Peroxisome Proliferator Activated Receptors

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Peroxisome Proliferator Activated Receptors

Raphaël Genolet, Liliane Michalik, Walter Wahli

22

22.1 Introduction

The metabolic activity of the liver plays a central role in coordinating the supply of energy to periph- eral organs, in particular the brain. It is at the heart of an adaptive system, which allows survival at times of food deprivation and governs the storage of excess energy when plenty of food is available. The liver controls the storage of energy first by convert- ing glucose into glycogen and, second, by esterify- ing excess fatty acids (FA) to triacylglycerol, which is released as a component of the very low density lipoproteins (VLDL), finally to be stored in the form of triglycerides (TG) mainly in the adipose tissue.

Conversely, during starvation, when plasma glycerol and free FA (FFA) levels are high due to TG lipolysis in the adipose tissue, the liver FA oxidative machin- ery participates in the production and release of ke- tone bodies that serve as lipid-derived fuel for the brain, muscle, kidney and other peripheral organs.

Furthermore, plasma glucose levels are maintained by the depletion of the hepatic glycogen stores and then by de novo glucose synthesis.

A dysfunction of the fine tuning of these meta- bolic pathways participates in ailments that affect millions of people, which are collectively known as metabolic syndrome. It is now well established that elevated circulating FFA levels, as seen in obesity, promote type 2 diabetes in part by stimulating he- patic gluconeogenesis and glucose output into the bloodstream.

During the past decade, it has been recognized that FAs are potent modulators of gene expression via the transcription factors peroxisome prolifera- tor activated receptors (PPARs). These receptors have been identified as key regulators of energy homeostasis when activated by FAs or FA deriva- tives that bind to them. Therefore, it is not surpris- ing that synthetic PPAR ligands are used as drugs to treat metabolic disorders. Although the action of PPARs is obviously not limited to the liver, herein

we will focus on their major hepatic effects in health and disease.

22.2 Peroxisome Proliferator Activated Receptors

The PPARs are ligand-dependent transcription fac- tors that belong to the nuclear receptor family, which comprises 48 members in man. Three isotypes en- coded in separate genes have been identified: PPAR α (NR1C1), PPAR β/δ (called PPARβ below) (NR1C2) and PPAR γ (NR1C3). They were initially identified as receptors that can be activated by compounds called peroxisome proliferators (PP), such as phtha- late plasticizers, various solvents, herbicides and degreasing agents, which cause peroxisome prolif- eration in rodent liver. The PPAR proteins are or- ganized in the same four classic domains, which de- termine their molecular mode of action, as the other members of the family [17].

As mentioned above, PPARs are FA and FA de- rivative sensors. Polyunsaturated FAs (PUFAs) are potent ligands of all the three PPAR isotypes, where- as eicosanoids like leukotrienes and prostaglandins may be more isotype selective. In addition to these biological ligands, selective synthetic ligands have been developed for each isotype, which are used as experimental activators or drugs [67]. Once ac- tivated by a ligand, PPARs heterodimerize with the retinoid X receptor (RXR; receptor for 9-cis retinoic acid, NR2B) and bind to peroxisome proliferator response elements (PPRE) present in the promoter region of their target genes, where they recruit co- factors that modulate their transcriptional activity [17].

In addition to the ligands, changes in the phos- phorylation status can modify the activity of PPARs.

Phosphorylation of PPAR α by kinases, such as mi-

togen-activated protein kinase (MAPK) and protein

kinase A (PKA), increases its activity, while insu-

lin and growth factor-induced phosphorylation of

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PPAR γ increases and decreases its transcriptional activity, respectively [17].

Analysis of the PPAR expression profiles in dif- ferent species, the characterization of ligands, the identification of target genes and, more recently, the investigation of several PPAR mutant mouse models have unveiled many functions of the PPAR isotypes.

In addition to its major functions in energy home- ostasis, which include FA catabolism, glucose and amino acid metabolism, PPAR α is involved in the control of the inflammatory response, a function that is shared with the two other isotypes. PPAR β is implicated in skin wound repair, lipid homeos- tasis, development of the placenta and myelination in part of the brain. PPAR γ plays a crucial role in adipogenesis and lipid storage, and also participates in glucose homeostasis and placenta development.

The deep implication of these transcription fac- tors in different and complementary roles in energy homeostasis sets them apart both as potential cul- prits for metabolic disturbances and as drug-induc- ible therapeutic targets for the improvement of the metabolic syndrome.

