Chapter 1

PPARs: History and Advances

Jihan Youssef and Mostafa Z. Badr

Abstract
Peroxisome proliferator-activated receptors (PPARs) are members of the steroid hormone receptor
superfamily, discovered in 1990. To date, three PPAR subtypes have been identified; PPARa, PPAR b/d,
and PPARg. These receptors share a high degree of homology but differ in tissue distribution and ligand
specificity. PPARs have been implicated in the etiology as well as treatment of several important diseases
and pathological conditions such as diabetes, inflammation, senescence-related diseases, regulation of
fertility, and various types of cancer. Consequently, significant efforts to discover novel PPAR roles
and delineate molecular mechanisms involved in their activation and repression as well as develop safer and
more effective PPAR modulators, as therapeutic agents to treat a myriad of diseases and conditions, are
underway. This volume of Methods in Molecular Biology contains details of experimental protocols used in
researching these receptors.

Key words: Peroxisome proliferators, PPARs, RXRs, PPRE, TZDs

1. Discovery
of Peroxisome
Proliferator-
Activated Receptors Existence of a specific mediator for the effects of chemicals which
are known to cause peroxisome proliferation (peroxisome prolif-
erators) was suggested by the tissue and cell specificity of the pleio-
tropic effects of these chemicals. In attempting to identify such a
molecular target, a cytosolic protein displaying reversible
stereospecific binding to nafenopin was detected in rat liver and a
receptor-mediated mechanism for peroxisome proliferation was
consequently postulated (1). A peroxisome proliferator-binding
protein was later purified from rat liver cytosol and was identified
as a dimer protein with a molecular weight of 140,000–
160,000 KDa. This protein was capable of binding to peroxisome
proliferators structurally related to clofibrate (2). Further analysis
of the isolated protein revealed that it is homologous with the heat

Mostafa Z. Badr and Jihan Youssef (eds.), Peroxisome Proliferator-Activated Receptors (PPARs): Methods and Protocols,
Methods in Molecular Biology, vol. 952, DOI 10.1007/978-1-62703-155-4_1, © Springer Science+Business Media New York 2013

1
2 J. Youssef and M.Z. Badr

shock protein HSP70 but its role in the process of peroxisome

proliferation remained unclear at that time (3). Thus, efforts con-
tinued in order to identify the putative mediator of these observed
prominent effects.
The ability of peroxisome proliferators to modulate specific
gene transcription suggested that they could act via a mechanism
similar to that of steroid hormones. This assumption paved the way
to a significant discovery when a novel member of the steroid hor-
mone receptor superfamily of ligand-activated transcription factors
was isolated by screening a mouse cDNA library (4). The cloned
receptor was found to be structurally related to steroid hormone
receptors and was activated by a wide range of molecules including
fatty acids and fibrates. Since the pattern of expression of the recep-
tor mRNA mirrored the tissue-specific effects of peroxisome pro-
liferators and because the identified receptor was thought to
mediate the peroxisome proliferative response, it was named per-
oxisome proliferator-activated receptor (PPAR).
Following the initial discovery of mouse PPAR, the receptor
was identified in other species including rat (5) and human (6). In
addition, three related xenopus receptors belonging to nuclear hor-
mone receptor superfamily were cloned and named PPARa,
PPARb, and PPARg proving the existence of more than one form
of PPAR in a given species (7). PPARd was initially identified in
human as an additional form of PPAR (6) but was found later to
be closely related to PPARb described in xenopus.

2. Molecular
Aspects of PPAR
Functions
Subsequent studies indicated that the three PPAR subtypes,
PPARa, PPARb/d, and PPARg, share a high degree of homology
but differ in tissue distribution and ligand specificity and each sub-
type is encoded by a different gene (8). Human PPARa gene is
located on chromosome 22 slightly telomeric to a linkage group of
six genes and genetic markers existing in the general region 22q12–
q13.1 (9), while PPARb/d gene is located on chromosome 6 at
position 6p21.1–p21.2 (10) and PPARg gene is mapped to chro-
mosome 3 at position 3p25 (11). Alternate transcription start sites
and alternate splicing generate four types of PPARg mRNA g1, g2,
g3, and g4. It is believed that PPARg1, g3, and g4 mRNA translate
to an identical protein. Thus only two protein isoforms, PPARg1
and PPARg2 (which has an additional 30 amino acids at the
N-terminus) exist (12).
PPARs form heterodimers with retinoic acid receptors (RXRs)
which also belong to the nuclear hormones family of receptors
(13, 14). Upon ligand binding, PPAR/RXR heterodimers
 1 PPARs: History and Advances 3

