and K. M. Gautvik, “Dexamethasone and Insulin Demonstrate Marked and Opposite Regulation of the Steady‐State mRNA Level of the Peroxisomal Proliferator‐Activated Receptor (PPAR) in Hepatic Cells: Hormonal Modulation of Fatty‐Acid‐Induced Transcription,” European Journal of Biochemistry, vol. 225, no. 3, pp. 967–974, 1994. Publisher Full Text | Google Scholar
  88. T. Lemberger, B. Staels, R. Saladin, B. Desvergne, J. Auwerx, and W. Wahli, “Regulation of the peroxisome proliferator-activated receptor α gene by glucocorticoids,” The Journal of Biological Chemistry, vol. 269, no. 40, pp. 24527–24530, 1994.
  89. S. A. Kliewer, B. M. Forman, B. Blumberg et al., “Differential expression and activation of a family of murine peroxisome proliferator-activated receptors,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 91, no. 15, pp. 7355–7359, 1994. Publisher Full Text | Google Scholar
  90. O. Braissant and W. Wahli, “Differential expression of peroxisome proliferator-activated receptor-α, -β, and -γ during rat embryonic development,” Endocrinology, vol. 139, no. 6, pp. 2748–2754, 1998. Publisher Full Text | Google Scholar
  91. T. E. Cullingford, K. Bhakoo, S. Peuchen, C. T. Dolphin, R. Patel, and J. B. Clark, “Distribution of mRNAs encoding the peroxisome proliferator-activated receptor α, β, and γ and the retinoid X receptor α, β, and γ in rat central nervous system,” Journal of Neurochemistry, vol. 70, no. 4, pp. 1366–1375, 1998. Publisher Full Text | Google Scholar
  92. J. Granneman, R. Skoff, and X. Yang, “Member of the peroxisome proliferator-activated receptor family of transcription factors is differentially expressed by oligodendrocytes,” Journal of Neuroscience Research, vol. 51, no. 5, pp. 563–573, 1998. Publisher Full Text | Google Scholar
  93. G. Xing, L. Zhang, T. Heynen et al., “Rat PPARΔ contains a CGG triplet repeat and is prominently expressed in the thalamic nuclei,” Biochemical and Biophysical Research Communications, vol. 217, no. 3, pp. 1015–1025, 1995. Publisher Full Text | Google Scholar
  94. H. Lim, R. A. Gupta, W.-G. Ma et al., “Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPARδ,” Genes & Development, vol. 13, no. 12, pp. 1561–1574, 1999. Publisher Full Text | Google Scholar
  95. P. Tontonoz, E. Hu, R. A. Graves, A. I. Budavari, and B. M. Spiegelman, “mPPARγ2: tissue-specific regulator of an adipocyte enhancer,” Genes & Development, vol. 8, no. 10, pp. 1224–1234, 1994. Publisher Full Text | Google Scholar
  96. A. Mansén, H. Guardiola-Diaz, J. Rafter, C. Branting, and J.-Å. Gustafsson, “Expression of the peroxisome proliferator-activated receptor (PPAR) in the mouse colonic mucosa,” Biochemical and Biophysical Research Communications, vol. 222, no. 3, pp. 844–851, 1996. Publisher Full Text | Google Scholar
  97. A. M. Lefebvre, I. Chen, P. Desreumaux et al., “Activation of the peroxisome proliferator-activated receptor γ promotes the development of colon tumors in C57BL/6J-APCMin/+ mice,” Nature Medicine, vol. 4, no. 9, pp. 1053–1057, 1998. Publisher Full Text | Google Scholar
  98. T. Lemberger, O. Braissant, C. Juge-Aubry et al., “PPAR tissue distribution and interactions with other hormone-signaling pathways,” Annals of the New York Academy of Sciences, vol. 804, pp. 231–251, 1996. Publisher Full Text | Google Scholar
  99. C. N. Palmer, M. H. Hsu, K. J. Griffin, J. L. Raucy, and E. F. Johnson, “Peroxisome Proliferator Activated Receptor-Alpha Expression in Human Liver,” Mol. Pharmacol, vol. 53, no. 11, pp. 14–22.
  100. R. Mukherjee, L. Jow, G. E. Croston, and J. R. Paterniti Jr., “Identification, characterization, and tissue distribution of human peroxisome proliferator-activated receptor (PPAR) isoforms PPARγ2 versus PPARγ1 and activation with retinoid X receptor agonists and antagonists,” The Journal of Biological Chemistry, vol. 272, no. 12, pp. 8071–8076, 1997. Publisher Full Text | Google Scholar
  101. M. E. Greene, B. Blumberg, O. W. McBride et al., “isolation of the human peroxisome proliferator activated receptor gamma cDNA: expression in hematopoietic cells and chromosomal mapping,” Gene Expression Patterns, vol. 4, no. 4-5, pp. 281–299, 1995.
  102. B. Desvergne and W. Wahli, “Peroxisome proliferator-activated receptors: nuclear control of metabolism,” Endocrine Reviews, vol. 20, no. 5, pp. 649–688, 1999.
  103. I. Takada, R. T. Yu, H. E. Xu et al., “Alteration of a single amino acid in peroxisome proliferator-activated receptor-α (PPARα) generates a PPARδ phenotype,” Molecular Endocrinology, vol. 14, no. 5, pp. 733–740, 2000. Publisher Full Text | Google Scholar
  104. M. Göttlicher, E. Widmark, Q. Li, and J. Gustafsson, “Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 89, no. 10, pp. 4653–4657, 1992. Publisher Full Text | Google Scholar
  105. R. Mukherjee, L. Jow, D. Noonan, and D. P. McDonnell, “Human and rat peroxisome proliferator activated receptors (PPARs) demonstrate similar tissue distribution but different responsiveness to PPAR activators,” The Journal of Steroid Biochemistry and Molecular Biology, vol. 51, no. 3-4, pp. 157–166, 1994. Publisher Full Text | Google Scholar
  106. T. Sher, H.-F. Yi, O. W. McBride, and F. J. Gonzalez, “cDNA cloning, chromosomal mapping, and functional characterization of the human peroxisome proliferator activated receptor,” Biochemistry, vol. 32, no. 21, pp. 5598–5604, 1993. Publisher Full Text | Google Scholar
  107. F. J. Gonzalez, “The Peroxisome Proliferator-Activated Receptor Alpha (PPAR Alpha): Role in Hepatocarcinogenesis,” Mol. Cell. Endocrinol, vol. 193, pp. 71–79, 2002.