22.3 PPARs and Control of Energy Homeostasis

Peroxisome proliferator activated receptors are cru- cial for energy homeostasis, in the junction of the pathways driving the utilization of FA, carbohy- drates, ketone bodies and amino acids as energy- providing molecules.

22.3.1

Role in Lipid Transport and Uptake

The regulation of circulating levels of FFA, TG and cholesterol is highly relevant since they constitute a major risk factor for atherosclerosis, diabetes and their associated diseases. Liver is a key site for this regulation, which involves a complex lipoprotein transport system that delivers cholesterol and FAs to the peripheral organs (Fig. 22.1). Lipoproteins are composed of apolipoproteins, phospholipids, cholesterol esters and TG. The roles of PPAR α in modulating this transport system were unveiled by the therapeutic benefits of fibrates that are ligands of this PPAR isotype. Fibrates increase the expres- sion of hepatic lipoprotein lipase (LPL) and repress the synthesis of hepatic apolipoprotein C-III (apo C- III), an inhibitor of LPL activity [59, 63] (Fig. 22.1).

These combined effects enhance the catabolism of TG-rich particles and reduce the hepatic secretion of

VLDL. Furthermore, it has been shown recently that apolipoprotein A5 (apo A-V), which is important in reducing plasma TG levels, is encoded in a gene that is highly responsive in liver to PPAR α activa- tors [65]. PPARs are also involved in reverse choles- terol transport (RCT), which brings back, from pe- ripheral cells to the liver, excess cholesterol that has been captured by high-density lipoprotein particles (HDL) (Fig. 22.1). In the liver, production of Apo A-I and Apo A-II, which are two major components of HDL particles, and of phospholipid transfer pro- tein (PLTP) is increased by ligand-activated PPAR α, which results in higher circulating concentrations of HDL and increased transfer of phospholipids from LDL/VLDL to HDL [6, 64]. Scavenger recep- tor class B type I (SR-BI) is able to mediate selective cholesterol uptake in hepatocytes and is therefore considered as a critical player in lipoprotein metab- olism. PPAR α upregulates its expression in periph- eral organs, but reduces its hepatic protein levels, which correlate with increased HDL particle size, unveiling a novel effect of fibrates on HDL metabo- lism [43]. Excess cholesterol taken up by the liver can be recycled by re-assemblage in VLDL particles, excreted directly into the bile or catabolized to bile acids (Fig. 22.1). Finally, PPAR α modulates hepatic uptake of circulating FA by upregulating the genes coding for the FA transport protein 1 (FATP1) and the FA translocase (FAT/CD36) [50]. PPAR α then

Fig. 22.1. Role of PPARα in lipid transport. Regulation of apolipoprotein synthesis results in a reduction of circulating FA and TG levels and a promotion of reverse cholesterol transport

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facilitates intracellular FA retention by increasing the liver FA-binding protein (L-FABP) and acyl CoA synthetase (ACS), allowing FA accumulation in the aqueous milieu of the cell [17].

Thus, PPAR α regulates a set of genes that reduce the risk of ailments associated with high circulating levels of FFA, TG and cholesterol.

22.3.2

Role in FA Catabolism

Formation of fatty acyl-CoA by ACS precedes either the incorporation of FA into triglycerides or their oxidation by peroxisomal β-oxidation and mito- chondrial β-oxidation (Table 22.1, Fig. 22.2). For each of these pathways key enzymes are upregu- lated by PPAR α [17]. Although the changes in the metabolic intermediates are chemically identical in both pathways, different and distinct enzymes are involved in peroxisomes and mitochondria. Peroxi-

somal β-oxidation is more versatile as it is able to metabolize a variety of FA analogs, such as dicarbo- xylic acids and eicosanoids. However, its main func- tion is in the chain-shortening of very long-chain FA (>20C), which predominantly come from the diet and cannot enter mitochondria via the carni- tine shuttle, in preparation for their subsequent oxi- dation in mitochondria [55]. The shortened chains that have lost two carbons at each oxidation round in the form of an acetyl-CoA molecule can then be degraded to completion by mitochondrial β-oxida- tion. The acetyl-CoA produced by the peroxisomal β-oxidation may be converted into acetate, acetyl- carnitine and acetoacetyl-CoA. Alternatively, it can be used by the FA chain-elongation system or serve other biosynthetic purposes in the cytosol, such as sterol synthesis, which illustrates the substantial role of peroxisomes in FA recycling. Mitochondrial FA oxidation is also stimulated by PPAR α, which upregulates enzymes of both the carnitine-depend- ent facilitated transport modulating the entry flux

