# Peroxisome Proliferators-Activated Receptor (PPAR) Modulators and Metabolic Disorders

Authors: Min-Chul Cho; Kyoung Lee; Sang-Gi Paik; Do-Young Yoon
Journal: PPAR Research
 (2008)

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## Abstract

Overweight and obesity lead to an increased risk for metabolic disorders such as impaired glucose regulation/insulin resistance, dyslipidemia, and hypertension. Several molecular drug targets with potential to prevent or treat metabolic disorders have been revealed. Interestingly, the activation of peroxisome proliferator-activated receptor (PPAR), which belongs to the nuclear receptor superfamily, has many beneficial clinical effects. PPAR directly modulates gene expression by binding to a specific ligand. All PPAR subtypes (α,γ, and
σ) are involved in glucose metabolism, lipid metabolism, and energy balance. PPAR agonists play an important role in therapeutic aspects of metabolic disorders. However, undesired effects of the existing PPAR agonists have been reported. A great deal of recent research has focused on the discovery of new PPAR modulators with more beneficial effects and more safety without producing undesired side effects. Herein, we briefly review the roles of PPAR in metabolic disorders, the effects of PPAR modulators in metabolic disorders, and the technologies with which to discover new PPAR modulators.


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## Body

## 1. Obesity, Adipocytes, and Adipokines

Obesity, which is
defined as excess adiposity for a given body size, results from an imbalance
between energy intake and energy expenditure. Body mass index (BMI), measured
as body weight in kilograms over the square of the height in meters (kg/m2),
represents a widely accepted measure of adiposity. Wealth in industrialized
societies, combined with an often-sedentary lifestyle and plentiful, high-calorie
diets, creates irreversible weight gain. This social phenomenon can adversely
impact well-being. Due to explosive concern for health and well-being, genes
associated with human obesity are currently being defined, and whole genome
scans will soon unveil its underlying genetic loci. The various causes of
obesity are grouped according to behavioral (activity levels, nutrition,
smoking status, and socioeconomic status), metabolic (physiological endocrine
factors), and biological (genetic, racial, gender, age, and pregnancy status)
influences [1]. Obesity has been recognized as a chronic disease since the
National Institutes of Health Consensus conference in 1985 [2]. The increase in
the prevalence of obesity has led the World Health Organization (WHO) to
recently refer to the obesity issue as a “global epidemic”.Chronic disruption of
the energy balance due to exceeding energy intake causes hypertrophy and
hyperplasia of fat cells, and this is representative of the pathology of
obesity. When the intake of energy chronically exceeds energy expenditure, most
of the excess energy is stored in the form of triglyceride in adipose tissue
(from Greekadip- or adipo, mean fat). Increased adipose tissue
mass can arise through an increase in cell size, cell number, or both.
Adipocytes are remarkably variable in size, which reflects the amount of stored
triglyceride. Mild obesity mainly reflects an increased adipose cell size
(hypertrophic obesity), while more severe obesity or obesity arising in
childhood typically also involves an increased number of fat cells
(hyperplastic obesity) [3]. As a key part of the homeostatic system that
controls energy balance, the molecular mechanisms that regulate preadipose cell
growth (proliferation), adipose differentiation (adipogenesis), and lipogenesis
have been subject to extensive scrutiny. An overview of cell types and
molecular events that occur during adipogenesis is presented in Figure 1.
Preadipocytes undergo growth arrest, postconfluent mitosis, and clonal
expansion following appropriate environmental and gene expression cues. The
committed preadipocytes must then withdraw from the cell cycle before adipose
conversion. During the differentiation of adipocytes, the adipocyte phenotypes are
characterized by sequential changes in the expression of numerous genes [4, 5].
The study of the cellular and molecular events of adipogenesis was facilitated
by the establishment of preadipose cell lines. Among these cell lines, some are
derived from embryonic cells such as the 3T3-L1 and 3T3-F442A cell lines, and
others such as the Ob17 cell line and its subclones, which originated from
adult animals [6]. When maintained in appropriate culture conditions, these
cells undergo an adipose conversion characterized by the transcriptional
activation of numerous genes. The process of adipose conversion is controlled
by external signals, and it has been found that adipogenic cocktails are
different depending on the cell systems used. For instance, the 3T3-L1
preadipose cells are induced to differentiate by a treatment with high
concentrations of cyclic AMP, dexamethasone, and insulin at the preadipose
stage. Hormones such as insulin, triiodothyronine, glucocorticoides, and growth
hormone exert positive actions on the differentiation of adipose cells.
Prostaglandins such as prostacyclin (PGI2), prostaglandin D2,
and 15-deoxy-Δ12, 14-PGJ2 (15d-PGJ2) have also been found to be strong activators of
adipogenesis. Several transcription factors have been shown to act
cooperatively and sequentially to control adipogenesis. These include members
of the transcription factors, such as CCAAT/enhancer-binding protein α (C/EBPα) [7], peroxisome proliferator-activated receptor-γ (PPAR-γ), and
adipocyte determination, as well as differentiation factor-1 (ADD-1). The last
stage of terminal differentiation corresponds to the activation of several
genes, including those for proteins involved in triglyceride metabolism [8].Figure 1
Adipocyte differentiation (adipogenesis) and transcriptional events in
adipogenesis. A pluripotent stem cell precursor gives rise to a multipotential
mesenchymal precursor cell with a potential to differentiate into an adipocyte.
The preadipocyte enters the adipogenesis stage via environmental and gene
expression signals. In an early stage of adipogenesis, major transcriptional
factors such as PPARγ and C/EBPα are expressed, and these factors strongly
regulate the expressions of adipogenesis-related genes. The adipocyte secretes
various factors, including adipokines, and the secreted factors play an important
role in glucose and lipid metabolism, immune system, appetite regulation, and
vascular disease. LCFAs: long-chain fatty acids; PGI2: prostacyclin;
LIF: leukemia inhibitory factor; GH: growth hormone; ADD-1: adipocyte
determination and differentiation factor-1; FABPs: fatty acid-binding proteins.Adipose tissue is partitioned
into a few large depots (subcutaneous and visceral locations), and many small
depots (heart, epicardium, pericardium, large blood vessels, major lymph nodes,
bone marrow, kidney, adrenal glands, and the brain) [9]. All adipocytes secrete
a large number of multifunctional molecules, including cytokines, growth
factors, enzymes, hormones, complement factors, matrix proteins, and so forth.
The proteins that are secreted from adipocytes are designated “adipokines” or
“adipocytokines”. Since the isolation of the first-known adipocyte-secreted
protein (the serine protease adipsin) in 1987 [10], the list of adipokines has
been greatly extended. Leptin (from Greek leptos,
means thin), encoded by the obese (ob)
gene [6, 11], adiponectin (also called Acrp30) [12, 13], Interleukin-6 (IL-6),
tumor necrosis factor-α (TNF-α) [14], resistin [15], and visfatin [16] are
candidates of great interest among the growing number of factors found to be
secreted by adipocytes. It has recently been shown that adipokines that are
secreted from adipocytes contribute to the development of obesity-associated
metabolic disorders, including insulin resistance, cardiovascular disease, and
cancer [17].