  108. T. S. Mikkelsen, Z. Xu, X. Zhang et al., “Comparative epigenomic analysis of murine and human adipogenesis,” Cell, vol. 143, no. 1, pp. 156–169, 2010. PubMed Abstract | Publisher Full Text | Google Scholar
  109. M. Ammerschlaeger, J. Beigel, K.-U. Klein, and S. O. Mueller, “Characterization of the species-specificity of peroxisome proliferators in rat and human hepatocytes,” Toxicological Sciences, vol. 78, no. 2, pp. 229–240, 2004. Publisher Full Text | Google Scholar
  110. G. Serviddio, A. M. Giudetti, F. Bellanti et al., “Oxidation of hepatic carnitine palmitoyl transferase-I (CPT-I) impairs fatty acid beta-oxidation in rats fed a methionine-choline deficient diet,” PLoS ONE, vol. 6, no. 9, Article ID e24084, 2011. Publisher Full Text | Google Scholar
  111. R. M. Evans, G. D. Barish, and Y.-X. Wang, “PPARs and the complex journey to obesity,” Nature Medicine, vol. 10, no. 4, pp. 355–361, 2004. Publisher Full Text | Google Scholar
  112. N. R. Coet, A. J. Smith, B. I. Frohnert, P. A. Watkins, and D. A. Bernlohr, “The fatty acid transport protein (FATP1) is a very long chain acyl-CoA synthetase,” The Journal of Biological Chemistry, vol. 274, no. 51, pp. 36300–36304, 1999. Publisher Full Text | Google Scholar
  113. J. E. Schaffer and H. F. Lodish, “Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein,” Cell, vol. 79, no. 3, pp. 427–436, 1994. Publisher Full Text | Google Scholar
  114. P. Dourlen, A. Sujkowski, R. Wessells, and B. Mollereau, “Fatty acid transport proteins in disease: New insights from invertebrate models,” Progress in Lipid Research, vol. 60, pp. 30–40, 2015. Publisher Full Text | Google Scholar
  115. G. Martin, K. Schoonjans, A.-M. Lefebvre, B. Staels, and J. Auwerx, “Coordinate regulation of the expression of the fatty acid transport protein and acyl-CoA synthetase genes by PPARα and PPARγ activators,” The Journal of Biological Chemistry, vol. 272, no. 45, pp. 28210–28217, 1997. Publisher Full Text | Google Scholar
  116. S. M. H. Chan, R.-Q. Sun, X.-Y. Zeng et al., “Activation of PPARα ameliorates hepatic insulin resistance and steatosis in high fructose-fed mice despite increased endoplasmic reticulum stress,” Diabetes, vol. 62, no. 6, pp. 2095–2105, 2013. Publisher Full Text | Google Scholar
  117. K. S. Frederiksen, E. M. Wulff, P. Sauerberg, J. P. Mogensen, L. Jeppesen, and J. Fleckner, “Prediction of PPAR-α ligand-mediated physiological changes using gene expression profiles,” Journal of Lipid Research, vol. 45, no. 3, pp. 592–601, 2004. Publisher Full Text | Google Scholar
  118. S. Haubenwallner, A. D. Essenburg, B. C. Barnett et al., “Hypolipidemic activity of select fibrates correlates to changes in hepatic apolipoprotein C-III expression: A potential physiologic basis for their mode of action,” Journal of Lipid Research, vol. 36, no. 12, pp. 2541–2551, 1995.
  119. N. Vu-Dac, S. Chopin-Delannoy, P. Gervois et al., “The nuclear receptors peroxisome proliferator-activated receptor α and rev-erbα mediate the species-specific regulation of apolipoprotein A-I expression by fibrates,” The Journal of Biological Chemistry, vol. 273, no. 40, pp. 25713–25720, 1998. Publisher Full Text | Google Scholar
  120. N. Vu-Dac, K. Schoonjans, V. Kosykh et al., “Fibrates increase human apolipoprotein A-II expression through activation of the peroxisome proliferator-activated receptor,” The Journal of Clinical Investigation, vol. 96, no. 2, pp. 741–750, 1995. Publisher Full Text | Google Scholar
  121. P. Delerive, P. Gervois, J.-C. Fruchart, and B. Staels, “Induction of IκBα expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor-α activators,” The Journal of Biological Chemistry, vol. 275, no. 47, pp. 36703–36707, 2000. Publisher Full Text | Google Scholar
  122. P. Delerive, K. De Bosscher, W. V. Berghe, J.-C. Fruchart, G. Haegeman, and B. Staels, “DNA binding-independent induction of IκBα gene transcription by PPARα,” Molecular Endocrinology, vol. 16, no. 5, pp. 1029–1039, 2002. Publisher Full Text | Google Scholar
  123. M. Pawlak, P. Lefebvre, and B. Staels, “Molecular mechanism of PPARα action and its impact on lipid metabolism, inflammation and fibrosis in non-alcoholic fatty liver disease,” Journal of Hepatology, vol. 62, no. 3, pp. 720–733, 2015. Publisher Full Text | Google Scholar
  124. B. Staels, W. Koenig, A. Habib et al., “Activation of human aortic smooth-muscle cells is inhibited by PPARα but not by PPARγ activators,” Nature, vol. 393, no. 6687, pp. 790–793, 1998. Publisher Full Text | Google Scholar
  125. N. Marx, G. Sukhova, C. Murphy, P. Libby, and J. Plutzky, “Macrophages in human atheroma contain PPARγ: differentiation-dependent peroxisomal proliferator-activated receptor γ (PPARγ) expression and reduction of MMP-9 activity through PPARγ activation in mononuclear phagocytes in vitro,” The American Journal of Pathology, vol. 153, no. 1, pp. 17–23, 1998. Publisher Full Text | Google Scholar
  126. S. M. Jackson, F. Parhami, X.-P. Xi et al., “Peroxisome proliferator-activated receptor activators target human endothelial cells to inhibit leukocyte-endothelial cell interaction,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 19, no. 9, pp. 2094–2104, 1999. Publisher Full Text | Google Scholar
  127. S. Sethi, O. Ziouzenkova, H. Ni, D. D. Wagner, J. Plutzky, and T. N. Mayadas, “Oxidized omega-3 fatty acids in fish oil inhibit leukocyte-endothelial interactions through activation of PPARα,” Blood, vol. 100, no. 4, pp. 1340–1346, 2002. Publisher Full Text | Google Scholar
  128. G. Chinetti, S. Lestavel, V. Bocher et al., “PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway,” Nature Medicine, vol. 7, no. 1, pp. 53–58, 2001. Publisher Full Text | Google Scholar
  129. P. R. Devchand, H. Keller, J. M. Peters, M. Vazquez, F. J. Gonzalez, and W. Wahli, “The PPARα-leukotriene B4 pathway to inflammation control,” Nature, vol. 384, no. 6604, pp. 39–43, 1996. Publisher Full Text | Google Scholar