Fig. 22.2. PPAR-regulated processes. a Fatty acid catabolism:

oxidation of FA via microsomal ω-oxidation and peroxisomal

and mitochondrial β-oxidation. b Overall effect of PPARs on dif- ferent hepatic pathways and processes

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Table 22.1. PPARα and γ target genes

PPARα PPARγ

Upregulated Downregulated Upregulated Downregulated Meta-

bolism

Lipid

FATP1

FAT/CD36 SRB-I FAT/CD36 Long-chain ACS

L-FABP FAS

ACS ACO

L-bifunctional protein 3-Ketoacyl-CoA thiolase CPTI CPTII VLCAD LCAD MCAD SCAD Trifunctional protein a/b subunit HMG-CoAS CYP4A1, A10, A14

CYPC2A5, C29 CYPC2A5, C29

CYP3A11 CYP3A11

APOAI, AII APOCIII Glucose

PDK4 GK PEPCK

G6Pase FBPase Amino

acid

Aspartate amino transferase Glyoxylate

reductase/

Alanine glyoxylate transaminase Hydroxypyruvate Glutaminase

Reductase Asparagine

synthetase Phenylalanine hydroxylase

Arginase Spermidine N1

acetyl transferase

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of FA in the mitochondria and the β-oxidative spiral [17]. The acetyl-CoA unit produced at each oxida- tive round can either be completely oxidized via the Krebs cycle or converted into ketone bodies that serve as an energy substrate for extrahepatic tissues, especially during fasting. The expression of hy- droxymethyl-glutaryl-CoA synthase (HMG-CoAS), which is the main enzyme of this conversion, is di- rectly regulated by PPAR α [17].

A third form of FA catabolism is provided by the cytochrome monoxygenase system of the endoplas-

mic reticulum. The CYP4A family of cytochrome P450 enzymes, which belongs to this system, cata- lyzes the microsomal ω-hydroxylation of FAs and eicosanoids and therefore contributes to their ca- tabolism. For instance, ω-hydroxylation is the first step in the neutralization of LTB4, a PPAR α ligand, which is then degraded by peroxisomal β-oxida- tion. Comparative studies of wild-type and PPAR α- deficient mice revealed that fibrates regulate, in a PPAR α-dependent manner, the liver expression of several different genes of the CYP4A family [4].

Table 22.1. Continued

PPARα PPARγ

Upregulated Downregulated Upregulated Downregulated Spermidine

synthase Carbamoyl phosphate synthase 1 Ornithine transcarbamoylase Arginino succinate synthase

Arginino succinate lyase

Inflammation

IkB CRP IL-1

Fibrinogen IL-6

P50 NF-κB TNFα

C/EBPβ MMP-9

MRS1 Cancer

Cyclin D1

CDK-1, CDK-4 Bak P21, P27 Bcl-2

c-myc, c-jun, c-fos Bak

JunB, erg-1, NUP475 Bax

Bcl-2 Caspase-3

CYPA, urate oxidase Glutathione S- transferase Catalase

Superoxide dismutase Epoxide hydrolase Glutathione peroxidase

Glutathione peroxidase

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Fig. 22.3. Liver in energy homeostasis. During feeding, glucose and FAs are stored as glycogen and triglycerides in the liver/

muscle and in the adipose tissue. Upon fasting, glycogen and

triglycerides are used by the liver to produce energy-rich glucose and ketone bodies to be used by peripheral organs

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Thus, PPAR α can stimulate the downregulation of the intracellular levels of its own ligands (FA and eicosanoids) by both microsomal and peroxisomal oxidation.

22.3.3

Role in Glucose Metabolism

Prolonged hypoglycemia causes acute brain damage, while chronic hyperglycemia is a major risk factor for neuropathy and vasculopathy, as seen in diabe- tes. These pathophysiologies underscore the crucial importance of the tight control system of glucose plasma levels in which liver plays an essential role.

