# PPARδ Agonism for the Treatment of Obesity and Associated Disorders: Challenges and Opportunities

Authors: Mylène Perreault; David V. Erbe; James F. Tobin
Journal: PPAR Research
 (2008)

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## Abstract

The prevalence of obesity in the USA and worldwide has reached epidemic proportions during the last two decades. Drugs currently available for the treatment of obesity provide no more than 5% placebo-adjusted weight loss and are associated with undesirable side effects. Peroxisome proliferator-activated receptor (PPAR) modulators offer potential benefits for the treatment of obesity and its associated complications but their development has been complicated by biological, technical, and regulatory challenges. Despite significant challenges, PPAR modulators are attractive targets for the treatment of obesity and could offer a viable alternative to the millions of patients who fail to lose weight following rigorous dieting and exercise protocols. In addition, PPAR modulators have the potential-added benefit of ameliorating the associated comorbidities.


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## Body

## 1. Introduction

During the last
two decades, the incidence of obesity has tripled in developing
countries, with
more than 1.1 billion adults overweight worldwide and 312 million of them obese
[1]. Obesity has been causally
linked to the development of certain forms of cancer, cardiovascular disease,
sleep apnea, psychiatric disorder, and type 2 diabetes (T2D) [2–6]. In parallel to the growing
rate of obesity, the incidence of T2D has also dramatically increased
worldwide, and it is believed that more than 366 million people will become
diabetic by 2030 [1]. Recent findings point toward visceral
adiposity as a causal link for the development of obesity-induced T2D. Visceral adipose tissue (VAT) is
metabolically more active than subcutaneous adipose tissue and secretes a
number of adipokines [7, 8]. The surgical removal of as little as 0.6 kg (0.8% total fat) of VAT significantly ameliorated insulin sensitivity in
obese patients [9], whereas removal of large amounts of
subcutaneous adipose tissue did not [10]. These findings suggested that adipose tissue
can no longer be considered an inert tissue strictly involved in lipid storage
but is rather an endocrine organ capable of regulating several aspects of
metabolism.Currently, a few
drugs are approved for the long-term treatment of obesity but their efficacy is
limited. Both sibutramine (Meridia) and orlistat (Xenical) have been available
for approximately 10 years for the treatment of obesity but these agents only
induce modest one-year weight loss of 4.2% and 2.9%, respectively [11]. More recently, the
cannabinoid receptor-1(CB1) inverse agonist, rimonabant, has been associated
with significant weight loss in several global clinical trials, including
RIO-Europe, RIO-Lipids, RIO-Diabetes, and RIO-North America [11]. Although rimonabant induced
an approximately 5% placebo-adjusted weight reduction in those studies, it was
not approved in the USA principally due to neurological and
psychiatric safety concerns [12]. Recently, the antidiabetic GLP-1
agonist exenatide (Byetta) was shown to significantly reduce mean body weight
(−4.7%) from baseline after 2 years of treatment [13]. This drug offers benefits
over existing therapies as it combines weight loss and improvements in glycosylated
hemoglobin (HbA1c), liver function, and blood pressure [13]. Side effects including
nausea and vomiting have been noted [14] and rare cases of acute
pancreatitis have been reported [15]. However, exenatide is currently
not approved for the treatment of obesity.With no more than
5% placebo-adjusted weight loss observed with currently available treatments,
more drugs are thus needed to provide patients with safe and efficacious
therapeutic options. Recent findings have suggested that peroxisome
proliferator-activated receptor (PPAR) delta agonists could induce body weight
loss by increasing energy expenditure and fatty acid oxidation [16]. In fact, PPARδ agonism either alone or in combination
with agonism of PPARα and/or PPARγcould not only induce body weight loss but could also potentially improve other
metabolic parameters including insulin sensitivity and lipid profiles, thus
producing benefits associated with the comorbidities of the obese state.


## 2. Mechanism of Action of PPARs

PPARs belong to
the nuclear receptor family of transcription factors and are master regulators
of genes involved in glucose and lipid metabolism. The PPARs are modular in
nature, containing an amino-terminal ligand-independent AF1 transactivation
domain, a DNA binding domain containing two zinc finger motifs, and a
carboxy-terminal domain that contains a dimerization domain and a ligand-dependent
AF-2 transactivation domain [17]. There are three members of
the PPAR family, PPARα, PPARγ, and PPARδ [18]. They are 60–80% conserved in
their DNA and ligand-binding domains and are activated by a number of natural
and synthetic ligands, including eicosanoids, free fatty acids (FFA), lipid
lowering drugs (fibrates), and the insulin sensitizers (thiazolidinediones).
PPARs heterodimerize with the retinoic X receptor (RXR) and bindspecific
promoter sequences termed PPAR responsive elements (PPREs) in their target
genes. In the absence of a ligand, PPARs form complexes with corepressors such
as NcoR, RIP140, and SMRT [19–21], which repress transcription
through the recruitment of histone deacetylases. Ligand binding induces a
conformational change that results in the dissociation of corepressors and the
recruitment of coactivators, such as PPARγ coactivator-1 (PGC-1), mediator
complex, P300/CBP [22–24]. The specificity of complexes
formed between the receptor and corepressors or coactivators appears to be
determined by cell type, conformational change induced by the ligand, and
sequence of the DNA binding element. This level of complexity allows
fine-tuning of the physiological response and explains the variability of gene
expression changes when a receptor is activated by different ligands.