  130. A. Shimaya, E. Kurosaki, K. Shioduka, R. Nakano, M. Shibasaki, and H. Shikama, “YM268 increases the glucose uptake, cell differentiation, and mRNA expression of glucose transporter in 3T3-L1 adipocytes,” Hormone and Metabolic Research, vol. 30, no. 9, pp. 543–548, 1998. Publisher Full Text | Google Scholar
  131. J. Aubert, O. Champigny, P. Saint-Marc et al., “Up-regulation of UCP-2 gene expression by PPAR agonists in preadipose and adipose cells,” Biochemical and Biophysical Research Communications, vol. 238, no. 2, pp. 606–611, 1997. Publisher Full Text | Google Scholar
  132. A. Valmaseda, M. C. Carmona, M. J. Barberá et al., “Opposite regulation of PPAR-α and -β gene expression by both their ligands and retinoic acid in brown adipocytes,” Molecular and Cellular Endocrinology, vol. 154, no. 1-2, pp. 101–109, 1999. Publisher Full Text | Google Scholar
  133. A. Chawla, E. J. Schwarz, D. D. Dimaculangan, and M. A. Lazar, “Peroxisome proliferator-activated receptor (PPAR) γ: adipose-predominant expression and induction early in adipocyte differentiation,” Endocrinology, vol. 135, no. 2, pp. 798–800, 1994. Publisher Full Text | Google Scholar
  134. W. C. Yeh, Z. Cao, M. Classon, and S. L. McKnight, “Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins,” Genes & Development, vol. 9, no. 2, pp. 168–181, 1995. Publisher Full Text | Google Scholar
  135. Z. Wu, E. D. Rosen, R. Brun et al., “Cross-regulation of C/EBPα and PPARγ controls the transcriptional pathway of adipogenesis and insulin sensitivity,” Molecular Cell, vol. 3, no. 2, pp. 151–158, 1999. Publisher Full Text | Google Scholar
  136. P. Cornelius, O. A. MacDougald, and M. D. Lane, “Regulation of adipocyte development,” Annual Review of Nutrition, vol. 14, pp. 99–129, 1994. Publisher Full Text | Google Scholar
  137. J. Wu, P. Boström, L. M. Sparks et al., “Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human,” Cell, vol. 150, no. 2, pp. 366–376, 2012. Publisher Full Text | Google Scholar
  138. M. Rosenwald, A. Perdikari, T. Rülicke, and C. Wolfrum, “Bi-directional interconversion of brite and white adipocytes,” Nature Cell Biology, vol. 15, no. 6, pp. 659–667, 2013. Publisher Full Text | Google Scholar
  139. V. Barquissau, D. Beuzelin, D. F. Pisani et al., “White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways,” Molecular Metabolism, vol. 5, no. 5, pp. 352–365, 2016. Publisher Full Text | Google Scholar
  140. B. M. Spiegelman, E. Hu, J. B. Kim, and R. Brun, “PPARγ and the control of adipogenesis,” Biochimie, vol. 79, no. 2-3, pp. 111–112, 1997. Publisher Full Text | Google Scholar
  141. P. Tontonoz, R. A. Graves, A. I. Budavari et al., “Adipocyte-specific transcription factor ARF6 is a heterodimeric complex of two nuclear hormone receptors, PPARγ and RXRα,” Nucleic Acids Research, vol. 22, no. 25, pp. 5628–5634, 1994. Publisher Full Text | Google Scholar
  142. B. I. Frohnert, T. Y. Hui, and D. A. Bernlohr, “Identification of a functional peroxisome proliferator-responsive element in the murine fatty acid transport protein gene,” The Journal of Biological Chemistry, vol. 274, no. 7, pp. 3970–3977, 1999. Publisher Full Text | Google Scholar
  143. P. C. Chui, H.-P. Guan, M. Lehrke, and M. A. Lazar, “PPARγ regulates adipocyte cholesterol metabolism via oxidized LDL receptor 1,” The Journal of Clinical Investigation, vol. 115, no. 8, pp. 2244–2256, 2005. Publisher Full Text | Google Scholar
  144. T. Hibuse, N. Maeda, T. Funahashi et al., “Aquaporin 7 deficiency is associated with development of obesity through activation of adipose glycerol kinase,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 102, no. 31, pp. 10993–10998, 2005. Publisher Full Text | Google Scholar
  145. A. Chawla, Y. Barak, L. Nagy, D. Liao, P. Tontonoz, and R. M. Evans, “PPAR-γ dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation,” Nature Medicine, vol. 7, no. 1, pp. 48–52, 2001. Publisher Full Text | Google Scholar
  146. N. Maeda, M. Takahashi, T. Funahashi et al., “PPARγ ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein,” Diabetes, vol. 50, no. 9, pp. 2094–2099, 2001. Publisher Full Text | Google Scholar
  147. B. A. Neuschwander-Tetri, E. M. Brunt, K. R. Wehmeier, D. Oliver, and B. R. Bacon, “Improved nonalcoholic steatohepatitis after 48 weeks of treatment with the PPAR-γ ligand rosiglitazone,” Hepatology, vol. 38, no. 4, pp. 1008–1017, 2003. Publisher Full Text | Google Scholar
  148. D. Kramer, R. Shapiro, A. Adler, E. Bush, and C. M. Rondinone, “Insulin-sensitizing effect of rosiglitazone (BRL-49653) by regulation of glucose transporters in muscle and fat of zucker rats,” Metabolism - Clinical and Experimental, vol. 50, no. 11, pp. 1294–1300, 2001. Publisher Full Text | Google Scholar
  149. A. Cabrero, J. C. Laguna, and M. Vázquez, “Peroxisome proliferator-activated receptors and the control of inflammation.,” Curr Drug Targets Inflamm Allergy, vol. 1, no. 3, pp. 243–248, 2002. Publisher Full Text | Google Scholar
  150. C. Jiang, A. T. Ting, and B. Seed, “PPAR-γ Agonists Inhibit Production Ofmonocyte Inflammatorycytokine,” Nature, vol. 391, pp. 82–86, 1998. Publisher Full Text | Google Scholar
  151. I. Inoue, S.-I. Goto, T. Matsunaga et al., “The ligands/activators for peroxisome proliferator-activated receptor α (PPARα) and PPARγ increase Cu2+,Zn2+-superoxide dismutase and decrease p22phox message expressions in primary endothelial cells,” Metabolism - Clinical and Experimental, vol. 50, no. 1, pp. 3–11, 2001. Publisher Full Text | Google Scholar
  152. J. Hwang, D. J. Kleinhenz, B. Lassègue, K. K. Griendling, S. Dikalov, and C. M. Hart, “Peroxisome proliferator-activated receptor-γ ligands regulate endothelial membrane superoxide production,” American Journal of Physiology-Cell Physiology, vol. 288, no. 4, pp. C899–C905, 2005. Publisher Full Text | Google Scholar