PPAR α, already discussed for its role in lipid me- tabolism, is involved in liver glucose metabolism. In PPAR α null mice, PPARα deficiency increases insu- lin sensitivity and protects from insulin resistance under high-fat diet [27]. Fasting in these animals, in addition to causing lipid accumulation in the liver, elevated plasma levels of FFA and hypoketonemia, also results in severe hypoglycemia [38]. Normally, the liver contributes to improve hypoglycemia by elevation of glycogenolysis, gluconeogenesis and release of glucose into the bloodstream (Fig. 22.3).

PPAR α participates directly in the regulation of gluconeogenesis by increasing the expression of the gene encoding pyruvate dehydrogenase kinase 4 (PDK4) [68]. This enzyme catalyzes the phospho- rylation and thus, the inactivation of the pyruvate dehydrogenase complex, leading to the utilization of pyruvate for gluconeogenesis instead of FA syn- thesis. PPAR α also influences substrate utilization for hepatic glucose production. In the fasted state, lactate production itself and glucose synthesis from lactate were strongly reduced in PPAR α null mice.

In contrast, glucose production from glycerol was increased in these animals and at the end of the fast- ing period, hepatic glucose production was higher in the PPAR α null mice than in wild-type animals [69]. Therefore, the severe hypoglycemia observed in these mutant mice during fasting may result from increased glucose disposal. Together, these observa- tions underscore the importance of PPAR α in the regulation of glucose production and disposal. Fi- nally, in several diabetic rodent models, synthetic PPAR α agonists improve glucose homeostasis by ef- fects on liver, skeletal muscle and pancreas [39, 40].

22.3.4

Role in Amino Acid Metabolism

Contrary to common understanding, oxidation of amino acids contributes significantly to energy pro-

duction in several organs, including liver. In addition to lipid and glucose metabolism, PPAR α was associ- ated with the regulation of amino acid metabolism [37]. Enzymes involved in transamination, deami- nation, oxidation of alpha keto acid, amino acid- derived product synthesis, amino-acid interconver- sion, and in the urea cycle are regulated by PPAR α, which represses a majority of these enzymes leading to a decrease of amino acid catabolism. The increase and reduction of plasma urea concentrations in fasted PPAR α null mice and fasted wild-type mice, respectively, support an overall inhibitory effect of PPAR α on amino acid metabolism. Amino acid oxi- dation is dramatically elevated during conditions such as sepsis and cachexia and after severe trauma and burns. These catabolic states are characterized by massive net body protein breakdown, leading to a negative nitrogen balance. So far, there is no infor- mation as to whether PPAR α ligands would improve these conditions.

22.3.5

Role During Fasting

The adaptive role of PPARs in situations of prolonged food deprivation, which is of major importance for survival, has already been mentioned (see Fig. 22.3).

Fasting is a situation where FA, glucose and amino acid metabolisms are thoroughly adapted with a simple main objective: keep the energy supply suf- ficient as long as possible, for the brain and other peripheral organs by providing ketone bodies and glucose. Hormonal cues induce glucose output and lipid breakdown in the liver and suppress the lipid storage and lipogenic pathways. PPARs largely con- tribute to promote this adaptation. PPAR γ, which is mainly involved in energy storage, is strongly re- pressed during fasting, in contrast to PPAR α, which is upregulated to increase energy production. In liver, its stimulation contributes to (a) the increase of glucose production via glycogenolysis or gluco- neogenesis (Fig. 22.3), (b) the increase of FA oxida- tion that provides the substrate for ketone body for- mation and the cofactors (acetyl-CoA, ATP, NADH) for gluconeogenesis (Figs. 22.2 and 22.3), and (c) the suppression of amino acid catabolism to optimize FA utilization and to prevent as long as possible the degradation of amino acids provided essentially by the catabolism of muscle proteins.

At the molecular level, food deprivation induces

a crosstalk between different transcription factors

and co-activators, which facilitates the necessary

adaptation of the organism, described above, to

feeding and starving. The coordinated induction

of the hormone receptor co-activator PGC-1 and

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repression of PPAR γ by the cAMP-responsive tran- scription factor CREB, which triggers gluconeogen- ic and fatty acid oxidation programs during fasting, participates in the hepatic antagonism between in- sulin and glucagon and cortisol [32]. Furthermore, if PPAR α is involved in FA degradation and drives the response to food deprivation, the LXR/SREBP-1c system is involved in FA synthesis and storage when plenty of food is available. Interestingly, a reciprocal suppression of activity between these factors facili- tates their opposite action. Indeed PPAR α represses LXR-induced SREBP-1c expression and conversely, LXR activity represses PPAR α expression and ac- tivity [54]. Thus, depending on the balance of these opposite activities defined by food availability, the general metabolic outcome will be energy expendi- ture by mobilization of fat stores or storage of excess energy in the form of glycogen and TG, mainly in liver and adipose tissue, respectively (Fig. 22.3).