### 2.1. PPARγ

PPARγ is the molecular target of
thiazolidinediones (TZDs) and has been well characterized in obese diabetic
rodent models and in diabetic patients. It is primarily expressed in adipocytes,
where it plays a pivotal role in the regulation of adipocyte differentiation
and lipid storage. Treatment of diabetic animals or patients with TZDs
increases subcutaneous fat mass area by inducing the number of small insulin
sensitive adipocytes in subcutaneous fat [25–28], while decreasing visceral
fat mass [29, 30]. This effect of TZDs most
likely contributes to the improvement of insulin sensitivity, although a direct
effect of PPARγ on skeletal muscle, liver, and
macrophages has also been demonstrated. For example, it has been reported that
the insulin sensitizing effect of rosiglitazone was attenuated in
macrophage-specific PPARγ knockout mice, suggesting that
macrophages are important for TZD-induced insulin sensitivity [31]. TZD treatment has also been
associated with significant improvement of skeletal muscle, liver, and adipose
tissue insulin signaling in both rodents and humans [30, 32, 33]. Additionally, PPARγ agonists increase the expression of
adiponectin, which decreases hepatic glucose production through activation of
AMP-activated protein kinase (AMPK) [34] and could thus contribute to
TZD-induced improvement in insulin sensitivity.


### 2.2. PPARα

PPARα is predominantly expressed in highly
oxidative tissues, including liver, skeletal muscle, brown adipose tissue, and
heart [35, 36] and is the target of the
fibrate class of drugs used for the treatment of dyslipidemia. Fibrates
decrease triglyceride levels in both rodents and humans by depleting the pool
of free fatty acids (FFAs) through peroxisomal and mitochondrial fatty acid
oxidation. PPARα activation has been associated with
increased expression of genes involved in all steps of fatty acid utilization,
including the breakdown of triglyceride particles to free fatty acids by
lipoprotein lipase (LPL), the transport of free fatty acids inside the cells by
fatty acid transport proteins (FATPs), and the peroxisomal and mitochondrial β-oxydation by activation of acyl CoA
oxydase (ACO) and medium chain acyl CoA decarboxylase (MCAD). In addition, PPARα is involved in the reverse cholesterol
transport pathway and contributes to HDL production in humans by increasing the
expression of the major constituent of high-density lipoprotein (HDL)
cholesterol, apolipoprotein A1 (ApoA1) [37], as well as increasing the
ATP-binding cassette transporter A1 (ABCA1) [37].Recently, PPARα has also been implicated in the
regulation of body weight through appetite suppression. In rodents, PPARα agonists, including WY-14643,
fenofibrate, and oleoylethanolamide (OEA), have been reported to induce body
weight loss in a PPARα-specific manner [38, 39]. One of the proposed mechanisms
involves stimulation of specific regions of the brain controlling satiety
through [40] vagal nerve activation.
Interestingly, PPARα agonists can no longer induce appetite
suppression in rats in which the vagal nerve has been severed [38]. It is also possible that
PPARα induces appetite suppression through
other mechanisms, including FGF-21 secretion [41–43] or increased fatty acid
oxidation and production of ketone bodies [44, 45].


### 2.3. PPARδ

PPARδ has recently been characterized in
rodents and humans and is more widely distributed than its two counterparts
with high level of expression in skeletal muscle, heart, kidney, adipose
tissue, liver, and macrophages [46]. The physiological role of
PPARδ has been elucidated not only  through genetic approaches, but also by
pharmacological activation of the receptor with specific agonists, including
GW501516. A major role for PPARδ in body weight regulation has been
reported in mice overexpressing a constitutively active form of PPARδ in adipose tissue. These mice are
resistant to diet-induced obesity (DIO) and are protected against the
development of genetically induced obesity through induction of energy
expenditure via mitochondrial fatty acid oxidation and energy uncoupling [16]. It is also possible that
PPARδ induces body weight loss by centrally
regulating appetite suppression since PPARδ was shown to mediate the hyperphagic
response following focal cerebral ischemia [47].In addition to
inducing body weight loss, PPARδ activation has been shown to improve
lipid profile by depleting the pool of fatty acids through mitochondrial fatty
acid oxidation [16, 48] and by increasing reverse cholesterol
transport [49]. Two-week treatment of
healthy volunteers with GW501516 significantly increased HDLc and decreased
triglycerides as compared to placebo-treated subjects [50]. PPARδ expression is upregulated in skeletal
muscle of T2D subjects and high-fat fed rats after bouts of exercise [46, 51–54] and may play a role in providing
a continuous source of energy to support the increasing energy demand.
Consistent with this hypothesis, skeletal muscle overexpression of PPARδ was associated with significant muscle-type
switching from glycolytic to oxidative fibers, further supporting its role in
lipid oxidation [55]. Interestingly, skeletal
muscle PPARδ transgenic mice were more resistant to
fatigue after intense exercise [55], suggesting a PPARδ-induced switch in substrate utilization
that helped maintain a higher level of
energy demand.In addition to
inducing weight loss and improving lipid profiles, PPARδ agonism has the potential to improve
insulin sensitivity and glucose metabolism [46, 56]. Recently, this has been shown to include a
crucial role for PPARδ expressed in tissue macrophages [57, 58]. Activation of resident
macrophages in metabolic tissues toward the inflammatory (M1) phenotype appears
to play a role in insulin resistance as a result of obesity. However, through
production of Th2 cytokines, both adipocytes and hepatocytes are able to induce
PPARδ expression in resident macrophages
leading to their alternative activation toward the anti-inflammatory M2
phenotype. This then results in increased insulin sensitivity in these tissues,
and could underlie some of the improvements in glucose metabolism seen in PPARδ-treated animals.