  153. S. Katoh, S. Hata, M. Matsushima et al., “Troglitazone prevents the rise in visceral adiposity and improves fatty liver associated with sulfonylurea therapy - A randomized controlled trial,” Metabolism - Clinical and Experimental, vol. 50, no. 4, pp. 414–417, 2001. Publisher Full Text | Google Scholar
  154. J. P. Berger, T. E. Akiyama, and P. T. Meinke, “PPARs: therapeutic targets for metabolic disease,” Trends in Pharmacological Sciences, vol. 26, no. 5, pp. 244–251, 2005. Publisher Full Text | Google Scholar
  155. T. Coll, R. Rodríguez-Calvo, E. Barroso et al., “Peroxisome proliferator-activated receptor (PPAR) β/δ: a new potential therapeutic target for the treatment of metabolic syndrome,” Current Molecular Pharmacology, vol. 2, no. 1, pp. 46–55, 2009. Publisher Full Text | Google Scholar
  156. K.-D. Wagner and N. Wagner, “Peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) acts as regulator of metabolism linked to multiple cellular functions,” Pharmacology & Therapeutics, vol. 125, no. 3, pp. 423–435, 2010. Publisher Full Text | Google Scholar
  157. L. Salvadó, E. Barroso, A. M. Gómez-Foix et al., “Pparβ/δ prevents endoplasmic reticulum stress-associated inflammation and insulin resistance in skeletal muscle cells through an ampk-dependent mechanism,” Diabetologia, vol. 57, no. 10, pp. 2126–2135, 2014. Publisher Full Text | Google Scholar
  158. L. Salvadó, L. Serrano-Marco, E. Barroso, X. Palomer, and M. Vázquez-Carrera, “Targeting PPARβ/δ for the treatment of type 2 diabetes mellitus,” Expert Opinion on Therapeutic Targets, vol. 16, no. 2, pp. 209–223, 2012. Publisher Full Text | Google Scholar
  159. U. Risérus, D. Sprecher, T. Johnson et al., “Activation of peroxisome proliferator-activated receptor (PPAR)delta promotes reversal of multiple metabolic abnormalities, reduces oxidative stress, and increases fatty acid oxidation in moderately obese men,” Diabetes, vol. 57, no. 2, pp. 332–339, 2008. Publisher Full Text | Google Scholar
  160. T.-C. He, T. A. Chan, B. Vogelstein, and K. W. Kinzler, “PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs,” Cell, vol. 99, no. 3, pp. 335–345, 1999. Publisher Full Text | Google Scholar
  161. E. Klimcakova, C. Moro, A. Mazzucotelli et al., “Profiling of adipokines secreted from human subcutaneous adipose tissue in response to PPAR agonists,” Biochemical and Biophysical Research Communications, vol. 358, no. 3, pp. 897–902, 2007. Publisher Full Text | Google Scholar
  162. S. Liu, B. Hatano, and M. Zhao, “Role of peroxisome proliferator-activated receptor δ/β in hepatic metabolic regulation,” The Journal of Biological Chemistry, vol. 286, no. 2, pp. 1237–1247, 2011. Publisher Full Text | Google Scholar
  163. L. D. Roberts, D. G. Hassall, D. A. Winegar, J. N. Haselden, A. W. Nicholls, and J. L. Griffin, “Increased hepatic oxidative metabolism distinguishes the action of Peroxisome proliferator-activated receptor δ from Peroxisome proliferator-activated receptor γ in the ob/ob mouse,” Genome Medicine, vol. 1, no. 12, p. 115, 2009. Publisher Full Text | Google Scholar
  164. D. Pan, M. Fujimoto, A. Lopes, and Y.-X. Wang, “Twist-1 is a PPARδ-inducible, negative-feedback regulator of PGC-1α in brown fat metabolism,” Cell, vol. 137, no. 1, pp. 73–86, 2009. Publisher Full Text | Google Scholar
  165. Z. Nahlé, M. Hsieh, T. Pietka et al., “CD36-dependent regulation of muscle FoxO1 and PDK4 in the PPAR delta/beta-mediated adaptation to metabolic stress,” The Journal of Biological Chemistry, vol. 283, no. 21, pp. 14317–14326, 2008. Publisher Full Text | Google Scholar
  166. U. Dressel, T. L. Allen, J. B. Pippal, P. R. Rohde, P. Lau, and G. E. O. Muscat, “The peroxisome proliferator-activated receptor β/δ agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells,” Molecular Endocrinology, vol. 17, no. 12, pp. 2477–2493, 2003. Publisher Full Text | Google Scholar
  167. A. G. Dulloo and S. Samec, “Uncoupling proteins: Their roles in adaptive thermogenesis and substrate metabolism reconsidered,” British Journal of Nutrition, vol. 86, no. 2, pp. 123–139, 2001. Publisher Full Text | Google Scholar
  168. H. P. Jiang and G. Serrero, “Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein.,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 89, no. 17, pp. 7856–7860, 1992. Publisher Full Text | Google Scholar
  169. H. Staiger, C. Haas, J. Machann et al., “Muscle-derived angiopoietin-like protein 4 is induced by fatty acids via peroxisome proliferator-activated receptor (PPAR)-δ and is of metabolic relevance in humans,” Diabetes, vol. 58, no. 3, pp. 579–589, 2009. Publisher Full Text | Google Scholar
  170. G. Santulli, “Angiopoietin-Like Proteins: A Comprehensive Look,” Frontiers in Endocrinology, vol. 5, pp. 1–6, 2014. Publisher Full Text | Google Scholar
  171. C. Gaudel, C. Schwartz, C. Giordano, N. A. Abumrad, and P. A. Grimaldi, “Pharmacological activation of PPARβ promotes rapid and calcineur-independent fiber remodeling and angiogenesis in mouse skeletal muscle,” American Journal of Physiology-Renal Physiology, vol. 295, no. 2, pp. E297–E304, 2008. Publisher Full Text | Google Scholar
  172. N. Wagner, C. Jehl-Piétri, P. Lopez et al., “Peroxisome proliferator-activated receptor β stimulation induces rapid cardiac growth and angiogenesis via direct activation of calcineurin,” Cardiovascular Research, vol. 83, no. 1, pp. 61–71, 2009. Publisher Full Text | Google Scholar
  173. M. D. Leibowitz, C. Fiévet, N. Hennuyer et al., “Activation of PPARδ alters lipid metabolism in db/db mice,” FEBS Letters, vol. 473, no. 3, pp. 333–336, 2000. Publisher Full Text | Google Scholar