## 2. Metabolic Disorders and Therapeutic Targets

Overweight and obesity
lead to increased risk for noninsulin-dependent diabetes, hypertension,
coronary artery disease, dyslipidemia, gallstones, osteoarthritis, sleep apnea,
certain forms of cancer, and degenerative arthritis. As the prevalence of
obesity has increased, the heterogeneous clinical disorder strongly associated
with abdominal obesity and insulin resistance has been identified as a major
risk factor for atherosclerotic cardiovascular disease. This disorder,
previously termed “syndrome X” by Reaven, and “insulin-resistance syndrome” by
others, is now considered to be metabolic syndrome or metabolic disorder [18].
This disorder shares similar cardiovascular risk factors, including abdominal
obesity, impaired glucose regulation/insulin resistance, dyslipidemia, and
hypertension. Accordingly, these factors define the clustering of findings
typical of the metabolic disorders, and establish diagnostic criteria. A number
of studies have shown that the excess body fat that is stored in the deep
abdominal area is associated with metabolic complications [19]. Recently,
several molecular drug targets with potential to prevent or treat metabolic
disorders have been revealed.The excess
glucocorticoid action by the enzyme 11β-HSD
(hydroxysteroid dehydrogenase) type 1 induces obesity and features of metabolic
disorders. Transgenic mice which are selectively  over-expressing 11β-HSD1 in adipose tissue lead to  increased food
intake and body weight, as well as the development of visceral obesity. In
addition, insulin-resistant diabetes, hyperlipidemia, and hyperphagia were
observed in 11β-HSD1
transgenic mice [20]. On the other hand, 11β-HSD1 deficiency causes favorably altered fat
distribution and adipose insulin sensitization. Even with high-fat and
cholesterogenic diets, lipid profiles are also improved [21]. 11β-HSD1 inhibitors might have beneficial
consequences in metabolic disorder. For instance, carbenoxolone, an 11β-HSD1 inhibitor, reduced total cholesterol in
healthy subjects, and decreased the glucose production rate during hyperglucagonemia
in diabetic patients [22]. AMP-activated protein kinase (AMPK) is a major
regulator of lipid and glucose metabolism, and AMPK activation appears as a
benefit of exercise in diabetic patients. Activation of AMPK by metformin
decreased the level of plasma glucose and plasma triglycerides by promoting muscle
glucose uptake and inhibiting hepatic glucose output [23]. SCD-1 (stearoyl CoA
desaturase-1) is required for the biosynthesis of the monounsaturated fatty
acids from saturated fatty acids, and SCD-1-deficient mice appear visibly lean compared
to their littermates. SCD-1 deficiency in ob/ob mice ameliorates obesity and completely corrects the excessive hepatic lipid
storage and VLDL production of the hypometabolic phenotype in leptin deficiency
[24]. An SCD-1 inhibitor that reduces SCD-1 activity may serve as a therapeutic
strategy for metabolic disorders, but very few reports are available for the use
of SCD-1 inhibitor. IkB kinase β (IKKβ) plays a key role in the activation of NF-kB by phosphorylating IkBα. It has recently been reported to act as a key
role in obesity-linked insulin resistance. In obese rodents, increased IKK
activity or overexpressed IKK promotes insulin resistance, whereas reduction of
IKK activity or IKKβ expression
improves insulin sensitivity. In addition, high doses of IKKβ inhibitors such as aspirin and salicylate
reverse insulin resistance by sensitizing insulin signaling in obese rodents
[25]. Protein tyrosine phosphatase 1B (PTP1B) is closely associated with insulin
signaling through the dephosphorylation of activated insulin receptor or
insulin receptor substrates. PTP1B deficiency and its heterozygote
significantly reduce glucose concentrations in the blood, and PTP1B deficiency causes
a significant reduction of circulating insulin concentration compared to
wild-type mice. When on a high-fat diet, PTP1B-deficient mice were resistant to
diet-induced weight gain, and remained insulin-sensitive [26]. Because PTP1B
inhibition provides attractive therapies against metabolic disorders, various studies
for the inhibition mechanism of inhibitors against PTP1B, the structure-activity
relationship, and synthetic and pharmacological materials have been performed
by different groups. Acetyl-CoA carboxylase (ACC) is a key determinant of
energy homeostasis because increased malonyl-CoA by ACC activation inhibits
mitochondrial fatty acid uptake and oxidation. A lack of malonyl-CoA in the muscle
and heart of ACC2-deficient mice show increased oxidation of fatty acid,
decreased fat in adipose and liver tissue, and decreased the storage of
glycogen in the liver [27]. CP-640186, an isozyme-nonselective ACC inhibitor,
inhibits fatty acid and TG synthesis in HepG2 cells, as well as fatty acid
synthesis in obese rodents. CP-640186 also stimulates fatty acid oxidation in
C2C12 cells [28]. These effects of the ACC inhibitor may provide novel
therapeutic potential for treatment of the metabolic disorder. Interestingly,
the activation of PPARs by their ligands has many beneficial effects in the
improvement of glucose homeostasis and lipid homeostasis.


## 3. PPARs and Metabolic Disorders

### 3.1. PPARs as a Nuclear Receptor Family

Peroxisomes are
subcellular organelles that perform diverse metabolic functions, including H2O2-derived
respiration, β-oxidation
of fatty acids, and cholesterol metabolism. Rodents exposed to peroxisome
proliferators lead to hepatocellular hypertrophy, hyperplasia, and
transcriptional induction of fatty acid-metabolizing enzymes that are regulated
in parallel with peroxisome proliferation [29]. Peroxisome proliferators may
activate PPARs by binding directly to the receptors, and the activated PPARs
may regulate the expression of genes involved in lipid metabolism and peroxisome
proliferation. Recent research on PPARs has moved toward their pivotal roles
comprising one family of nuclear receptors [30]. Nuclear receptors, which are
present in multicellular organisms, directly control the expression of genes in
response to a wide range of developmental, physiological, and environmental
signals.The PPARs of nuclear
receptors mainly consist of three subtypes (PPARα, PPARγ, and PPARδ/β). All three
PPAR isoforms possess similar structural and functional features. Principally,
four functional domains have been
identified, and are referred to as A/B, C, D, and E/F. The N-terminal A/B
domain contains ligand-independent activation function 1 (AF-1). The
ligand-independent activation region can confer constitutive activity on the
receptor, and is negatively regulated by phosphorylation [31]. The DNA-binding
domain (DBD) or C domain consists of two zinc fingers, and is directly involved
with the binding of PPAR to the peroxisome proliferator response element (PPRE)
in the promoter regions of target genes. PPREs are direct repeat (DR)-1
elements consisting of two hexanucleotides with the AGGTCA consensus sequence
separated by a single nucleotide spacer. Such a sequence, or a similar one, has
been found in numerous PPAR-inducible genes, including acyl-CoA oxidase (ACO)
and adipocyte fatty acid-binding protein (aP2) [32]. The D site is a hinge
region and a docking domain for corepressors. The E/F domain or ligand-binding
domain (LBD) is responsible for ligand specificity and the activation of PPAR
binding to the PPRE, which increases the expression of the targeted gene. Upon
the binding of a specific ligand to LBD of the E/F domain, the conformation of
a PPAR is altered and stabilized. The ligand-bound LBD results in the
recruitment of transcriptional coactivators, resulting in gene transcription. Although
three of the PPAR isoforms possess similar structures, it is clear that these
receptors perform distinct functions according to the specific ligands and their
expression patterns in the tissues.