## 2.1. PPARγ

PPARγ is the molecular target of
thiazolidinediones (TZDs) and has been well characterized in obese diabetic
rodent models and in diabetic patients. It is primarily expressed in adipocytes,
where it plays a pivotal role in the regulation of adipocyte differentiation
and lipid storage. Treatment of diabetic animals or patients with TZDs
increases subcutaneous fat mass area by inducing the number of small insulin
sensitive adipocytes in subcutaneous fat [25–28], while decreasing visceral
fat mass [29, 30]. This effect of TZDs most
likely contributes to the improvement of insulin sensitivity, although a direct
effect of PPARγ on skeletal muscle, liver, and
macrophages has also been demonstrated. For example, it has been reported that
the insulin sensitizing effect of rosiglitazone was attenuated in
macrophage-specific PPARγ knockout mice, suggesting that
macrophages are important for TZD-induced insulin sensitivity [31]. TZD treatment has also been
associated with significant improvement of skeletal muscle, liver, and adipose
tissue insulin signaling in both rodents and humans [30, 32, 33]. Additionally, PPARγ agonists increase the expression of
adiponectin, which decreases hepatic glucose production through activation of
AMP-activated protein kinase (AMPK) [34] and could thus contribute to
TZD-induced improvement in insulin sensitivity.


## 2.2. PPARα

PPARα is predominantly expressed in highly
oxidative tissues, including liver, skeletal muscle, brown adipose tissue, and
heart [35, 36] and is the target of the
fibrate class of drugs used for the treatment of dyslipidemia. Fibrates
decrease triglyceride levels in both rodents and humans by depleting the pool
of free fatty acids (FFAs) through peroxisomal and mitochondrial fatty acid
oxidation. PPARα activation has been associated with
increased expression of genes involved in all steps of fatty acid utilization,
including the breakdown of triglyceride particles to free fatty acids by
lipoprotein lipase (LPL), the transport of free fatty acids inside the cells by
fatty acid transport proteins (FATPs), and the peroxisomal and mitochondrial β-oxydation by activation of acyl CoA
oxydase (ACO) and medium chain acyl CoA decarboxylase (MCAD). In addition, PPARα is involved in the reverse cholesterol
transport pathway and contributes to HDL production in humans by increasing the
expression of the major constituent of high-density lipoprotein (HDL)
cholesterol, apolipoprotein A1 (ApoA1) [37], as well as increasing the
ATP-binding cassette transporter A1 (ABCA1) [37].Recently, PPARα has also been implicated in the
regulation of body weight through appetite suppression. In rodents, PPARα agonists, including WY-14643,
fenofibrate, and oleoylethanolamide (OEA), have been reported to induce body
weight loss in a PPARα-specific manner [38, 39]. One of the proposed mechanisms
involves stimulation of specific regions of the brain controlling satiety
through [40] vagal nerve activation.
Interestingly, PPARα agonists can no longer induce appetite
suppression in rats in which the vagal nerve has been severed [38]. It is also possible that
PPARα induces appetite suppression through
other mechanisms, including FGF-21 secretion [41–43] or increased fatty acid
oxidation and production of ketone bodies [44, 45].