  174. W. R. Oliver Jr., J. L. Shenk, M. R. Snaith et al., “A selective peroxisome proliferator-activated receptor δ agonist promotes reverse cholesterol transport,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 98, no. 9, pp. 5306–5311, 2001. Publisher Full Text | Google Scholar
  175. D. L. Sprecher, C. Massien, G. Pearce et al., “Triglyceride: High-density lipoprotein cholesterol effects in healthy subjects administered a peroxisome proliferator activated receptor δ agonist,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 27, no. 2, pp. 359–365, 2007. Publisher Full Text | Google Scholar
  176. T. Tanaka, J. Yamamoto, S. Iwasaki et al., “Activation of peroxisome proliferator-activated receptor δ induces fatty acid β-oxidation in skeletal muscle and attenuates metabolic syndrome,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 100, no. 26, pp. 15924–15929, 2003. Publisher Full Text | Google Scholar
  177. J. I. Odegaard, R. R. Ricardo-Gonzalez, A. Red Eagle et al., “Alternative M2 activation of Kupffer cells by PPARdelta ameliorates obesity-induced insulin resistance,” Cell Metabolism, vol. 7, no. 6, pp. 496–507, 2008. Publisher Full Text | Google Scholar
  178. N. E. Buroker, J. Barboza, and J.-Y. Huang, “The IκBα gene is a peroxisome proliferator-activated receptor cardiac target gene,” FEBS Journal, vol. 276, no. 12, pp. 3247–3255, 2009. Publisher Full Text | Google Scholar
  179. R. Rodríguez-Calvo, L. Serrano, T. Coll et al., “Activation of peroxisome proliferator-activated receptor β/δ inhibits lipopolysaccharide-induced cytokine production in adipocytes by lowering nuclear factor-κB activity via extracellular signal-related kinase 1/2,” Diabetes, vol. 57, no. 8, pp. 2149–2157, 2008. Publisher Full Text | Google Scholar
  180. J. Pollinger and D. Merk, “Therapeutic applications of the versatile fatty acid mimetic WY14643,” Expert Opinion on Therapeutic Patents, vol. 27, no. 4, pp. 517–525, 2017. Publisher Full Text | Google Scholar
  181. O. Rau, Y. Syha, H. Zettl, M. Kock, A. Bock, and M. Schubert-Zsilavecz, “α-alkyl substituted pirinixic acid derivatives as potent dual agonists of the peroxisome proliferator activated receptor alpha and gamma,” Archiv der Pharmazie, vol. 341, no. 3, pp. 191–195, 2008. Publisher Full Text | Google Scholar
  182. E. Ip, G. C. Farrell, G. Robertson, P. Hall, R. Kirsch, and I. Leclercq, “Central role of PPARα-dependent hepatic lipid turnover in dietary steatohepatitis in mice,” Hepatology, vol. 38, no. 1, pp. 123–132, 2003. Publisher Full Text | Google Scholar
  183. E. Ip, G. Farrell, P. Hall, G. Robertson, and I. Leclercq, “Administration of the potent pparα agonist, Wy-14,643, reverses nutritional fibrosis and steatohepatitis in mice,” Hepatology, vol. 39, no. 5, pp. 1286–1296, 2004. Publisher Full Text | Google Scholar
  184. J. C. Fruchart, B. Staels, and P. Duriez, “The role of fibric acids in atherosclerosis,” Current Atherosclerosis Reports, vol. 3, no. 1, pp. 83–92, 2001. Publisher Full Text | Google Scholar
  185. R. Shiri-Sverdlov, K. Wouters, P. J. V. Gorp et al., “Early diet-induced non-alcoholic steatohepatitis in APOE2 knock-in mice and its prevention by fibrates,” Journal of Hepatology, vol. 44, no. 4, pp. 732–741, 2006. Publisher Full Text | Google Scholar
  186. N. Zhang, Y. Lu, X. Shen et al., “Fenofibrate treatment attenuated chronic endoplasmic reticulum stress in the liver of nonalcoholic fatty liver disease mice,” Pharmacology, vol. 95, no. 3-4, pp. 173–180, 2015. Publisher Full Text | Google Scholar
  187. E. A. Abd El-Haleim, A. K. Bahgat, and S. Saleh, “Resveratrol and fenofibrate ameliorate fructose-induced nonalcoholic steatohepatitis by modulation of genes expression,” World Journal of Gastroenterology, vol. 22, no. 10, pp. 2931–2948, 2016. Publisher Full Text | Google Scholar
  188. J. Laurin, K. D. Lindor, J. S. Crippin et al., “Ursodeoxycholic acid or clofibrate in the treatment of non-alcohol-induced steatohepatitis: A pilot study,” Hepatology, vol. 23, no. 6, pp. 1464–1467, 1996. Publisher Full Text | Google Scholar
  189. M. Basaranoglu, O. Acbay, and A. Sonsuz, “A controlled trial of gemfibrozil in the treatment of patients with nonalcoholic steatohepatitis,” Journal of Hepatology, vol. 31, no. 2, p. 384, 1999. Publisher Full Text | Google Scholar
  190. C. Fernández-Miranda, M. Pérez-Carreras, F. Colina, G. López-Alonso, C. Vargas, and J. A. Solís-Herruzo, “A pilot trial of fenofibrate for the treatment of non-alcoholic fatty liver disease,” Digestive and Liver Disease, vol. 40, no. 3, pp. 200–205, 2008. Publisher Full Text | Google Scholar
  191. J. M. Peters, T. Aoyama, A. M. Burns, and F. J. Gonzalez, “Bezafibrate is a dual ligand for PPARα and PPARβ: Studies using null mice,” Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, vol. 1632, no. 1-3, pp. 80–89, 2003. Publisher Full Text | Google Scholar
  192. T. Nagasawa, Y. Inada, S. Nakano et al., “Effects of bezafibrate, PPAR pan-agonist, and GW501516, PPARδ agonist, on development of steatohepatitis in mice fed a methionine- and choline-deficient diet,” European Journal of Pharmacology, vol. 536, no. 1-2, pp. 182–191, 2006. Publisher Full Text | Google Scholar
  193. C. H. Chung, M. Y. Lee, R. Choi et al., “Peroxisome proliferator-activated receptor δ agonist attenuates hepatic steatosis by anti-inflammatory mechanism,” Experimental & Molecular Medicine, vol. 44, no. 10, pp. 578–585, 2012. Publisher Full Text | Google Scholar
  194. H. E. Bays, S. Schwartz, T. Littlejohn III et al., “MBX-8025, a novel peroxisome proliferator receptor-δ agonist: lipid and other metabolic effects in dyslipidemic overweight patients treated with and without atorvastatin,” The Journal of Clinical Endocrinology & Metabolism, vol. 96, no. 9, pp. 2889–2897, 2011. Publisher Full Text | Google Scholar
  195. FDA. Updated, FDA Review Concludes That Use of Type 2 Diabetes Medicine Pioglitazone May Be Linked to an Increased Risk of Bladder Cancer,.