### 3.2. PPARs and Their Ligands

Ligand-induced
activation of the PPAR, by means of low-affinity binding to natural lipid
ligands, stimulates an array of molecular responses that aim at maintaining
lipid and glucose homeostasis. Ligand-unbound PPAR is associated with chaperon
in the cytosol, and the association induces the PPAR to be held in a
conformation that allows for high-affinity binding of the ligand [33]. The
translocation of a hydrophobic ligand into the cell is facilitated in intra- and
extracellular fluids by intracellular lipid-binding
proteins (iLBPs) that are members of the family of fatty acid-binding proteins
(FABPs). The iLBPs with relatively small sizes (15-16kDa) play important roles
in the solubilization and protection of ligands in aqueous spaces.
Ligand-loaded iLBP in the cytosol translocates into the nucleus by free
diffusion, and they form a short-lived complex with PPAR [34–36]. Ligand is
then transferred to the PPAR, and the ligand-bound PPAR forms a heterodimer
with the partner nuclear receptor, retinoid X receptor (RXRα). Upon binding to a ligand, the conformation
of PPAR is altered and stabilized, and the PPAR-RXR heterodimer then recruits
transcriptional coactivators [37–39]. The transcription machinery is bound to PPRE,
and directly controls the expression of the target gene (Figure 2) [40].Figure 2
Signaling pathways activating PPAR and regulating the biological effects of
PPAR in different organs. PPAR activity can be regulated by the direct binding
of small lipophilic ligands. Ligand-unbound PPAR in the cytosol is associated
with chaperons, and the association changes the conformation of PPAR that
allows for high-affinity binding to the ligand. Ligand-bound PPAR forms a heterodimer
with RXR, and the PPAR-RXR heterodimer constructs the transcriptional machinery
through the recruitment of coregulators. The transcriptional machinery
regulates gene expression by binding to specific DNA sequence elements, termed
PPAR response elements (PPRE). PPARα is strongly
expressed in the liver, heart, and blood vessels, and regulates the expressions
of genes related to lipid metabolism and inflammation control. PPARγ exerts its effects on insulin sensitivity and
glucose homeostasis in adipocytes and skeletal muscles. PPARδ is expressed ubiquitously, and controls the expressions
of genes that are involved in glucose and lipid metabolism. FA: fatty acid; HDL:
high-density lipoprotein; VDL: very low-density lipoprotein; ABCA1: ATP-binding
cassette transporter A1; UCP: uncoupling protein; TG: triglyceride.PPARα was first cloned from the rodent liver in 1990
[41], and PPARβ and PPARγ were first identified in Xenopus [42]. Several groups subsequently reported the cloning of
mammalian orthologs of PPARα, PPARβ, and PPARγ. Although PPARα and PPARγ are highly conserved across species, PPARβ varies considerably between Xenopus and mammals. The murine clone
was named PPARδ because of
this divergence [43]. PPARα is
predominantly expressed in the liver, and is involved in peroxisome
proliferation and regulation of fatty acid catabolism. The expression of PPARδ is ubiquitous and abundant in the brain,
intestine, skeletal muscle, spleen, macrophages, lung, and adrenals [44]. PPARδ is activated by a large variety of ligands,
and has been implicated in developmental and metabolic regulation in several
tissues. PPARγ is
expressed in adipose tissue, promoting adipogenesis and increasing lipid
storage. PPARγ has at
least two promoters, and results in the production of two isoforms, 1 and 2.
These isoforms are expressed in a tissue-specific pattern. The PPARγ1 isoform is expressed in the spleen,
intestine, and white adipose tissue, while the PPARγ2 is preferentially expressed in white and
brown fat. PPARγ2 is most
abundantly expressed in fat cells, and plays a pivotal role in fat cell
differentiation and lipid storage [45]. The
distinct physiological roles of each subtype have been shown to be determined
by binding to a discrete set of ligands. Although fatty acids could activate
PPARs, PPARα activity
was induced by eicosanoids [46], cabaprostacyclin [47], and nonsteroidal
anti-inflammatory drugs (NSAIDs) [48]. PPARδ was activated by several polyunsaturated fatty
acids [49] and eicosanoids [50]. PPARγ
specifically binds to thiazolidinediones (TZDs), a class of antidiabetic drugs.
Other PPARγ ligands
include the natural prostaglandin metabolite 15-deoxy-Δ12.14-prostaglandin J2(PGJ2), polyunsaturated fatty acids, and NSAIDs such as ibuprofen
and indomethacin [51, 52].


### 3.3. Post-Translational Regulation of PPARs

PPARs and other nuclear
receptors modulate their transcriptional activity via phosphorylation by
various kinases, including the mitogen-activated protein kinase (ERK MAPK and
p38 MAPK), protein kinase A and C (PKA and PKC), AMP kinase (AMPK), and glycogen
synthase kinase 3 (GSK3) [53]. Several mechanisms have been described to
explain the modulation of PPAR transcriptional function. First, phosphorylation
modulates the affinity of PPARs for their ligand, as well as the coactivator
recruitment abilities of PPARs. Although the main phosphorylation site of PPARγ (Ser 112) is located far from the
ligand-binding domain, mutated PPARγ (S112D)
exhibits a decreased ligand-binding affinity and decreased coactivator
recruitment [54]. Second, the phosphorylation of PPARs modulates binding to
PPRE. In gel retardation experiments, PPARα phosphorylation via PKA enhances gene
expression due to the stabilization of the binding of PPARα to DNA [55]. Finally, phosphorylation plays an
important role in ubiquitination and proteasomal catabolism of PPARs.
Phosphorylation of the PPARγ AF-1 domain
by IFNγ-ERK-regulated
serine phosphorylation promotes the degradation of PPARγ by the ubiqutin-proteasome-dependent degradation
in response to ligand activation [56]. PPARα is also degraded by the
ubiquitin-proteasome-dependent degradation. However, in contrast to PPARγ, phosphorylation of PPARα induces the stabilization of PPARα by reducing ubiquitination. The phosphorylation
and interaction of PPARα with a corepressor
stabilize PPARα protein by
decreasing its ubiquitination in order to keep a pool of PPARα available for ligand binding and activation
[57].SUMOylation
consists of the covalent and reversible conjugation of small ubiquitin-related
modifiers (SUMOs) to target protein and regulate biological processes. The
number of known SUMO targets is growing, and SUMOylation of PPARγ has recently been reported. SUMOylation of
PPARγ mainly occurs at a lysine residue within a
ligand-independent activating function domain (AF-1). PPARγ is SUMOylated by SUMO-1 and PIAS proteins that
function as E3 ligases [58]. Potential SUMOylation sites of PPARγ include K77 (equivalent to K107 of PPARγ2) and K365. SUMOylation of PPARγ at K77 and K365 occurs in a ligand-dependent
manner. SUMOylation of K107 inhibits PPARγ-dependent gene induction, but does not affect transrepression, whereas mutation of K365
eliminates the ability of agonist-activated PPARγ to repress iNOS and to be recruited to its
promoter [59]. Phosphorylation at S112 of PPARγ2 promotes K107 SUMOylation and exerts more
potent repressive effects. The SUMOylation-defective mutation of PPARγ at K77R promotes adipocyte differentiation. The
potential SUMOylation site of PPARα has one
K185 within the D region, and PPARδ/β has one K104 in the C region, but in vivo SUMOylation is specific for
PPARγ among the PPARs [60]. Relatively few studies of
post-translational regulation of PPARs have been reported.