## 2.3. PPARδ

PPARδ has recently been characterized in
rodents and humans and is more widely distributed than its two counterparts
with high level of expression in skeletal muscle, heart, kidney, adipose
tissue, liver, and macrophages [46]. The physiological role of
PPARδ has been elucidated not only  through genetic approaches, but also by
pharmacological activation of the receptor with specific agonists, including
GW501516. A major role for PPARδ in body weight regulation has been
reported in mice overexpressing a constitutively active form of PPARδ in adipose tissue. These mice are
resistant to diet-induced obesity (DIO) and are protected against the
development of genetically induced obesity through induction of energy
expenditure via mitochondrial fatty acid oxidation and energy uncoupling [16]. It is also possible that
PPARδ induces body weight loss by centrally
regulating appetite suppression since PPARδ was shown to mediate the hyperphagic
response following focal cerebral ischemia [47].In addition to
inducing body weight loss, PPARδ activation has been shown to improve
lipid profile by depleting the pool of fatty acids through mitochondrial fatty
acid oxidation [16, 48] and by increasing reverse cholesterol
transport [49]. Two-week treatment of
healthy volunteers with GW501516 significantly increased HDLc and decreased
triglycerides as compared to placebo-treated subjects [50]. PPARδ expression is upregulated in skeletal
muscle of T2D subjects and high-fat fed rats after bouts of exercise [46, 51–54] and may play a role in providing
a continuous source of energy to support the increasing energy demand.
Consistent with this hypothesis, skeletal muscle overexpression of PPARδ was associated with significant muscle-type
switching from glycolytic to oxidative fibers, further supporting its role in
lipid oxidation [55]. Interestingly, skeletal
muscle PPARδ transgenic mice were more resistant to
fatigue after intense exercise [55], suggesting a PPARδ-induced switch in substrate utilization
that helped maintain a higher level of
energy demand.In addition to
inducing weight loss and improving lipid profiles, PPARδ agonism has the potential to improve
insulin sensitivity and glucose metabolism [46, 56]. Recently, this has been shown to include a
crucial role for PPARδ expressed in tissue macrophages [57, 58]. Activation of resident
macrophages in metabolic tissues toward the inflammatory (M1) phenotype appears
to play a role in insulin resistance as a result of obesity. However, through
production of Th2 cytokines, both adipocytes and hepatocytes are able to induce
PPARδ expression in resident macrophages
leading to their alternative activation toward the anti-inflammatory M2
phenotype. This then results in increased insulin sensitivity in these tissues,
and could underlie some of the improvements in glucose metabolism seen in PPARδ-treated animals.


## 3. Challenges to the Development of PPARδ Modulators

Following the
publication of several high-profile studies implicating PPARδ as a potential target for obesity and
associated metabolic disorders, multiple pharmaceutical companies have
initiated drug discovery efforts to identify specific PPARδ, PPARα/δ, PPARγ/δ, and pan PPAR modulators. However,
development of these PPAR agonists faces significant challenges including major
differences in PPAR biology between rodents and humans, difficulties in
predicting in vivo efficacy from simple in vitro models of activity,
toxicological findings arising from weight reduction through targeting β-oxidation of fatty acids, and safety
requirements unique to PPAR biology and drug development.


### 3.1. Differences
in PPAR Pharmacology Between Rodents and Humans

Despite very
high homology between human and rodent PPAR receptors, major biological and
physiological differences exist between these species. One clear and
well-understood example relates to peroxisome proliferation and liver
enlargement with PPARα agonists. In rodents, but not humans,
PPARα activation leads to hepatic peroxisome
proliferation associated with liver enlargement and cell necrosis. One
potential biological explanation for these differences is that hepatic PPARα expression is approximately 10 times
higher in rodents versus humans [59].Similarly, it has been
reported that PPARδ
pharmacology differs between higher species and rodents. Contradicting reports
suggest that pharmacological PPARδ
activation inconsistently regulates body weight [55, 60, 61] and cholesterol or triglyceride levels in rodents [55, 60–62]. However, significant elevation of HDL cholesterol
and/or reduction of LDL cholesterol and triglyceride have been reported in
obese rhesus monkeys and humans after chronic PPARδ agonist
administration [50, 63]. One potential explanation may reside in the
observation that the rodent ApoA-1 promoter differs
from the human promoter by 3 nucleotides, resulting in a nonfunctional PPRE
site [64, 65]. Interestingly, PPARδ
agonists have been shown to decrease triglyceride and increase HDL cholesterol
in humanized ApoA-1 transgenic mice [66] suggesting that mice can positively respond to PPARδ
activation once provided with a functional ApoA1 gene. Overall, these species
differences could make the observation of PPARδ driven
outcomes more difficult in rodents, and thus complicate the development of PPARδ
modulators.