22.4 Metabolic Disorders

Due to their implication in the different metabolic pathways mentioned so far, PPARs and their ligands are instrumental in the treatment of metabolic per- turbations associated with premature atherosclero- sis and an elevated risk of cardiovascular complica- tion. Due to their action in lipid catabolism, fibrates have been commonly used to treat dyslipidemia [36]. Via PPAR α, they modulate lipid transport and catabolism, downregulate inflammatory cytokines and acute phase proteins, elevate HDL cholesterol, reduce TG-rich lipoproteins and induce a shift to receptor-active buoyant LDL [3]. This pleiotropic action of fibrates attenuates the atherosclerotic bur- den in dyslipidemia. Thiazolidinediones (TZDs) are a group of PPAR γ agonists characterized by their ability to decrease insulin resistance. They decrease plasma glucose levels, but in addition they mark- edly lower plasma TG and FFAs, which is in general accompanied by body fat gain. Until the effects of long-term TZD treatment on weight gain and water retention are better known, the use of such “adi- pogenic compounds” has to be followed carefully.

More information about the role of the liver in the development of metabolic disorders and its charac- teristics as target of the above-mentioned drugs will shortly be gained from animal models in which the different PPAR isotypes can be invalidated using the tissue-specific gene knockout technology.

22.4.1

Obesity and Fatty Liver

Obesity is often associated with the development of a fatty liver (steatosis), a process dependent on the balance between FA uptake, oxidation, synthesis, esterification, and TG secretion [12]. Steatosis is also observed in mice lacking PPAR α, which together with the known roles of PPAR α in lipid metabolism, suggest the possibility of a PPAR α ligand-based treatment against obesity-dependent steatosis. Re- cent studies in mice with increased TG content in the liver indeed show that PPAR α ligands reduce he- patic TG levels [13].

In contrast to PPAR α, the expression of PPARγ is normally low in the liver, but its expression was found to be upregulated in genetically obese mice with fatty liver. The major role of PPAR γ is to pro- mote adipocyte differentiation and lipid storage and to mediate the effects of the insulin-sensitizing TZDs, as mentioned above. In obese animals, TZDs induce the liver expression of several PPAR γ target genes involved in lipid uptake and storage, usually expressed in the adipose tissue, such as the FA- binding protein (ap2), LPL, FAT/CD36, whose added effects result in a fatty liver [47]. In parallel, FA oxi- dation is decreased due to the inhibition of the long- chain acyl-CoA synthetase (ACS) [60], while de novo synthesis of FA is increased by upregulation of FA synthase (FAS) [46]. These changes in enzymatic activities, together with the augmentation of insu- lin-stimulated hepatic conversion of glucose into FA can explain to a large extent the increase of FA pro- duction and content in the liver of obese mice.

The lipoatrophic A-ZIP/F-1 mouse has been used as a model to study the function of liver PPAR γ, which is elevated in these animals [10]. The abla- tion of liver PPAR γ in these mice led to the conclu- sion that the liver is a major site of TZD action in the absence of adipose tissue. However, rosiglita- zone remained effective in mice with adipose tis- sue, but which lacked liver PPAR γ, suggesting that adipose tissue is the major site of TZD action in ani- mals with normal adipose tissue [25]. In conditions where it is expressed in liver, PPAR γ can participate in the regulation of TG homeostasis, contributing to hepatic steatosis, but protecting other tissues from TG accumulation and insulin resistance. Elevated expression of PPAR γ and a concomitant fatty liver phenotype have also been observed in mice deficient in CREB [32]. Thus, CREB modulators may present a potential for modulating lipid metabolism in liver and enhancing insulin sensitivity.

A recent pilot study on adult humans with non-

alcoholic steatohepatitis, treated for 48 weeks with

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rosiglitazone resulted in a significant improve- ment of the necroinflammatory score, mean serum alanine aminotransferase levels and insulin sensi- tivity [51]. These results suggest that increasing in- sulin sensitivity via PPAR γ action may be important in treating non-alcoholic steatohepatitis. However, the mechanism by which such an effect would be ob- tained remains to be determined.