### 3.4. Role of PPAR Ligands in Metabolic Isorders

The
activation of PPARα upregulates
the expressions of several catabolic enzymes that are involved in mitochondrial
and peroxisomal β-oxidation
and microsomal ω-oxidation,
as well as in the transcriptional regulation of genes that are necessary for
the maintenance of the redox balance during the oxidative catabolism of fatty
acids. The derivatives from fatty acids and fibrates, including gemfibrozil,
fenofibrate, cofibrate, bezafibrate, and ciprofibrate, can activate PPARα. These fibrates are used in the treatment of
hypertriglyceridemia. PPARα agonists
fundamentally regulate β-oxidation
of fatty acids, and promote the expression of cytochrome P450 enzymes, which
catalyze the ω-hydroxylation
of fatty acid [61]. WY14,643, a well-known specific PPARα agonist, increases fatty acid oxidation by increasing
the expressions of peroxisomal and mitochondrial fatty acid β-oxidation
enzymes. WY14,643 reduces liver insulin resistance more efficiently than muscle
insulin resistance by normalizing the circulating triglyceride levels and blood
glucose levels in lipoatrophic mice [62]. PPARα agonists also activate the expression of
apolipoprotein A-1 (ApoA-1) and ATP-binding cassette transporter A1 (ABCA1)
[63, 64]. The increased ApoA-1 and ABCA1 proteins enhance cholesterol efflux by the reverse  cholesterol
transport (RCT) pathway. In addition, PPARα agonists have anti-inflammatory effects in
vascular cells. WY14,643 or bezafibrate induces PPARα-mediated inhibition of osteopontin (OPN)
expression in human macrophages of atherosclerotic lesions, where they are abundantly
synthesized. Bezafibrate significantly decreases OPN plasma levels in type 2
diabetic patients [65]. Therefore, the PPARα agonist reduces the progression of
atherosclerosis and decreases the incidence of coronary heart disease [66]. However,
fibrates are contraindicated in patients with renal insufficiency, gallstones,
abnormal liver function tests, and pregnancy [67].The activation of PPARγ promotes the storage of fat by increasing
adipocyte differentiation and enhancing the transcription of genes that are
important for lipogenesis. The activation of either PPARα or PPARγ in macrophages promotes the cellular efflux of
phospholipids and cholesterol in the form of high-density lipoproteins by upregulating
the expression of the liver X-receptor
(LXR), an oxysterol-activated nuclear hormone receptor that increases
expression of the lipid transporter ABCA1 (ATP-binding cassette, subfamily A,
member 1) [68]. PPARγ has been the focus of intense research during
the past decade because ligands for this receptor have emerged as potent
insulin sensitizers that can be used in the treatment of type 2 diabetes [69].
Increased levels of circulating free fatty acids and lipid accumulation in
non-adipose tissue have been implicated in the development of insulin
resistance. This situation is improved by the PPARγ agonist, which promotes fatty acid storage in
fat depots and regulates the expression of adipocyte-secreted hormones that
impact glucose homeostasis [70]. The net result of the pleiotropic effects of
the PPARγ agonist is
improvement of insulin sensitivity, although undesired side effects limit the
utility of this therapy. In fact, TZD, a synthetic agonist of PPARγ, appears to be ideally suited for the
treatment of this cluster of metabolic abnormalities, which has been termed the
insulin resistance or cardiovascular dysmetabolic syndrome as a whole [71]. Two
compounds in this class are currently approved for use in the United States. They are Rosiglitazone
(Avandia), approved by the US Food and Drug Administration (FDA) in May 1999,
and Pioglitazone (Actos), which was approved in July 1999. Historically, the
first agent in this class, Troglitazone (Rezulin), was marketed in the United States
from March 1997 to March 2000. Troglitazone was banned because the FDA
determined that the risk of idiosyncratic hepatoxicity associated with Troglitazone
therapy outweighed its potential benefits [72, 73].The activation of PPARδ in macrophages also upregulates the expression
of the ABCA1 transporter. Recent evidence indicates that PPARδ can also promote cellular lipid accumulation
by increasing the expressions of genes that are involved in lipid uptake, and
by repressing key genes that are involved in lipid metabolism, inflammation,
atherosclerosis, obesity, fertility, and cancer [74, 75]. Several 14- to
18-carbon saturated fatty acids as well as 16- to 20-carbon polyunsaturated
fatty acids are screened as PPARδ-binding
chemicals in ligand screening and competition binding assays [50, 76, 77]. As
physiological ligands of PPARδ, these
fatty acids or eicosanoids are unsettled. However, Chawla et al. hypothesized that PPARδ acts as a
lipid sensor, where fatty acids derived from very-low-density lipoprotein
(VLDL) can activate PPARδ [78]. A
PPARδ-specific agonist, GW501516, decreases plasma
triglyceride levels in obese monkeys, raises high-density lipoprotein levels,
and prompts the initiation of clinical trials to assess its efficacy in
hyperlipidemic patients [79]. GW501516 also attenuates weight gain and insulin
resistance in mice fed high-fat diets by increasing the expressions of genes that
promote lipid catabolism and mitochondrial uncoupling in skeletal muscle,
thereby increasing β-oxidation
of the fatty acids in skeletal muscle [80]. PPARδ agonists also have anti-inflammatory
properties. The PPARδ agonist
inhibits LPS-inducible genes, such as COX-2 and iNOS in murine peritoneal
macrophages [81]. These reports indicate that the PPARδ-specific agonist is a potential therapeutic
interest for the treatment of metabolic disorder.


## 3.1. PPARs as a Nuclear Receptor Family

Peroxisomes are
subcellular organelles that perform diverse metabolic functions, including H2O2-derived
respiration, β-oxidation
of fatty acids, and cholesterol metabolism. Rodents exposed to peroxisome
proliferators lead to hepatocellular hypertrophy, hyperplasia, and
transcriptional induction of fatty acid-metabolizing enzymes that are regulated
in parallel with peroxisome proliferation [29]. Peroxisome proliferators may
activate PPARs by binding directly to the receptors, and the activated PPARs
may regulate the expression of genes involved in lipid metabolism and peroxisome
proliferation. Recent research on PPARs has moved toward their pivotal roles
comprising one family of nuclear receptors [30]. Nuclear receptors, which are
present in multicellular organisms, directly control the expression of genes in
response to a wide range of developmental, physiological, and environmental
signals.The PPARs of nuclear
receptors mainly consist of three subtypes (PPARα, PPARγ, and PPARδ/β). All three
PPAR isoforms possess similar structural and functional features. Principally,
four functional domains have been
identified, and are referred to as A/B, C, D, and E/F. The N-terminal A/B
domain contains ligand-independent activation function 1 (AF-1). The
ligand-independent activation region can confer constitutive activity on the
receptor, and is negatively regulated by phosphorylation [31]. The DNA-binding
domain (DBD) or C domain consists of two zinc fingers, and is directly involved
with the binding of PPAR to the peroxisome proliferator response element (PPRE)
in the promoter regions of target genes. PPREs are direct repeat (DR)-1
elements consisting of two hexanucleotides with the AGGTCA consensus sequence
separated by a single nucleotide spacer. Such a sequence, or a similar one, has
been found in numerous PPAR-inducible genes, including acyl-CoA oxidase (ACO)
and adipocyte fatty acid-binding protein (aP2) [32]. The D site is a hinge
region and a docking domain for corepressors. The E/F domain or ligand-binding
domain (LBD) is responsible for ligand specificity and the activation of PPAR
binding to the PPRE, which increases the expression of the targeted gene. Upon
the binding of a specific ligand to LBD of the E/F domain, the conformation of
a PPAR is altered and stabilized. The ligand-bound LBD results in the
recruitment of transcriptional coactivators, resulting in gene transcription. Although
three of the PPAR isoforms possess similar structures, it is clear that these
receptors perform distinct functions according to the specific ligands and their
expression patterns in the tissues.