recognize and bind to a specific DNA sequence known as PPAR

response element (PPRE) consisting of a direct repeat of six nucle-
otides separated by a single spacer nucleotide (15). The 5¢ flanking
nucleotides of the core PPRE may play an important role in PPAR
subtype specificity (16). PPRE is found in a large number of genes
encoding proteins involved in a variety of cellular functions includ-
ing lipid and carbohydrate metabolism. The three PPARs activate
both overlapping and distinct sets of target genes. Both PPARa
and PPARb/d activate genes involved in lipid oxidation (17, 18),
while PPARg activates lipogenic genes (19).
Intrinsic properties of each PPAR subtype, including post-
translational modifications, are key determinant of the genes that
can be activated by a certain PPAR subtype. PPAR subtype
specificity may also be partly imposed by differential affinity of the
receptors towards cofactors where distinct conformations could be
induced by the individual coactivator resulting in differential tran-
scriptional activity or specificity (20). With regards to receptor acti-
vators, each PPAR ligand induces a specific change in receptor
conformation, resulting in the differential recruitment of cofactors
and gene-specific transcriptional regulation. Thus, in addition to a
panel of common genes regulated in a similar manner by all PPAR
agonists, each agonist regulates its unique profile of genes, result-
ing in specific biological effects. This concept inspired the creation
of new compounds with differential gene regulating properties as
novel therapeutic agents without significant adverse effects.

2.1. Recent Advances PPARs have been implicated in several important diseases and
in PPAR Research pathological conditions. Recent discoveries suggest that PPAR
subtypes present a valuable target for the treatment of several
significant diseases and pathological conditions in various organs.
In the central nervous system, PPARs mediate neuroprotective
effects where PPAR agonists have been proven useful in animal
models of several CNS diseases (21). These receptors are expressed
in the heart and PPAR agonists have shown promising effects in
preventing progression of atherosclerosis in experimental models
as well as in clinical trials (22, 23). In addition, the discovery of
anti-inflammatory and immunomodulatory roles of PPARs has
prompted the investigation of these receptors as potential targets
for treatment of asthma and other inflammatory lung diseases (24),
as well as for several gastrointestinal diseases (25–27).
PPARg ligands, thiazolidinediones (TZDs), are effective oral
antidiabetic drugs. These agonists improve glycemic control in
part by increasing insulin action in adipose tissue, skeletal muscles,
and liver. TZDs also increase insulin biosynthesis and release as
well as glucose transport in b-cells by up-regulating expression of
genes involved in these processes (28, 29). In addition, studies
revealed that the ability of TZDs to reduce b-cell apoptosis and
enhance their proliferation may be responsible for the increase in
4 J. Youssef and M.Z. Badr

b-cell mass with consequent improvement in glucose metabolism

(30, 31). Ligands of PPARb/d have also been proposed as insulin
sensitizers (32).
In the musculoskeletal system, PPARs are reported to play an
interesting role. It appears that PPARa and PPARg activation have
opposite regulatory effects in bone formation (33). The role of
PPARb/d, however, is not yet defined although studies suggest
that it may contribute to bone anabolism (34). In the skeletal mus-
cle, PPARb/d is the most abundant PPAR isotype with a higher
expression level in oxidative type I muscle fibers compared to gly-
colytic type II muscle fibers (35, 36). Evidence suggests that
PPARb/d plays an important role in regulation of skeletal muscle
metabolism particularly lipid oxidation by acting as an activator of
fat burning with subsequent beneficial effects in metabolic disease
(37). Activation of PPARd has been shown to increase physical
performance and improve endurance performance (38). PPARd
agonists are, therefore, characterized as exercise mimetics (38), and
are claimed to be, therefore, abused by athletes (39).
Among the most consequential involvements of PPARs is their
role in cell differentiation and cancer. The literature is replete with
contradictory evidence implicating PPARs in the promotion and
development of cancer, as well as for a protective role against can-
cer. While numerous studies report that the expression level of
these receptors and/or their activation correlates with a positive
outcome against cancer, this does not appear to be a universal phe-
nomenon (40).
Based on the above mentioned PPAR roles, in addition to numer-
ous others, significant efforts to discover and develop safer and more
effective PPAR modulators, as therapeutic agents to treat a myriad of
diseases and conditions, are underway. The following chapters pres-
ent details of state-of-the-art experimental protocols essential to the
successful conduction of research in this expanding field.

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