In addition to metabolic disorders, alcohol abuse is a common cause of steatosis [14]. A link between alcohol and PPAR α has been revealed by the obser- vation that ethanol and its metabolite acetaldehyde inhibit the transcriptional and DNA-binding activi- ties of PPAR α, suggesting that alcohol-induced stea- tosis might be meditated through PPAR α inactiva- tion [24]. Interestingly, and in line with the above, treatment of mice with the PPAR α agonist Wy 14,643 restored PPAR α activity and reduced the hepatic li- pid accumulation due to ethanol exposure [22].

22.4.2

Insulin Resistance

Glucose homeostasis, a fine-tuned balance between glucose production and disposal, is controlled by pancreatic β cell insulin secretion. Insulin resist- ance is the inability of the body to respond to in- creased insulin levels by reducing serum glucose concentrations. In this situation, lipolysis and he- patic glucose production are maintained, leading to the deleterious effects of excess circulating glu- cose. Obesity is associated with insulin resistance in 80% of people with type 2 diabetes. The circulating FFA resulting from TG lipolysis, levels of which are elevated in obesity, contribute to this association [5]. In the liver, these FA increase the production of glucose by stimulating gluconeogenesis, possibly via PPAR α, aggravating hyperglycemia [11]. In ad- dition, elevated FFA decrease insulin secretion and hepatic insulin clearance.

The specific PPAR γ agonists TZDs have been used since 1997 to treat type 2 diabetes. One major action of these compounds is to lower the plasmatic FFA concentrations by increasing the ability of adi- pose tissue to store them as TG. This partial deple- tion in serum reduces FFA accumulation in other organs such as muscle and liver, which improves their insulin sensitivity and glucose disposal in skeletal muscle. It is commonly accepted that en- dogenous glucose production is increased in type 2 diabetes mainly due to an increase in gluconeogen- esis [5]. Therefore, the repressing action of PPAR γ in gluconeogenesis may help severe hyperglycemic type 2 diabetes patients. Muscle-specific PPAR γ de- pletion in mice revealed that muscle PPAR γ is not

required for the antidiabetic effects of TZDs, but is important for the maintenance of normal adiposity, as the mutant mice develop excess adipose tissue mass [33]. Furthermore, PPAR γ is required for mus- cle and liver insulin action and whole-body insulin sensitivity, suggesting an organ crosstalk that is un- derscored by the observation that adipose-specific PPAR γ knockout caused increased hepatic gluco- genesis and hepatic insulin resistance [30].

22.5 Liver Injury

During liver injury, activation of hepatic stellate cells (HSC) plays an important role in the regula- tion of the inflammatory response and wound re- pair. Therefore, HSC are commonly used as a model to study their role in hepatic tissue repair and fibro- genesis.

22.5.1 Inflammation

The first evidence implicating PPARs in the control of the inflammatory response came from a study on PPAR α null mice, which presented a prolonged inflammatory reaction in response to the inflam- matory cytokine leukotriene B4 (LTB4) [18]. It was proposed that LTB4 increases PPAR α activity and, consequently, induces the oxidation pathway that neutralizes LTB4 itself, thereby attenuating the inflammatory reaction. Additional studies have further highlighted the anti-inflammatory role of PPAR α. Patients with hyperlipidemia show a de- crease in the plasma level of interleukin-6 (IL-6), in- terferon- γ (INF-γ), tumor necrosis factor-α (TNF- α), fibrinogen, and the C-reactive protein (CRP) after fibrate administration [42, 62]. At least part of the anti-inflammatory effects of PPAR α is mediated via its transrepressional action of various transcrip- tion factors [15]. In addition to transrepression, PPAR α upregulates direct target genes that exert an anti-inflammatory action such as that of I κB, a pro- tein inhibiting NF- κB activity [16]. Liver is meant to participate in these effects, but its specific contribu- tion is difficult to assess in studies involving whole animals or patients.

The effect of PPAR γ on inflammation is less

clear. Rosiglitazone has been shown to reduce the

plasma concentrations of CRP and matrix metallo-

proteinase-9 (MMP-9) in patients treated for type 2

diabetes [28]. PPAR γ is expressed in macrophages,

including Kupffer cells, which play an important

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role in the production of cytokines and other proin- flammatory mediators, whose production is down- regulated by PPAR γ-specific ligands.