## 3.2. PPARs and Their Ligands

Ligand-induced
activation of the PPAR, by means of low-affinity binding to natural lipid
ligands, stimulates an array of molecular responses that aim at maintaining
lipid and glucose homeostasis. Ligand-unbound PPAR is associated with chaperon
in the cytosol, and the association induces the PPAR to be held in a
conformation that allows for high-affinity binding of the ligand [33]. The
translocation of a hydrophobic ligand into the cell is facilitated in intra- and
extracellular fluids by intracellular lipid-binding
proteins (iLBPs) that are members of the family of fatty acid-binding proteins
(FABPs). The iLBPs with relatively small sizes (15-16kDa) play important roles
in the solubilization and protection of ligands in aqueous spaces.
Ligand-loaded iLBP in the cytosol translocates into the nucleus by free
diffusion, and they form a short-lived complex with PPAR [34–36]. Ligand is
then transferred to the PPAR, and the ligand-bound PPAR forms a heterodimer
with the partner nuclear receptor, retinoid X receptor (RXRα). Upon binding to a ligand, the conformation
of PPAR is altered and stabilized, and the PPAR-RXR heterodimer then recruits
transcriptional coactivators [37–39]. The transcription machinery is bound to PPRE,
and directly controls the expression of the target gene (Figure 2) [40].Figure 2
Signaling pathways activating PPAR and regulating the biological effects of
PPAR in different organs. PPAR activity can be regulated by the direct binding
of small lipophilic ligands. Ligand-unbound PPAR in the cytosol is associated
with chaperons, and the association changes the conformation of PPAR that
allows for high-affinity binding to the ligand. Ligand-bound PPAR forms a heterodimer
with RXR, and the PPAR-RXR heterodimer constructs the transcriptional machinery
through the recruitment of coregulators. The transcriptional machinery
regulates gene expression by binding to specific DNA sequence elements, termed
PPAR response elements (PPRE). PPARα is strongly
expressed in the liver, heart, and blood vessels, and regulates the expressions
of genes related to lipid metabolism and inflammation control. PPARγ exerts its effects on insulin sensitivity and
glucose homeostasis in adipocytes and skeletal muscles. PPARδ is expressed ubiquitously, and controls the expressions
of genes that are involved in glucose and lipid metabolism. FA: fatty acid; HDL:
high-density lipoprotein; VDL: very low-density lipoprotein; ABCA1: ATP-binding
cassette transporter A1; UCP: uncoupling protein; TG: triglyceride.PPARα was first cloned from the rodent liver in 1990
[41], and PPARβ and PPARγ were first identified in Xenopus [42]. Several groups subsequently reported the cloning of
mammalian orthologs of PPARα, PPARβ, and PPARγ. Although PPARα and PPARγ are highly conserved across species, PPARβ varies considerably between Xenopus and mammals. The murine clone
was named PPARδ because of
this divergence [43]. PPARα is
predominantly expressed in the liver, and is involved in peroxisome
proliferation and regulation of fatty acid catabolism. The expression of PPARδ is ubiquitous and abundant in the brain,
intestine, skeletal muscle, spleen, macrophages, lung, and adrenals [44]. PPARδ is activated by a large variety of ligands,
and has been implicated in developmental and metabolic regulation in several
tissues. PPARγ is
expressed in adipose tissue, promoting adipogenesis and increasing lipid
storage. PPARγ has at
least two promoters, and results in the production of two isoforms, 1 and 2.
These isoforms are expressed in a tissue-specific pattern. The PPARγ1 isoform is expressed in the spleen,
intestine, and white adipose tissue, while the PPARγ2 is preferentially expressed in white and
brown fat. PPARγ2 is most
abundantly expressed in fat cells, and plays a pivotal role in fat cell
differentiation and lipid storage [45]. The
distinct physiological roles of each subtype have been shown to be determined
by binding to a discrete set of ligands. Although fatty acids could activate
PPARs, PPARα activity
was induced by eicosanoids [46], cabaprostacyclin [47], and nonsteroidal
anti-inflammatory drugs (NSAIDs) [48]. PPARδ was activated by several polyunsaturated fatty
acids [49] and eicosanoids [50]. PPARγ
specifically binds to thiazolidinediones (TZDs), a class of antidiabetic drugs.
Other PPARγ ligands
include the natural prostaglandin metabolite 15-deoxy-Δ12.14-prostaglandin J2(PGJ2), polyunsaturated fatty acids, and NSAIDs such as ibuprofen
and indomethacin [51, 52].


## 3.3. Post-Translational Regulation of PPARs

PPARs and other nuclear
receptors modulate their transcriptional activity via phosphorylation by
various kinases, including the mitogen-activated protein kinase (ERK MAPK and
p38 MAPK), protein kinase A and C (PKA and PKC), AMP kinase (AMPK), and glycogen
synthase kinase 3 (GSK3) [53]. Several mechanisms have been described to
explain the modulation of PPAR transcriptional function. First, phosphorylation
modulates the affinity of PPARs for their ligand, as well as the coactivator
recruitment abilities of PPARs. Although the main phosphorylation site of PPARγ (Ser 112) is located far from the
ligand-binding domain, mutated PPARγ (S112D)
exhibits a decreased ligand-binding affinity and decreased coactivator
recruitment [54]. Second, the phosphorylation of PPARs modulates binding to
PPRE. In gel retardation experiments, PPARα phosphorylation via PKA enhances gene
expression due to the stabilization of the binding of PPARα to DNA [55]. Finally, phosphorylation plays an
important role in ubiquitination and proteasomal catabolism of PPARs.
Phosphorylation of the PPARγ AF-1 domain
by IFNγ-ERK-regulated
serine phosphorylation promotes the degradation of PPARγ by the ubiqutin-proteasome-dependent degradation
in response to ligand activation [56]. PPARα is also degraded by the
ubiquitin-proteasome-dependent degradation. However, in contrast to PPARγ, phosphorylation of PPARα induces the stabilization of PPARα by reducing ubiquitination. The phosphorylation
and interaction of PPARα with a corepressor
stabilize PPARα protein by
decreasing its ubiquitination in order to keep a pool of PPARα available for ligand binding and activation
[57].SUMOylation
consists of the covalent and reversible conjugation of small ubiquitin-related
modifiers (SUMOs) to target protein and regulate biological processes. The
number of known SUMO targets is growing, and SUMOylation of PPARγ has recently been reported. SUMOylation of
PPARγ mainly occurs at a lysine residue within a
ligand-independent activating function domain (AF-1). PPARγ is SUMOylated by SUMO-1 and PIAS proteins that
function as E3 ligases [58]. Potential SUMOylation sites of PPARγ include K77 (equivalent to K107 of PPARγ2) and K365. SUMOylation of PPARγ at K77 and K365 occurs in a ligand-dependent
manner. SUMOylation of K107 inhibits PPARγ-dependent gene induction, but does not affect transrepression, whereas mutation of K365
eliminates the ability of agonist-activated PPARγ to repress iNOS and to be recruited to its
promoter [59]. Phosphorylation at S112 of PPARγ2 promotes K107 SUMOylation and exerts more
potent repressive effects. The SUMOylation-defective mutation of PPARγ at K77R promotes adipocyte differentiation. The
potential SUMOylation site of PPARα has one
K185 within the D region, and PPARδ/β has one K104 in the C region, but in vivo SUMOylation is specific for
PPARγ among the PPARs [60]. Relatively few studies of
post-translational regulation of PPARs have been reported.