### 3.2. Complexity Induced by Ligand-Specific Effects

Traditionally, the
potency and affinity of PPAR modulators have been determined using multiple
assays, including displacement of radiolabeled compound, displacement and
recruitment of corepressors and coactivators, and cell-based transcriptional
activation assays. Often, the in vitro potency in binding and transcriptional
activation assays correlates with in vivo efficacy, a notable example is the
hypoglycemic effects of TZDs. TZDs with more potent in vitro activities are
often more active in rodent models [67, 68]. In some cases, these correlations do not hold,
reflecting a more complex relationship between simple in vitro measures and in
vivo pharmacology that is best understood in the context of the selective PPAR
modulator (SPPARM) hypothesis. The SPPARM concept emerged to describe the
complete spectrum of PPAR conformation/activation states leading to each
specific biological response. This model stipulates that PPAR modulators turn
on and off specific genes by recruiting or releasing a complex assortment of
coactivators and corepressors. The coactivators and corepressors are likely
tissue specific and different modulators are believed to recruit different sets
of proteins leading to various degree of gene activation/repression. This level
of complexity provides a potential strategy to eliminate the known liabilities
associated with PPAR activation by selecting modulators that are
recruiting/releasing specific sets of coactivators and corepressors. However,
this complexity also complicates the development of PPAR agonists because
species differences can exist concerning coactivator expression and
recruitment, tissue specificity and availability.Consistent with the
SPPARM hypothesis, Berger et al. reported that PPARγ/δ agonists
L-165461 and L-783483 had similar binding affinities and transcriptional
activities against PPARγ and
PPARδ while
the glucose lowering effect was more pronounced with L-783483 in db/db mice [67]. We have observed that PPAR pan modulators with
similar profiles in transcriptional cell-based assays can yield significantly
different in vivo activities (Table 1) despite similar pharmacokinetic
properties. These differences likely stem from differential interactions with
the ligand-binding pocket of each PPAR resulting in modulator-specific
differential recruitment of coactivators/corepressors.Table 1
Compounds with similar in vitro profiles induced
different in vivo efficacy.
CompoundIn vitro potentcy (μM)Weight lossInsulin (% reduction)Adiponectin (fold increase)αγδ1 [69, 70]6.00.15.0Weight neutral30%2.12 [69, 70]3.00.11.014%80%3.2


### 3.3. Challenges
Associated with the Development of Weight-Reducing Agents Targeting Oxidation of Fatty Acids

It is well
understood that body weight is physiologically regulated through two major
mechanisms: food intake and energy expenditure. Energy expenditure can be
positively or negatively modulated by regulating metabolic rate, body
temperature, or level of physical activity. Targeting metabolic rate through
oxidation of free fatty acids and/or energy uncoupling are effective ways to
induce body weight loss. For example, it has been shown that targeted deletion
of acetyl-CoA carboxylase-2 (ACC2), an enzyme responsible for the synthesis of
malonyl-CoA, is associated with approximately 10–20% body weight
loss caused by significant increase in total energy expenditure, with no change
[71] or increased food consumption
[72]. The increased energy
expenditure was mostly explained by a significant increase in fatty acid
oxidation and not an elevation of the level of physical activity [71, 72]. Similarly, transgenic mice
with muscle-specific activation of PPARα are resistant to diet-induced obesity
despite consuming similar amounts of food compared to nontransgenic mice [73]. As expected, the rate of
palmitate oxidation and the level of expression of genes regulating
mitochondrial and peroxisomal β-oxidation were increased in muscle-specific
PPARα transgenic mice [73]. Overall, these
studies provided convincing evidence that specifically targeting oxidation of
fatty acids is a relevant mechanism to induce body weight loss.PPARδ is known to influence all of the
parameters regulating energy expenditure by increasing the transport and
oxidation of free fatty acids in adipose tissue and skeletal muscle, increasing
thermogenesis and energy uncoupling in adipose tissue and inducing muscle fiber-type
switching to promote endurance and resistance to fatigue. Recently, PPARδ has been suggested as a potential
target for body weight loss because of its role in fatty acid oxidation. In
mouse and human skeletal muscle, PPARδ activation increases the expression of
several genes involved in mitochondrial oxidation of free fatty acids,