22.5.2 Fibrosis

Several forms of chronic liver injury induce a wound-healing process known as hepatic fibrogen- esis. During injury, quiescent HSC become active and proliferative. HSC are recruited to the dam- aged area, where they start to produce collagen and components of the extracellular matrix as well as chemotactic factors. During HSC activation, PPAR γ and PPAR β expression is decreased and increased, respectively [31]. Treatment of HCS with PPAR β lig- ands, or with a PPAR β anti-sense probe, revealed that PPAR β promotes the proliferation of the HSC.

This effect was confirmed in a model of acute liver injury, where administration of PPAR β ligand in- creased the number of HSC and the mRNA level of collagen type 1, α-smooth muscle actin (α-SMA) and lysyl oxidase, which are markers of activated HSC [31]. The elucidation of a possible role of PPAR β as mediator of HSC proliferation during acute and chronic liver inflammation is of importance for the search for innovative therapeutics against these pathologies.

In contrast, activation of PPAR γ reduced the profibrogenic and proinflammatory processes in these cells. Indeed, activation of stellate cells, which includes platelet-derived growth factor-induced proliferation, DNA synthesis, expression of α1-col- lagen, α-SMA and monocyte chemotactic protein 1 (MCP-1), and chemotaxis, can be attenuated by PPAR γ agonists, opening a new possible therapeutic application for PPAR γ ligands in diseases associated with liver fibrosis or inflammation [23, 49].

22.6 Hepatocarcinogenesis in Rodents

Chronic administration of PPs, such as di(2- ethylhexyl)phthalate (DEHP) to mice and rats, pro- duces liver hypertrophy and hyperplasia. Long-term administration induces the development of hepato- cellular carcinomas, by a mechanism independent of DNA damage, but involving peroxisome prolifer- ation [21]. PPAR α null mice do not respond to PPs, showing that the peroxisome proliferation these compounds induce is mediated by PPAR α [54].

At least two theories attempt to explain the role of PPAR α in rodent hepatocarcinogenesis. The first

one concentrates on the control by PPAR α of cell proliferation and apoptosis. In line with this propos- al, PP treatment induces an increase in liver weight by stimulating DNA replication and hepatocyte proliferation and growth [34, 44]. Several proteins involved in the cell cycle are regulated by PPAR α, although evidence for a direct effect is still lacking.

Wy 14,643 treatment increases the mRNA levels of cyclin D1, cyclin-dependent kinase (CDK)-1, CDK-4 and c-myc in a PPAR α-dependent manner [2, 53]. In addition, c-fos, c-jun, junB, erg-1 and NUP475, which are other cell-cycle genes, are also induced by PPs in rodents, causing a progression of the cell cycle [41].

Coincident with this induction, PPAR α suppresses apoptosis in the liver [26], which may also be caused by PPAR β. Indeed, PPARβ exerts anti-apoptotic functions in keratinocytes through phosphoryla- tion and activation of the main survival factor Akt1 (protein kinase B- α) that in turn phosphorylates and inactivates Bad and FOXO in the mitochondrial and death receptor apoptosis pathways, respectively [19]. However, there is no evidence so far to indicate that the same anti-apoptotic mechanism is used in hepatocytes.

In rodent liver, Kupffer cells are thought to play a role in the effect of PPs on both proliferation and apoptosis. Two PPs, Wy 14,643 and nafenopin, have been shown to activate Kupffer cells that secrete a number of cytokines, including TNF- α, which is able to induce S phase and suppress hepatic apopto- sis [57, 58]. However, since PPAR α is expressed at a low level in Kupffer cells, the question remains open as to its role in these cells in mediating the PP effect [26].

The second theory is based on an increase of DNA damage, due to oxidative stress as an indirect consequence of PPs action. PPs induce peroxisome proliferation and strongly increase peroxisomal β-oxidation and therefore the production of H

2

O

₂

. Coincidently, PPs also upregulate H

₂

O

₂

-degener- ating enzymes, but the overall regulatory effect of PPARs on these enzymes does not seem sufficient to counterbalance the strongly increased generation of H

₂

O

₂

by the oxidative pathways. Therefore, it is plausible that the increasing concentrations of H

₂

O

₂

may generate DNA damage and participate in the tumorigenic effect of PPs.