  196. FDA, FDA Drug Safety Communication: FDA Eliminates the Risk Evaluation and Mitigation Strategy (REMS) for Rosiglitazone-Containing Diabetes Medicines. 2015.
  197. K. Kawaguchi, I. Sakaida, M. Tsuchiya, K. Omori, T. Takami, and K. Okita, “Pioglitazone prevents hepatic steatosis, fibrosis, and enzyme-altered lesions in rat liver cirrhosis induced by a choline-deficient L-amino acid-defined diet,” Biochemical and Biophysical Research Communications, vol. 315, no. 1, pp. 187–195, 2004. Publisher Full Text | Google Scholar
  198. I. A. Leclercq, C. Sempoux, P. Stärkel, and Y. Horsmans, “Limited therapeutic efficacy of pioglitazone on progression of hepatic fibrosis in rats,” Gut, vol. 55, no. 7, pp. 1020–1029, 2006. Publisher Full Text | Google Scholar
  199. I. A. Leclercq, V. A. Lebrun, P. Stärkel, and Y. J. Horsmans, “Intrahepatic insulin resistance in a murine model of steatohepatitis: effect of PPARγ agonist pioglitazone,” Laboratory Investigation, vol. 87, no. 1, pp. 56–65, 2007. Publisher Full Text | Google Scholar
  200. P.-J. Hsiao, T.-J. Hsieh, K.-K. Kuo et al., “Pioglitazone retrieves hepatic antioxidant DNA repair in a mice model of high fat diet,” BMC Molecular Biology, vol. 9, article no. 82, 2008. Publisher Full Text | Google Scholar
  201. S. H. Caldwell, E. E. Hespenheide, J. A. Redick, J. C. Iezzoni, E. H. Battle, and B. L. Sheppard, “A pilot study of a thiazolidinedione, troglitazone, in nonalcoholic steatohepatitis,” American Journal of Gastroenterology, vol. 96, no. 2, pp. 519–525, 2001. Publisher Full Text | Google Scholar
  202. J. M. Lehmann, L. B. Moore, T. A. Smith-Oliver, W. O. Wilkison, T. M. Willson, and S. A. Kliewer, “An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ),” The Journal of Biological Chemistry, vol. 270, no. 22, pp. 12953–12956, 1995. Publisher Full Text | Google Scholar
  203. F. H. Luyckx, C. Desaive, A. Thiry et al., “Liver abnormalities in severely obese subjects: effect of drastic weight loss after gastroplasty,” International Journal of Obesity and Related Metabolic Disorders, vol. 22, no. 3, pp. 222–226, 1998. Publisher Full Text | Google Scholar
  204. E. T. Poehlman, “Abdominal obesity: The metabolic multi-risk factor,” Coronary Artery Disease, vol. 9, no. 8, pp. 469–471, 1998. Publisher Full Text | Google Scholar
  205. K. Promrat, G. Lutchman, G. I. Uwaifo et al., “A pilot study of pioglitazone treatment for nonalcoholic steatohepatitis,” Hepatology, vol. 39, no. 1, pp. 188–196, 2004. Publisher Full Text | Google Scholar
  206. R. Belfort, S. A. Harrison, K. Brown et al., “A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis,” The New England Journal of Medicine, vol. 355, no. 22, pp. 2297–2307, 2006. Publisher Full Text | Google Scholar
  207. G. Lutchman, K. Promrat, D. E. Kleiner et al., “Changes in Serum Adipokine Levels During Pioglitazone Treatment for Nonalcoholic Steatohepatitis: Relationship to Histological Improvement,” Clinical Gastroenterology and Hepatology, vol. 4, no. 8, pp. 1048–1052, 2006. Publisher Full Text | Google Scholar
  208. G. P. Aithal, J. A. Thomas, P. V. Kaye et al., “Randomized, placebo-controlled trial of pioglitazone in nondiabetic subjects with nonalcoholic steatohepatitis,” Gastroenterology, vol. 135, no. 4, pp. 1176–1184, 2008. Publisher Full Text | Google Scholar
  209. V. Ratziu, P. Giral, S. Jacqueminet et al., “Rosiglitazone for nonalcoholic steatohepatitis: one-year results of the randomized placebo-controlled Fatty Liver Improvement with Rosiglitazone Therapy (FLIRT) Trial,” Gastroenterology, vol. 135, no. 1, pp. 100–110, 2008. Publisher Full Text | Google Scholar
  210. V. Ratziu, F. Charlotte, C. Bernhardt et al., “Long-term efficacy of rosiglitazone in nonalcoholic steatohepatitis: results of the fatty liver improvement by rosiglitazone therapy (FLIRT 2) extension trial,” Hepatology, vol. 51, no. 2, pp. 445–453, 2010. Publisher Full Text | Google Scholar
  211. A. J. Sanyal, N. Chalasani, K. V. Kowdley et al., “Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis,” The New England Journal of Medicine, vol. 362, no. 18, pp. 1675–1685, 2010. Publisher Full Text | Google Scholar
  212. D. M. Torres, F. J. Jones, and J. C. Shaw, “Rosiglitazone versus rosiglitazone and metformin versus rosiglitazone and losartan in the treatment of nonalcoholic steatohepatitis in humans: a 12-month randomized, prospective, open- label trial,” Hepatology, vol. 54, no. 5, pp. 1631–1639, 2011. Publisher Full Text | Google Scholar
  213. B. C. Sharma, A. Kumar, V. Garg, R. S. Reddy, P. Sakhuja, and S. K. Sarin, “A Randomized Controlled Trial Comparing Efficacy of Pentoxifylline and Pioglitazone on Metabolic Factors and Liver Histology in Patients with Non-alcoholic Steatohepatitis,” Journal of Clinical and Experimental Hepatology, vol. 2, no. 4, pp. 333–337, 2012. Publisher Full Text | Google Scholar
  214. K. Cusi, B. Orsak, F. Bril et al., “Long-term pioglitazone treatment for patients with nonalcoholic steatohepatitis and prediabetes or type 2 diabetes mellitus a randomized trial,” Annals of Internal Medicine, vol. 165, no. 5, pp. 305–315, 2016. Publisher Full Text | Google Scholar
  215. E. Boettcher, G. Csako, and F. Pucino, “Meta-analysis: pioglitazone improves liver histology and fibrosis in patients with non-alcoholic steatohepatitis,” Alimentary Pharmacology & Therapeutics, vol. 35, no. 1, pp. 66–75, 2012. Publisher Full Text | Google Scholar
  216. S. A. Polyzos and C. S. Mantzoros, “Adiponectin as a target for the treatment of nonalcoholic steatohepatitis with thiazolidinediones: a systematic review,” Metabolism - Clinical and Experimental, vol. 65, no. 9, pp. 1297–1306, 2016. Publisher Full Text | Google Scholar
  217. S. Nakano, T. Nagasawa, T. Ijiro et al., “Bezafibrate prevents hepatic stellate cell activation and fibrogenesis in a murine steatohepatitis model, and suppresses fibrogenic response induced by transforming growth factor-β1 in a cultured stellate cell line,” Hepatology Research, vol. 38, no. 10, pp. 1026–1039, 2008. Publisher Full Text | Google Scholar