including carnitine palmitoyltransferase-1 (CPT-1), medium chain acyl-CoA
dehydrogenase (MCAD), long chain acyl-CoA dehydrogenase (LCAD), and PGC-1α [16, 48, 55, 74] and its pharmacological
activation has been associated with body weight loss in rodents [48, 55].Currently,
controversies exist as to whether an increased rate of fatty acid oxidation is
beneficial. Numerous studies have been published demonstrating that an elevated
rate ofβ-oxidation is beneficial because it
depletes the pool of fatty acids required to synthesize triglyceride particles,
prevents accumulation of fatty acid metabolites in skeletal muscle and other
nonfat tissues, and induces body weight loss. However, it has been shown that
sustained level of fatty acid oxidation, through long-term fibrate treatment or
PPARα overexpression, is associated with
skeletal and heart muscle degeneration [75], cardiomyopathy [76, 77], and insulin resistance [73] in mice. Consistent with
these reports, we have previously observed that pan PPAR activation induced
significant levels of skeletal muscle degeneration and liver vacuolation and
necrosis in mice, but these observations were completely absent in PPARα knockout animals (M. Perreault et al.,
unpublished observations). Collectively, these results suggest that sustained
levels of fatty acid oxidation through PPARα and potentially PPARδ could significantly improve some
metabolic parameters but with potential deleterious effects on muscle and
liver.It has been
proposed that the detrimental effects of sustained fatty acid oxidation result
from the production of reactive oxygen species produced during peroxisomal and
mitochondrial oxidation of fatty acids. The first and rate-limiting step of
peroxisomalβ-oxidation, ACO, generates hydrogen
peroxide during oxidation of acyl-CoAs. It has been reported that hydrogen
peroxide levels are significantly increased in the heart of cardiac-specific
PPARα transgenic mice and this effect is
exacerbated on a high-fat diet, where the substrates for fatty acid oxidation
are more abundant [76]. Similarly, oxidation of
fatty acids through mitochondrial β-oxidation has been associated with an
increased production of reactive oxygen species (ROSs) as long-chain fatty
acids provide reducing equivalents that fuel the electron transport chain.While the role of PPARα in skeletal and cardiac muscle
degeneration has been established in rodents, the effect of PPARδ activation on these parameters is less
clear. It has been reported that PPARδ activates distinct metabolic programs
in the mouse heart as compared to PPARα, leading to cardioprotection in the
setting of myocardial ischemia/reperfusion injury [77]. The exact mechanism for the
cardioprotection is currently unknown, but in contrast to PPARα, PPARδ overexpression induced cardiac glucose
oxidation as opposed to fatty acid oxidation [77]. Normal hearts exhibit
substrate flexibility by switching between lipid and glucose to match the
metabolic state. However, diabetic hearts mostly rely on fatty acids, leading
to excessive rates of myocardial fatty acid oxidation concomitant with reduced
glucose oxidation. In the heart of cardiac-specific PPARα transgenic mice, genes involved in
fatty acid uptake, lipogenesis, and triglyceride synthesis (fatty acid
transport protein (FATP), CD36, glycerol-3-phosphate acyltransferase (GPAT),
acyl-CoA synthetase (ACS), fatty acid synthase (FAS), microsomal triglyceride
transfer protein (MTP)) were significantly elevated while genes involved in
glucose metabolism (glucose transporter 4 (GLUT4), phosphofructokinase (PFK))
were not. In contrast, the expression of fatty acid transport and
esterification genes was unchanged in cardiac-specific PPARδ transgenic mice while genes regulating
glucose metabolism were significantly elevated. These results further
contribute to the hypothesis suggesting that increased and sustained rate of
fatty acid oxidation could be detrimental for cardiac functions and suggest
that this mechanism is PPARα-specific. Furthermore, it has been
reported that PPARδ activation is associated with elevated
expression of catalase [78], an enzyme responsible for
hydrogen peroxide degradation potentially providing an additional protective
effect.Recently, the concept of incomplete fatty acid oxidation
has emerged as another potential explanation for fatty acid oxidation-induced
metabolic disturbances. It was proposed that obesity results in an excessive
fatty acid load on mitochondria causing accumulation of incompletely oxidized
intermediates, including acylcarnitine esters, while decreasing levels of
metabolites of the tricarboxylic acid (TCA) cycle [79]. The exact mechanism leading
to metabolic disturbances is still unknown and whether acylcarnitine esters
directly induce insulin resistance and metabolic disorders remains to be
determined.