## 3.4. Role of PPAR Ligands in Metabolic Isorders

The
activation of PPARα upregulates
the expressions of several catabolic enzymes that are involved in mitochondrial
and peroxisomal β-oxidation
and microsomal ω-oxidation,
as well as in the transcriptional regulation of genes that are necessary for
the maintenance of the redox balance during the oxidative catabolism of fatty
acids. The derivatives from fatty acids and fibrates, including gemfibrozil,
fenofibrate, cofibrate, bezafibrate, and ciprofibrate, can activate PPARα. These fibrates are used in the treatment of
hypertriglyceridemia. PPARα agonists
fundamentally regulate β-oxidation
of fatty acids, and promote the expression of cytochrome P450 enzymes, which
catalyze the ω-hydroxylation
of fatty acid [61]. WY14,643, a well-known specific PPARα agonist, increases fatty acid oxidation by increasing
the expressions of peroxisomal and mitochondrial fatty acid β-oxidation
enzymes. WY14,643 reduces liver insulin resistance more efficiently than muscle
insulin resistance by normalizing the circulating triglyceride levels and blood
glucose levels in lipoatrophic mice [62]. PPARα agonists also activate the expression of
apolipoprotein A-1 (ApoA-1) and ATP-binding cassette transporter A1 (ABCA1)
[63, 64]. The increased ApoA-1 and ABCA1 proteins enhance cholesterol efflux by the reverse  cholesterol
transport (RCT) pathway. In addition, PPARα agonists have anti-inflammatory effects in
vascular cells. WY14,643 or bezafibrate induces PPARα-mediated inhibition of osteopontin (OPN)
expression in human macrophages of atherosclerotic lesions, where they are abundantly
synthesized. Bezafibrate significantly decreases OPN plasma levels in type 2
diabetic patients [65]. Therefore, the PPARα agonist reduces the progression of
atherosclerosis and decreases the incidence of coronary heart disease [66]. However,
fibrates are contraindicated in patients with renal insufficiency, gallstones,
abnormal liver function tests, and pregnancy [67].The activation of PPARγ promotes the storage of fat by increasing
adipocyte differentiation and enhancing the transcription of genes that are
important for lipogenesis. The activation of either PPARα or PPARγ in macrophages promotes the cellular efflux of
phospholipids and cholesterol in the form of high-density lipoproteins by upregulating
the expression of the liver X-receptor
(LXR), an oxysterol-activated nuclear hormone receptor that increases
expression of the lipid transporter ABCA1 (ATP-binding cassette, subfamily A,
member 1) [68]. PPARγ has been the focus of intense research during
the past decade because ligands for this receptor have emerged as potent
insulin sensitizers that can be used in the treatment of type 2 diabetes [69].
Increased levels of circulating free fatty acids and lipid accumulation in
non-adipose tissue have been implicated in the development of insulin
resistance. This situation is improved by the PPARγ agonist, which promotes fatty acid storage in
fat depots and regulates the expression of adipocyte-secreted hormones that
impact glucose homeostasis [70]. The net result of the pleiotropic effects of
the PPARγ agonist is
improvement of insulin sensitivity, although undesired side effects limit the
utility of this therapy. In fact, TZD, a synthetic agonist of PPARγ, appears to be ideally suited for the
treatment of this cluster of metabolic abnormalities, which has been termed the
insulin resistance or cardiovascular dysmetabolic syndrome as a whole [71]. Two
compounds in this class are currently approved for use in the United States. They are Rosiglitazone
(Avandia), approved by the US Food and Drug Administration (FDA) in May 1999,
and Pioglitazone (Actos), which was approved in July 1999. Historically, the
first agent in this class, Troglitazone (Rezulin), was marketed in the United States
from March 1997 to March 2000. Troglitazone was banned because the FDA
determined that the risk of idiosyncratic hepatoxicity associated with Troglitazone
therapy outweighed its potential benefits [72, 73].The activation of PPARδ in macrophages also upregulates the expression
of the ABCA1 transporter. Recent evidence indicates that PPARδ can also promote cellular lipid accumulation
by increasing the expressions of genes that are involved in lipid uptake, and
by repressing key genes that are involved in lipid metabolism, inflammation,
atherosclerosis, obesity, fertility, and cancer [74, 75]. Several 14- to
18-carbon saturated fatty acids as well as 16- to 20-carbon polyunsaturated
fatty acids are screened as PPARδ-binding
chemicals in ligand screening and competition binding assays [50, 76, 77]. As
physiological ligands of PPARδ, these
fatty acids or eicosanoids are unsettled. However, Chawla et al. hypothesized that PPARδ acts as a
lipid sensor, where fatty acids derived from very-low-density lipoprotein
(VLDL) can activate PPARδ [78]. A
PPARδ-specific agonist, GW501516, decreases plasma
triglyceride levels in obese monkeys, raises high-density lipoprotein levels,
and prompts the initiation of clinical trials to assess its efficacy in
hyperlipidemic patients [79]. GW501516 also attenuates weight gain and insulin
resistance in mice fed high-fat diets by increasing the expressions of genes that
promote lipid catabolism and mitochondrial uncoupling in skeletal muscle,
thereby increasing β-oxidation
of the fatty acids in skeletal muscle [80]. PPARδ agonists also have anti-inflammatory
properties. The PPARδ agonist
inhibits LPS-inducible genes, such as COX-2 and iNOS in murine peritoneal
macrophages [81]. These reports indicate that the PPARδ-specific agonist is a potential therapeutic
interest for the treatment of metabolic disorder.


## 4. Various Strategies for Safer PPAR Modulators

Each PPAR subtype
regulates a distinct metabolic pathway, and the agonists of each of the PPAR
subtypes have distinct effects with undesired side effects such as weight gain,
hepatotoxicity, and heart failure. In the case of the TZD class as PPARγ agonists, the major side effect is weight gain.
A Pro12Ala substitution in PPARγ2 decreases
PPARγ activity, BMI, and insulin resistance [82]. Because
of these undesirable effects caused by PPARγ agonists, new therapeutic solutions have been
investigated in order to reduce their side effects. Various compounds have been
reported to be PPAR antagonists, including Bisphenol A diglycidyl ether
(BADGE), PD068235, LG100641, GW9662, SR-202, GW6741, and Compound A and B [83].
A potent selective PPARγ antagonist,
GW9662, does not recruit PPAR coactivators such as SRC-1 and p300, and it
suppresses rosiglitazone-induced adipocyte differentiation in 3T3-L1 adipocytes.
GW9662 prevents high-fat diet-induced obesity without affecting food intake,