PPAR γ ligands have been reported to induce cell

growth arrest and apoptosis in various malignant

cells, especially on different hepatocellular carcino-

ma cell lines [71]. Together, these findings suggest

that in the future, after further investigation in the

laboratory and clinic, PPARs might become interest-

ing targets for the treatment of various tumors [48].

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22.7 Conclusions

The development of selective ligands and mutant mice has provided powerful tools to elucidate the multiple functions of PPARs (Fig. 22.2b). PPAR α and PPAR γ are involved in lipid and glucose me- tabolism where they play complementary and oppo- site roles in the control of energy homeostasis. The implication of PPARs in these metabolic pathways is supported by the identification of single point mutations in the PPARs, which are related to meta- bolic disorders (Table 22.2). The fine-tuned balance between energy storage and expenditure, which is largely controlled by PPARs, requires a crosstalk

between different organs, recently highlighted by the tissue-specific knockout technology, where the role of liver is central. The knowledge that has ac- cumulated over the past 10 years on PPAR biology has allowed a better utilization of fibrates and the introduction of TZDs to treat disorders associated with the metabolic pathways controlled by PPAR α and PPAR γ. The optimized clinical application of these lipid- and glucose-modulating and anti-in- flammatory drugs represents an effective approach to the clinical management of a wide spectrum of disorders such as dyslipidemias, type 2 diabetes and metabolic syndrome.

In contrast to the well-defined roles of PPAR α in the liver, the functions of PPAR β and γ in this organ remain more elusive, although evidence is accumu-

Table 22.2. Polymorphisms in human PPAR genes

Mutation Allele frequency Population Consequences References

PPARα

Leu162Val 0.066 Diabetic Caucasian Influences plasma lipid concentration (higher cholesterol, HDL cholesterol and Apo A1)

[52]

0.13 Caucasian Associated with Alzheimer‘s disease [7]

Val227Ala 0.051 Japanese Decreases total cholesterol [70]

PPARγ

Pro12Ala 0.11 Caucasian Lowers BMI, decreases risk factor

of type 2 diabetes, correlated with hypertension

[9, 45, 56]

0.11 Japanese Decreases carotid artery IMT [35]

0.9 Diabetes Decreases diabetic nephropathy [8]

Cys1431Thr 0.119 Type 2 diabetes Increases BMI [20]

Pro115Gln 0.033 Caucasian Obesity [29]

Arg425Cys Partial lipodystrophy [1]

Cys161Thr 0.173 IgAN Japanese Increases survival of IgAN patients [61]

Cys161Thr 0.163 Caucasian Reduces CAD risk [66]

ApoA1 apolipoprotein A1, BMI body mass index, CAD coronary artery disease, IgAN immunoglobulin A nephropathy, IMT intima- media thickness.

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lating for an association with injury repair, fibrino- genesis and inflammation and probably indirectly with liver insulin sensitivity. Pursuing the genera- tion of mice allowing a spatio-temporal ablation of the PPAR genes for each of the three isotypes in the liver, either alone or in combination, will greatly improve our knowledge on the hepatic functions of these receptors. How do they regulate basic bio- logical processes depending on the many intricate metabolic routes and signals that are merging in liver? These tools will help to answer this question and develop new sets of more effective drugs for specific applications within the spectrum of mani- festations of metabolic and inflammatory diseases.

There is little doubt that there will be an important extension of the therapeutic roles of PPARs in the near future.

Selected Reading

Bocher V, Pineda-Torra I, Fruchart JC, Staels B. PPARs: transcription factors controlling lipid and lipoprotein metabolism.

Ann NY Acad Sci 2002;967:7–18. (This review describes genes regulated by PPARs in a subtype- and tissue-specific manner, which are involved in intracellular lipid metabolism, lipoprotein metabolism, and reverse cholesterol transport.) Lee CH, Olson P, Evans RM. Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology 2003;144:2201–2207. (This paper reviews the roles of PPARs in lipid metabolism.)

Daynes RA, Jones DC. Emerging roles of PPARs in inflammation and immunity. Nat Rev Immunol 2002;2:748–759. (This review gives a broad overview of PPARs in the inflammatory process.)

Michalik L, Desvergne B, Wahli W. Peroxisome-proliferator-activated receptors and cancers: complex stories. Nat Rev Can- cer 2004;4(1):61–70. (The implication in a subtype- and tissue-specific manner of PPARs in carcinogenesis is discussed in view of the most recent findings.)

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