### 3.4. Safety Study Requirements Unique to PPAR Biology
and Drug Development

PPAR gamma (TZDs) and alpha (fibrates) agonists have been
used for many years to treat type 2 diabetic and dyslipidemic patients,
respectively. TZDs (Avandia and Actos) are very well characterized in rodents
and humans and result in significant improvements in insulin sensitivity and
glycemic control but are associated with increases in body weight and fluid
retention that can exacerbate congestive heart failure. The liabilities
associated with PPARγ agonists are observed in a small but
significant number of patients and are very closely related to their efficacy,
as reduction in HbA1c is directly correlated to body weight gain [80]. PPARγ-associated weight gain and fluid
retention liabilities are well understood and can be reversed by discontinuing
drug administration or by treating with diuretics.Recently, the PPARγ activator rosiglitazone has been
suggested to directly affect cardiac function independent from its effects on
fluid retention. A number of meta-analyses concluded that rosiglitazone
significantly increased the rates of myocardial infarction and death by
cardiovascular causes [81, 82]. However, controversies exist
as several recent studies refuting those results have been published [83, 84]. Interestingly, a significant
lower risk of death, myocardial infarction and stroke was observed with
pioglitazone (Actos) [85], suggesting a difference
between rosiglitazone and pioglitazone in relation to cardiovascular effects.
Pioglitazone has been previously shown to improve lipid profile (triglyceride,
LDL and HDL cholesterol) by weakly activating PPARα, while rosiglitazone seems to worsen
these parameters which could contribute to the deleterious effects on cardiac
functions [86–88]. The findings from these
meta-analyses will have to be confirmed with cardiovascular outcome studies, as
the clinical trials included in these analyses were not originally designed to
evaluate cardiovascular endpoints. The RECORD study is a clinical trial
evaluating the effects of rosiglitazone in approximately 2000 patients to
specifically address cardiovascular effects as primary outcomes. The interim
analysis indicated no significant increased myocardial infarction and death
with rosiglitazone, however, the authors admitted that their study might be
underpowered due to lower cardiovascular events in this patient population and
that the analysis was performed before completion of the study [89–92].Whether the cardiovascular effects of rosiglitazone and
pioglitazone are confirmed or not with clinical outcome studies, these recent
findings have changed the way regulatory agencies review and approve drugs and
protein therapeutics for the treatment of T2D. Earlier this year, the FDA
published their draft guidance for the development of new diabetes
therapeutics. The guidance affirms that in the absence of cardiovascular
signal, long-term cardiovascular studies could be conducted postapproval in a
reasonable timeframe [93]. However, large outcome
trials should be conducted prior to submission of regulatory dossiers for drugs
that show nonclinical or clinical evidence of increasing cardiovascular risk [93].In addition to the cardiovascular effects, multiple PPAR
modulators have been discontinued over the last several years due to
carcinogenicity findings in rodents. Because of the prevalence of positive
carcinogenicity findings with PPAR agonists and the lack of complete
understanding of PPAR-induced tumor development, 2-year carcinogenicity studies
in mice and rats are now required before clinical trials longer than 6 months
in duration can be initiated [93, 94]. Moreover, PPARγ activation has recently been linked to
an increased risk of fracture in humans [95–97]. It has been proposed that PPARγ activators could have a direct effect
on osteoblastogenesis [98, 99] and osteoclastogenesis [100] in rodents, but further
clinical studies will be necessary to understand if these mechanisms are also
involved in TZDs-induced bone loss in humans. Despite an increased risk of
fracture with PPARγ activators in humans, the role of other
PPAR isoforms on bone formation and resorption is less clear. The presence of
PPARδ both at protein and mRNA levels has
been reported in rat bone tissue sections, preosteoblasts, rodent and human
osteoblastic cell lines as well as rabbit osteoclasts [101–104], while PPARα has been found in preosteoblasts,
chondrocytes, and human peripheral blood mononuclear cell (PBMC)-derived
osteoclasts [101]. The role of PPARα and PPARδ in bone formation and resorption needs
to be further determined.


## 3.1. Differences
in PPAR Pharmacology Between Rodents and Humans

Despite very
high homology between human and rodent PPAR receptors, major biological and
physiological differences exist between these species. One clear and
well-understood example relates to peroxisome proliferation and liver
enlargement with PPARα agonists. In rodents, but not humans,
PPARα activation leads to hepatic peroxisome
proliferation associated with liver enlargement and cell necrosis. One
potential biological explanation for these differences is that hepatic PPARα expression is approximately 10 times
higher in rodents versus humans [59].Similarly, it has been
reported that PPARδ
pharmacology differs between higher species and rodents. Contradicting reports
suggest that pharmacological PPARδ
activation inconsistently regulates body weight [55, 60, 61] and cholesterol or triglyceride levels in rodents [55, 60–62]. However, significant elevation of HDL cholesterol
and/or reduction of LDL cholesterol and triglyceride have been reported in
obese rhesus monkeys and humans after chronic PPARδ agonist
administration [50, 63]. One potential explanation may reside in the
observation that the rodent ApoA-1 promoter differs
from the human promoter by 3 nucleotides, resulting in a nonfunctional PPRE
site [64, 65]. Interestingly, PPARδ
agonists have been shown to decrease triglyceride and increase HDL cholesterol
in humanized ApoA-1 transgenic mice [66] suggesting that mice can positively respond to PPARδ
activation once provided with a functional ApoA1 gene. Overall, these species
differences could make the observation of PPARδ driven
outcomes more difficult in rodents, and thus complicate the development of PPARδ
modulators.


## 3.2. Complexity Induced by Ligand-Specific Effects

Traditionally, the
potency and affinity of PPAR modulators have been determined using multiple
assays, including displacement of radiolabeled compound, displacement and
recruitment of corepressors and coactivators, and cell-based transcriptional
activation assays. Often, the in vitro potency in binding and transcriptional
activation assays correlates with in vivo efficacy, a notable example is the
hypoglycemic effects of TZDs. TZDs with more potent in vitro activities are
often more active in rodent models [67, 68]. In some cases, these correlations do not hold,
reflecting a more complex relationship between simple in vitro measures and in
vivo pharmacology that is best understood in the context of the selective PPAR
modulator (SPPARM) hypothesis. The SPPARM concept emerged to describe the
complete spectrum of PPAR conformation/activation states leading to each
specific biological response. This model stipulates that PPAR modulators turn
on and off specific genes by recruiting or releasing a complex assortment of
coactivators and corepressors. The coactivators and corepressors are likely
tissue specific and different modulators are believed to recruit different sets
of proteins leading to various degree of gene activation/repression. This level
of complexity provides a potential strategy to eliminate the known liabilities
associated with PPAR activation by selecting modulators that are
recruiting/releasing specific sets of coactivators and corepressors. However,
this complexity also complicates the development of PPAR agonists because
species differences can exist concerning coactivator expression and
recruitment, tissue specificity and availability.Consistent with the
SPPARM hypothesis, Berger et al. reported that PPARγ/δ agonists
L-165461 and L-783483 had similar binding affinities and transcriptional
activities against PPARγ and
PPARδ while
the glucose lowering effect was more pronounced with L-783483 in db/db mice [67]. We have observed that PPAR pan modulators with
similar profiles in transcriptional cell-based assays can yield significantly
different in vivo activities (Table 1) despite similar pharmacokinetic
properties. These differences likely stem from differential interactions with
the ligand-binding pocket of each PPAR resulting in modulator-specific
differential recruitment of coactivators/corepressors.Table 1
Compounds with similar in vitro profiles induced
different in vivo efficacy.
CompoundIn vitro potentcy (μM)Weight lossInsulin (% reduction)Adiponectin (fold increase)αγδ1 [69, 70]6.00.15.0Weight neutral30%2.12 [69, 70]3.00.11.014%80%3.2