  218. T. Harrity, D. Farrelly, A. Tieman et al., “Muraglitazar, a novel dual (α/γ) peroxisome proliferator-activated receptor activator, improves diabetes and other metabolic abnormalities and preserves β-cell function in db/db mice,” Diabetes, vol. 55, no. 1, pp. 240–248, 2006. Publisher Full Text | Google Scholar
  219. S. E. Nissen, K. Wolski, and E. J. Topol, “Effect of muraglitazar on death and major adverse cardiovascular events in patients with type 2 diabetes mellitus,” The Journal of the American Medical Association, vol. 294, no. 20, pp. 2581–2586, 2005. Publisher Full Text | Google Scholar
  220. R. R. Henry, A. M. Lincoff, S. Mudaliar, M. Rabbia, C. Chognot, and M. Herz, “Effect of the dual peroxisome proliferator-activated receptor-α/γ agonist aleglitazar on risk of cardiovascular disease in patients with type 2 diabetes (SYNCHRONY): a phase II, randomised, dose-ranging study,” The Lancet, vol. 374, no. 9684, pp. 126–135, 2009. Publisher Full Text | Google Scholar
  221. B. Hamrén, K. P. Öhman, M. K. Svensson, and M. O. Karlsson, “Pharmacokinetic-pharmacodynamic assessment of the interrelationships between tesaglitazar exposure and renal function in patients with type 2 diabetes mellitus,” Clinical Pharmacology and Therapeutics, vol. 52, no. 9, pp. 1317–1327, 2012. Publisher Full Text | Google Scholar
  222. A prospective randomized trial to compare saroglitazar with pioglitazone in nonalcoholic fatty liver disease ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT02265276?term=glitazar&rank=1 (accessed Sep 5, 2017).
  223. B. Cariou, Y. Zaïr, B. Staels, and E. Bruckert, “Effects of the new dual PPARα/δ agonist GFT505 on lipid and glucose homeostasis in abdominally obese patients with combined dyslipidemia or impaired glucose metabolism,” Diabetes Care, vol. 34, no. 9, pp. 2008–2014, 2011. Publisher Full Text | Google Scholar
  224. Phase 3 study to evaluate the efficacy and safety of elafibranor versus placebo in patients with nonalcoholic steatohepatitis (NASH) ClinicalTrials.gov, https://clinicaltrials.gov/ct2/show/record/NCT02704403?term=RESOLVE-IT&rank=1 (accessed Sep 5, 2017).
  225. V. Ratziu, F. Charlotte, A. Heurtier et al., “Sampling variability of liver biopsy in nonalcoholic fatty liver disease,” Gastroenterology, vol. 128, no. 7, pp. 1898–1906, 2005. Publisher Full Text | Google Scholar
  226. Y. Rotman and A. J. Sanyal, “Current and upcoming pharmacotherapy for non-alcoholic fatty liver disease,” Gut, vol. 66, no. 1, pp. 180–190, 2017. Publisher Full Text | Google Scholar
  227. G. Musso, M. Cassader, and R. Gambino, “Non-alcoholic steatohepatitis: emerging molecular targets and therapeutic strategies,” Nature Reviews Drug Discovery, vol. 15, no. 4, pp. 249–274, 2016. Publisher Full Text | Google Scholar
  228. A. Bertola, S. Bonnafous, R. Anty et al., “Hepatic expression patterns of inflammatory and immune response genes associated with obesity and nash in morbidly obese patients,” PLoS ONE, vol. 5, no. 10, Article ID e13577, 2010. Publisher Full Text | Google Scholar
  229. S. Friedman, A. Sanyal, Z. Goodman et al., “Efficacy and safety study of cenicriviroc for the treatment of non-alcoholic steatohepatitis in adult subjects with liver fibrosis: CENTAUR Phase 2b study design,” Contemporary Clinical Trials, vol. 47, pp. 356–365, 2016. Publisher Full Text | Google Scholar
  230. F. J. Barreyro, S. Holod, P. V. Finocchietto et al., “The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis,” Liver International, vol. 35, no. 3, pp. 953–966, 2015. Publisher Full Text | Google Scholar
  231. P. G. Traber, H. Chou, E. Zomer et al., “Regression of fibrosis and reversal of cirrhosis in rats by galectin inhibitors in thioacetamide-induced liver disease,” PLoS ONE, vol. 8, no. 10, Article ID e75361, 2013. Publisher Full Text | Google Scholar
  232. P. G. Traber and E. Zomer, “Therapy of experimental NASH and fibrosis with galectin inhibitors,” PLoS ONE, vol. 8, no. 12, Article ID e83481, 2013. Publisher Full Text | Google Scholar
  233. G. Musso, M. Cassader, E. Paschetta, and R. Gambino, “Thiazolidinediones and Advanced Liver Fibrosis in Nonalcoholic Steatohepatitis,” JAMA Internal Medicine, vol. 177, no. 5, p. 633, 2017. Publisher Full Text | Google Scholar
  234. M.-K. Kim, Y. N. Chae, S.-H. Choi et al., “PAM-1616, a selective peroxisome proliferator-activated receptor γ modulator with preserved anti-diabetic efficacy and reduced adverse effects,” European Journal of Pharmacology, vol. 650, no. 2-3, pp. 673–681, 2011. Publisher Full Text | Google Scholar
  235. K. R. Kim, J. H. Lee, S. J. Kim et al., “KR-62980: A novel peroxisome proliferator-activated receptor γ agonist with weak adipogenic effects,” Biochemical Pharmacology, vol. 72, no. 4, pp. 446–454, 2006. Publisher Full Text | Google Scholar
  236. C. H. Chang, L. A. McNamara, M. S. Wu et al., “A novel selective peroxisome proliferator-activator receptor-γ modulator-SPPARγM5 improves insulin sensitivity with diminished adverse cardiovascular effects,” European Journal of Pharmacology, vol. 584, no. 1, pp. 192–201, 2008. Publisher Full Text | Google Scholar
  237. J. P. Taygerly, L. R. McGee, S. M. Rubenstein et al., “Discovery of INT131: a selective PPARγ modulator that enhances insulin sensitivity,” Bioorganic & Medicinal Chemistry, vol. 21, no. 4, pp. 979–992, 2013. Publisher Full Text | Google Scholar
  238. A. Motani, Z. Wang, J. Weiszmann et al., “INT131: a selective modulator of PPARγ,” Journal of Molecular Biology, vol. 386, no. 5, pp. 1301–1311, 2009. Publisher Full Text | Google Scholar
  239. X. Xie, X. Zhou, W. Chen et al., “L312, a novel PPARγ ligand with potent anti-diabetic activity by selective regulation,” Biochimica et Biophysica Acta (BBA) - General Subjects, vol. 1850, no. 1, pp. 62–72, 2015. Publisher Full Text | Google Scholar