and has no effect on high-fat diet-induced glucose intolerance [84]. The
phosphonophosphate SR-202, a PPARγ antagonist,
inhibits BRL 49653-mediated recruitment of SRC-1 and troglitazone-induced
transcriptional activity. SR-202 inhibits PPARγ-induced adipocyte differentiation of 3T3-L1
and prevents weight gain and adipose tissue deposition in mice given a standard
diet or high-fat diet. In addition, SR-202 markedly reduces hyperglycemia and
hyperinsulinemia in ob/ob mice [85]. A few PPARα antagonists have been reported, but in vivo data have not been disclosed.
Several PPARγ antagonists
may have therapeutic availability as antiobesity drugs. However, further
studies of the molecular effects of PPARγ antagonists are necessary.The combination agonist
strategy, which uses a combination of agonists, has been designed to activate
each receptor subtype. In terms of its pharmacological aspects, this strategy
may provide more efficacious effects and more safety for undesired side
effects. The possible combinations are PPARα/γ dual
agonist, PPARγ/δ dual agonist, PPARα/δ dual
agonist, and PPARpan (PPARα/γ/δ) agonist. The
initial combination agonist strategy was focused on the development of PPARα/γ dual
agonists. PPARγ agonists
such as rosiglitazone and pioglitazone provide undesired side effects of TZDs,
including weight gain. By contrast, PPARα agonists such as fibrate decreased adiposity
through the stimulation of lipid oxidation. Dual PPARα/γ stimulation
with a combination of rosiglitazone and fenofibrate in type 2 diabetic patients
effectively improved the atherogenic dyslipidemic profile, which plays a key
role in the occurrence of cardiovascular mortality [86]. Applications of
structurally various PPARα/γ dual agonists have recently been reported.
Among these dual PPARα/γ agonists, compounds belonging to the glitazar
class have been advanced to clinical development (Phases II and III). These PPARα/γ dual
agonists commonly reduce triglycerides and total cholesterol, increase HDL
levels, and consequently improve insulin sensitivity. However, the use of a few
PPARα/γ dual
agonists, including muraglitazar, tesaglitazar, ragaglitazar, farglitazar,
TAK559, and KRP297, has been discontinued due to various safety liabilities
compared to selective agonists. All glitazars significantly increase weight
gain and edema, because of higher PPARγ affinity
than PPARα affinity
although their affinity for PPARα is higher
than fibrates. Muraglitazar increases cardiovascular risks, tesaglitazar
impairs glomerular filteration rate, and some have carcinogenic effects in mice
[87]. The safety liabilities may be the result of their imbalanced activities
on PPARγ and PPARα. Therefore, the best solution would be to
screen candidates with appropriate affinity for PPARα and selective PPARγ-modulating activity [88].Both PPARγ and PPARδ play important roles in glucose and lipid
metabolism. A PPARγ/δ dual agonist with a properly controlled γ/δ ratio could
attenuate undesired weight gain, improve insulin sensitivity, and stimulate
fatty acid oxidation. The dual PPARγ/δ agonist
(R)-3-{2-ethyl-4-[3-(4-ethyl-2-pyridin-2-yl-phenoxy)-butoxy]-phenyl}-propionic
acid has been shown to lower the glucose level and cause less weight gain than
rosiglitazone in hyperglycemic male Zucker diabetic fatty (ZDF) rats [89]. The
other dual PPARγ/δ agonist, (R)-3-{4-[3-(4-chloro-2-phenoxy-phenoxy)-butoxy]-2-ethyl-phenyl}-propionic
acid, improves insulin sensitivity and reverses diabetic hyperglycemia with
less weight gain relative to rosiglitazone in female ZDF rats [90]. PPARα/δ dual
agonists (T659 and Compound 24) have recently been reported. T659 has had
beneficial effects on HDL-C in experimental primates [91]. Compound 24 has also
shown significant effects on HDL-C, TG, and FFA levels in male hApoA1
transgenic mice [92]. PPARα/δ dual agonists may improve hyperlipidemia,
insulin resistance, and risk of atherosclerosis. The development of PPARα/δ and PPARγ/δ dual
agonists is currently being pursued.Another strategy to
reduce the adverse effects of previous PPARγ agonists is the identification of partial
agonists, also referred to as selective PPARγ modulators (SPPARγMs). SPPARγMs are PPARγ ligands with insulin-sensitizing activity and
lower stimulation of adipogenesis. Because SPPARγMs bind to the ligand-binding pocket of the
PPARγ receptor in distinct manners, SPPARγM-bound PPARγ induces the displacement of the differential
cofactor and the specific gene expression in a tissue-specific manner. Although
several PPARγ agonists
have been classified as SPPARγMs, the
majority of these synthetic ligands remain to be characterized at the molecular
level, and need to be evaluated in in
vivopreclinical models to
assess their relationships with weight gain [93]. Halofenate (HA) and PA-082,
new SPPARγMs, were
recently developed. HA causes displacement of corepressors (N-CoR and SMRT),
but does not cause efficient recruitment of coactivators (p300, CBP, and TRAP
220). Moreover, HA selectively regulates the expression of multiple PPARγ responsive genes in 3T3-L1 adipocytes, and has
acute antidiabetic properties in diabetic ob/ob mice [94]. The isoquinoline derivative PA-082, a prototype of a novel class of
non-TZD partial PPARγ agonists,
causes preferential recruitment of PPARγ-coactivator-1α (PGC1α) to the
receptor compared with rosiglitazone. PA-082 antagonizes rosiglitazone-driven
transactivation and TG accumulation in C2H10T1/2 mesenchymal stem cells.
However, PA-082 induces mRNAs of genes that encode components of insulin
signaling pathways. It also facilitates glucose uptake and insulin signaling in
mature adipocytes [95]. The functional study of SPPARγMs will provide more information about effective
antidiabetic agents to reduce the side effects of weight gain.The PPARpan agonists
can activate all three PPAR subtypes, and they can potentially exert various
effects on metabolic disorders such as insulin resistance, obesity,
dyslipidemia, and hypertension. The well-known lipid-lowering bezafibrate is
the first clinically-tested PPARpan agonist. Though bezafibrate is a PPAR
ligand with a relatively low potency, it considerably raises HDL cholesterol,
reduces triglycerides, improves insulin sensitivity, and reduces blood glucose
levels [96]. GW677954, a novel PPARpan agonist, is being investigated in Phase
II trials for the treatment of metabolic disorders [97]. PLX-204 and GW-625019
are also progressing in Phase I trials for the treatment of metabolic
disorders. In addition, LY-465608, DRF-11605, CS-204, and DRL-11605 are under
investigation, and may be potent therapeutic agents for the treatment of
metabolic disorders [88].