  240. J. J. Acton III, T. E. Akiyama, C. H. Chang et al., “Discovery of (2R)-2-(3-{3-[(4-methoxyphenyl)carbonyl]-2-methyl-6- (trifluoromethoxy)-1H-indol-1-yl}phenoxy)butanoic acid (MK-0533): A novel selective peroxisome proliferator-activated receptor γ modulator for the treatment of type 2 diabetes mellitus with a reduced potential to increase plasma and extracellular fluid volume,” Journal of Medicinal Chemistry, vol. 52, no. 13, pp. 3846–3854, 2009. Publisher Full Text | Google Scholar
  241. F. M. Gregoire, F. Zhang, H. J. Clarke et al., “MBX-102/JNJ39659100, a novel peroxisome proliferator-activated receptor-ligand with weak transactivation activity retains antidiabetic properties in the absence of weight gain and edema,” Molecular Endocrinology, vol. 23, no. 7, pp. 975–988, 2009. Publisher Full Text | Google Scholar
  242. A. M. DePaoli, L. S. Higgins, R. R. Henry, C. Mantzoros, F. L. Dunn, and INT131-007 Study Group, “Can a selective PPARγ modulator improve glycemic control in patients with type 2 diabetes with fewer side effects compared with pioglitazone?” Diabetes Care, vol. 37, no. 7, pp. 1918–1923, 2014. Publisher Full Text | Google Scholar
  243. K.-A. K. Sohn, C. Cruciani-Guglielmacci, N. Kassis et al., “S26948, a new specific peroxisome proliferator activated receptor gamma modulator improved in vivo hepatic insulin sensitivity in 48 h lipid infused rats,” European Journal of Pharmacology, vol. 608, no. 1-3, pp. 104–111, 2009. Publisher Full Text | Google Scholar
  244. T. Fujimura, H. Sakuma, S. Konishi et al., “FK614, a novel peroxisome proliferator-activated receptor γ modulator, induces differential transactivation through a unique ligand-specific interaction with transcriptional coactivators,” Journal of Pharmacological Sciences, vol. 99, no. 4, pp. 342–352, 2005. Publisher Full Text | Google Scholar
  245. H. Minoura, S. Takeshita, M. Ita et al., “Pharmacological characteristics of a novel nonthiazolidinedione insulin sensitizer, FK614,” European Journal of Pharmacology, vol. 494, no. 2-3, pp. 273–281, 2004. Publisher Full Text | Google Scholar
  246. R. Mukherjee, P. A. Hoener, L. Jow et al., “A selective peroxisome proliferator-activated receptor-γ (PPARγ) modulator blocks adipocyte differentiation but stimulates glucose uptake in 3T3-L1 adipocytes,” Molecular Endocrinology, vol. 14, no. 9, pp. 1425–1433, 2000.
  247. J. H. Choi, A. S. Banks, T. M. Kamenecka et al., “Antidiabetic actions of a non-agonist PPARγ ligand blocking Cdk5-mediated phosphorylation,” Nature, vol. 477, no. 7365, pp. 477–481, 2011. Publisher Full Text | Google Scholar
  248. A. A. Amato, S. Rajagopalan, and J. Z. Lin, “GQ-16, a novel peroxisome proliferator-activated receptor γ (PPARγ) ligand, promotes insulin sensitization without weight gain,” The Journal of Biological Chemistry, vol. 287, no. 33, pp. 28169–28179, 2012. Publisher Full Text | Google Scholar
  249. J. H. Choi, A. S. Banks, J. L. Estall et al., “Anti-diabetic drugs inhibit obesity-linked phosphorylation of PPARγ 3 by Cdk5,” Nature, vol. 466, no. 7305, pp. 451–456, 2010. Publisher Full Text | Google Scholar
  250. A. S. Banks, F. E. McAllister, J. P. Camporez et al., “An ERK/Cdk5 axis controls the diabetogenic actions of PPARγ,” Nature, vol. 517, no. 7534, pp. 391–395, 2014. Publisher Full Text | Google Scholar
  251. S.-S. Choi, E. S. Kim, M. Koh et al., “A novel non-agonist peroxisome proliferator-activated receptor γ (PPARγ) ligand UHC1 blocks PPARγ phosphorylation by cyclin-dependent kinase 5 (CDK5) and improves insulin sensitivity,” The Journal of Biological Chemistry, vol. 289, no. 38, pp. 26618–26629, 2014. Publisher Full Text | Google Scholar
  252. K. F. Petersen, S. Dufour, D. Befroy, M. Lehrke, R. E. Hendler, and G. I. Shulman, “Reversal of nonalcoholic hepatic steatosis, hepatic insulin resistance, and hyperglycemia by moderate weight reduction in patients with type 2 diabetes,” Diabetes, vol. 54, no. 3, pp. 603–608, 2005. Publisher Full Text | Google Scholar
  253. K. Ueki, T. Kondo, Y.-H. Tseng, and C. R. Kahn, “Central role of suppressors of cytokine signaling proteins in hepatic steatosis, insulin resistance, and the metabolic syndrome in the mouse,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 101, no. 28, pp. 10422–10427, 2004. Publisher Full Text | Google Scholar
Research Article
Nuclear Receptor Research
Vol. 4 (2017), Article ID 101310, 52 pages
doi:10.11131/2017/101310

Therapeutic Potential of Peroxisome Proliferator-Activated Receptor Modulation in Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis

Leonie Gellrich and Daniel Merk

Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany


Received 13 September 2017; Accepted 26 October 2017

Editor: Li Wang

Copyright © 2017 Leonie Gellrich and Daniel Merk. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

A long neglected hepatic manifestation of the metabolic syndrome, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) arise as serious health burden with alarming global prevalence. The disease complex is currently attracting considerable interest of drug discovery and many experimental approaches are studied in all stages of clinical development. Peroxisome proliferator-activated receptors (PPARs) have a successful history as pharmaceutical targets in the treatment of several aspects of the metabolic syndrome and, therefore, a putative therapeutic value of PPAR modulators in NAFLD/NASH is obvious. However, so far only the PPARα/δ agonist elafibranor has revealed clear efficacy and reached an advanced stage of development while the far more established PPAR subtypes PPARα and PPARγ have disappointed. Still, clinical trial design and population might have obscured beneficial activities and, in addition, synergistic multi-target approaches as well as selective PPAR modulators could generate safer approaches with higher therapeutic efficacy.