## 5. Technologies to Discover New PPAR Modulators

The
development of new technology to discover PPAR modulator is significant in
functional study of the nuclear receptors and new potent drug discovery. In
general, transactivation and chimeric receptor transactivation assays have been
used as cell-based methods employing mammalian cells for the screening of new
PPAR modulators. Cell-based assays provide a more physiological relevance, but these
assays are costly, time-consuming, and difficult to apply to automated systems
used for high-throughput screening (HTS). Recently, Chen et al. introduced a yeast-based
method for screening PPAR modulators [98]. Cell-free assays for the screening
of PPAR modulators have been developed in numerous forms. The X-ray crystal
structure study revealed that the human apo-PPARγ ligand-binding domain (LBD) has a large
binding pocket, which may explain the diversity of the PPARγ ligands [99]. When binding to specific ligands
in LBD, PPAR changes its conformation. Glutamate and lysine residues that are
highly conserved in LBDs of PPAR form a “charge clamp” that contacts the
backbone atoms of the LXXLL helices of coactivators such as steroid receptor coactivator-1
(SRC-1). In the case of SRC-1, four consecutive LXXLL motifs make identical
contacts with both subunits of a PPAR-RXR heterodimer [100]. Such allosteric
conformational changes promote the recruitment of nuclear receptor coactivators
and effectively stimulate the transcription of their target genes. Different
PPAR ligands may elicit distinct downstream biological effects due to unique
conformational changes in the nuclear receptor.A cell-free competition
radioreceptor assay using competitive interaction between a recombinant PPAR
protein and a radioisotope-labeled ligand in the presence of competitor ligands
has been reported previously (Figure3(a)) [101, 102]. The coactivator-dependent
receptor ligand assay (CARLA) has been reported as a cell-free assay based on
the interaction between PPAR and the coactivator. CARLA is based on the
recruitment of a transcriptional coactivator by changes in the conformation of
ligand-bound PPAR (Figure 3(b)). In presence of PPAR ligands,35S-labeled
SRC-1 has stronger interaction with GST-fused PPAR proteins immobilized on
glutathione Sepharose beads. Autoradiograms and the quantification of the
effect of candidates are dependent on the retention of SRC-1 by the GST-PPAR
LBD [76]. The major advantage of CARLA is that it does not require radioactive
labeling of candidate modulators, which makes it possible to screen a large
number of compounds with this assay; this has simultaneous economic advantages
in terms of materials and time.Various cell-free assays to discover PPAR modulator. A. Competition radioreceptor assays are performed by incubating recombinant PPAR protein and radioisotope-labeled ligand in the presence of competitor ligands. Bound ligands are separated from free forms by filtration. The amount of bound radioisotope-labeled ligand is determined by liquid scintillation counting. B. Coactivator-dependent receptor ligand assays (CARLAs) based on the recruitment of a coactivator due to a conformational change of specific ligand-bound PPAR. CARLA is carried out by incubating GST-PPAR and radioisotope-labeled coactivator with a ligand candidate or by incubating radioisotope-labeled PPAR and GST-coactivator with ligand candidates. The amount of ligand-bound PPAR-coactivator complex is determined by pull-down assay. C. In the scintillation proximity assay (SPA), the receptor-ligand complex is bound to the SPA bead through interaction between the biotinylated receptor and the streptavidin moiety located on the surface of the SPA bead. Because no separation step is required, SPA has benefits in its application to HTS. D. In the fluorescence resonance energy transfer (FRET)-based in vitro recruitment assay, GST-PPAR LBD proteins are indirectly linked to EU cryptate, (Eu)K, through (Eu)K-labeled anti-GST antibody,α-GST-(Eu)K. Purified recombinant coactivator is biotinylated, and is indirectly linked to XL665, which is produced only when there is a ligand-induced change in receptor conformation that results in binding to the coactivator. The extent of the FRET is measured as a ratio of 665 nm/620 nm X 10,000. E. A simple ELISA based on binding between PPAR and coactivators. The ligand unbound-PPAR weakly binds to the LXXLL motifs of coactivator, whereas ligand loaded-PPAR strongly binds to the LXXLL motifs of coactivator due to the conformational change of PPAR by specific agonists. This binding is detected by a specific anti-PPAR antibody, followed by horseradish peroxidase-conjugated secondary antibody. F. Schematic representation of SPR technology. One of the interacting partners is immobilized on a gold or waveguide layer of the sensor chip using the standard amine-coupling protocol. The other flows over the surface of the sensor chip, allowing interaction with the immobilized interacting partners. The interaction of immobilized partners with interacting molecules gives rise to an increase in mass. The refractive index and the angle of reflected light is thereby changed. As soon as the injection is stopped, the complex is washed with a washing buffer. The interacting molecules are dissociated from the immobilized interacting partner, resulting in a decrease in the signal due to a shift in the angle of the reflected light to its original position.
(a)(b)(c)(d)(e)(f)Scintillation proximity
has been developed as a tool for measuring the interaction between a receptor
and a ligand. The scintillation proximity assay (SPA) bead is impregnated with a
scintillant and coated with a capture molecule such as streptavidin. After
preincubation of SPA beads and biotinylated PPAR LBD, radiolabeled ligands were
added to a complex of SPA bead-PPAR LBD. Unbound free ligands were eliminated
from the SPA-PPAR complex. When SPA-bead and radiolabeled ligands come into
close proximity, radioactive counts are determined by theβ⁢emission
from the radioisotope to be absorbed by the scintillant, which will then shift
this energy to produce light (Figure 3(c)) [103, 104]. The advantages of the SPA are as follows: low cost,
high sensitivity, high reliability, and simplicity, that is, no separation step
is required. The simplicity of SPA is an important benefit in its application
to HTS. However, unsuitability for kinetic determination and the limited number
of useful isotopes were perceived as potential disadvantages.Fluorescence has been
considered as an analytical technique with which to study for the detection and
quantitation of interacting molecules. There are several advantages to this
technique, including high sensitivity, the relative ease of handling and
disposal compared to radioactivity, and the diversity of available fluorophores.
Thus, fluorescence resonance energy transfer (FRET) has been applied in the
probing of molecular interactions [105]. As shown in Figure 3(d), GST-PPAR LBD
was indirectly linked to Eu(K) through an anti-GST antibody, which was
covalently linked to Eu(K). Coactivator was also indirectly linked to XL665
through a streptavidin (SA)-biotin adapter. The conformational change of PPAR
by the PPAR agonist induces the recruitment of coactivator, and the interaction
between PPAR and a coactivator will result in the close proximity of the
fluorescence donor and acceptor. Consequently, the fluorescence donor
(anti-GST-Eu(K)) is excited, and inputted energy will be transferred to the
acceptor (streptavidin-XL665). Homogenous time-resolved fluorescence (HTRF)
energy transfer technology takes advantage of fluorescence, as well as the
homogenous and time-resolved detection mode. These specificities of HTRF enable
it to overcome most of the drawbacks encountered in FRET [106].Previous cell-free
methods with which to screen PPAR agonists have used isotope or fluorescence
labeling agonists or proteins. We established very simple ELISA systems based
on the ligand-dependent binding between PPAR and coactivators. In brief, the
purified recombinant LXXLL motif of coactivators was applied into a 96-well
plate, andE. coli lysates containing
recombinant PPAR proteins were then added with candidate PPAR agonists. The
complex consisting of PPAR and coactivator was then identified with the
anti-PPAR antibody (Figure 3(e)). Major advantages of this simple method are its
simplicity and its low cost, as these systems do not require any labeling of
candidate modulators and proteins. This makes it possible to screen a large
number of compounds, with simultaneous economic advantages in terms of materials
and time. On the other hand, this method has relatively low sensitivity and has
to use a suitable anti-PPAR antibody [107–109].In
the 1980s, surface plasmon resonance (SPR) and related techniques that
exploited evanescent waves were applied for the study of biological and
chemical interactions. SPR technology has also been successfully employed to
study the interactions between ligands and nuclear receptors [110–112], the
effects of ligand-binding on nuclear receptor dimerization [113], and ligand
screening based on interactions between ligand-bound nuclear receptors and coactivators
[114, 115] (Figure 3(f)). In the interaction analysis between PPAR LBD and ligand,
PPAR LBD is immobilized on the sensor chip by a standard primary amine-coupling
reaction, and the ligand is injected over the immobilized PPAR LBD. In the
investigation of ligand binding on receptor dimerization, the partner nuclear
receptor is immobilized on the sensor chip by the standard amine-coupling
reaction, and the nuclear receptor pre-incubated with its ligand is injected
over the immobilized partner nuclear receptor. In ligand screening based on
interactions between ligand-bound nuclear receptors and coactivator, the coactivator
or LXXLL peptide is immobilized on the sensor using same methods, and the
nuclear receptor that was preincubated with a candidate chemical is injected
over the immobilized coactivator peptide. The association (ka) and dissociation (kd) rate constants and the dissociation equilibrium constants
(kDs) for the bindings were determined using the Biacore biosensor.
The binding responses in resonance units (RUs) were continuously recorded, and
were presented graphically as a function of time. SPR technology has the
advantages in that it requires no labeling, can be performed in real-time, and
utilizes noninvasive measurements.


## 6. Summary

Obesity mainly reflects
an increased adipose cell size, an increased adipocyte cell number, and an imbalance
between energy intake and energy expenditure. Excess body fat is associated
with metabolic disorders. As a molecular drug target for metabolic disorders,
the activation of PPAR by specific ligands has many beneficial clinical effects
in the improvement of glucose and lipid homeostasis. The PPARs mainly consist
of three subtypes (PPARα, PPARγ, and PPARδ), and all three PPAR isoforms possess similar
structural and functional features involving glucose metabolism, lipid
metabolism, and energy balance. PPARs directly modulate gene expression upon
binding to specific ligands transferred into PPAR via iLBP-mediated
translocation. PPAR agonists play an important role in therapeutic aspects of
metabolic disorders, whereas undesired effects for the existing PPAR agonists
prescribed as therapeutic agents have been reported. To discover new PPAR
modulators with more efficacious effects and more safety against undesired side
effects, a novel PPAR antagonist or the combination of agonists such as PPARα/γ dual
agonist, PPARγ/δ dual agonist, PPARα/δ dual
agonist, and PPARpan (PPARα/γ/δ) agonist has been applied to
activate each receptor subtype and selective PPARγ modulators (SPPARγMs). In addition, various technologies have
been developed in attempts to discover PPAR modulators as therapeutic agents
for the treatment of metabolic disorders. Because cell-based assays have more
physiological relevance, the transactivation assay, chimeric receptor
transactivation, and yeast two-hybrid methods have also been used. Since cell-free
assays are based on direct interaction between PPAR and their specific ligands,
a new concept for competing radioreceptor assays has been developed by making
the best use of competitive interactions between recombinant PPAR protein and
radioisope-labeled ligands. Later, cell-free assays (CARLA and SPA) were
developed based on conformational changes in PPARs caused by their ligands, and
the simplicity of SPA permitted application to high-throughput screening (HTS).
Radioisotope-free assays like FRET (HTRF), ELISA, and SPR methods are relatively
simple in terms of handling and disposal. Thus, HTRF and SPR assays can be
applied to a homogenous and time-resolved detection mode.Interestingly, prior to
the discovery of the PPARα, it was
reported that Wy-14,643, a well-known synthetic agonist of PPARα, decreased serum cholesterol and triglyceride
levels in mice [116]. TZD derivatives, well-known synthetic agonists of PPARγ, were reported as antidiabetes agents prior to
the discovery of the PPARγ [117, 118].
An antidiabetes agent, pioglitazone, a TZD derivative, has recently been shown
to bind specifically to a protein named mitoNEET [119]. These studies of PPAR,
PPAR modulators, and technologies to discover PPAR modulators will elicit the
development of drugs with more efficacious effects and more safety for the treatment
of metabolic disorders.



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*Source: 679137-2008-06-15.xml*