## 3.3. Challenges
Associated with the Development of Weight-Reducing Agents Targeting Oxidation of Fatty Acids

It is well
understood that body weight is physiologically regulated through two major
mechanisms: food intake and energy expenditure. Energy expenditure can be
positively or negatively modulated by regulating metabolic rate, body
temperature, or level of physical activity. Targeting metabolic rate through
oxidation of free fatty acids and/or energy uncoupling are effective ways to
induce body weight loss. For example, it has been shown that targeted deletion
of acetyl-CoA carboxylase-2 (ACC2), an enzyme responsible for the synthesis of
malonyl-CoA, is associated with approximately 10–20% body weight
loss caused by significant increase in total energy expenditure, with no change
[71] or increased food consumption
[72]. The increased energy
expenditure was mostly explained by a significant increase in fatty acid
oxidation and not an elevation of the level of physical activity [71, 72]. Similarly, transgenic mice
with muscle-specific activation of PPARα are resistant to diet-induced obesity
despite consuming similar amounts of food compared to nontransgenic mice [73]. As expected, the rate of
palmitate oxidation and the level of expression of genes regulating
mitochondrial and peroxisomal β-oxidation were increased in muscle-specific
PPARα transgenic mice [73]. Overall, these
studies provided convincing evidence that specifically targeting oxidation of
fatty acids is a relevant mechanism to induce body weight loss.PPARδ is known to influence all of the
parameters regulating energy expenditure by increasing the transport and
oxidation of free fatty acids in adipose tissue and skeletal muscle, increasing
thermogenesis and energy uncoupling in adipose tissue and inducing muscle fiber-type
switching to promote endurance and resistance to fatigue. Recently, PPARδ has been suggested as a potential
target for body weight loss because of its role in fatty acid oxidation. In
mouse and human skeletal muscle, PPARδ activation increases the expression of
several genes involved in mitochondrial oxidation of free fatty acids,
including carnitine palmitoyltransferase-1 (CPT-1), medium chain acyl-CoA
dehydrogenase (MCAD), long chain acyl-CoA dehydrogenase (LCAD), and PGC-1α [16, 48, 55, 74] and its pharmacological
activation has been associated with body weight loss in rodents [48, 55].Currently,
controversies exist as to whether an increased rate of fatty acid oxidation is
beneficial. Numerous studies have been published demonstrating that an elevated
rate ofβ-oxidation is beneficial because it
depletes the pool of fatty acids required to synthesize triglyceride particles,
prevents accumulation of fatty acid metabolites in skeletal muscle and other
nonfat tissues, and induces body weight loss. However, it has been shown that
sustained level of fatty acid oxidation, through long-term fibrate treatment or
PPARα overexpression, is associated with
skeletal and heart muscle degeneration [75], cardiomyopathy [76, 77], and insulin resistance [73] in mice. Consistent with
these reports, we have previously observed that pan PPAR activation induced
significant levels of skeletal muscle degeneration and liver vacuolation and
necrosis in mice, but these observations were completely absent in PPARα knockout animals (M. Perreault et al.,
unpublished observations). Collectively, these results suggest that sustained
levels of fatty acid oxidation through PPARα and potentially PPARδ could significantly improve some
metabolic parameters but with potential deleterious effects on muscle and
liver.It has been
proposed that the detrimental effects of sustained fatty acid oxidation result
from the production of reactive oxygen species produced during peroxisomal and
mitochondrial oxidation of fatty acids. The first and rate-limiting step of
peroxisomalβ-oxidation, ACO, generates hydrogen
peroxide during oxidation of acyl-CoAs. It has been reported that hydrogen
peroxide levels are significantly increased in the heart of cardiac-specific
PPARα transgenic mice and this effect is
exacerbated on a high-fat diet, where the substrates for fatty acid oxidation
are more abundant [76]. Similarly, oxidation of
fatty acids through mitochondrial β-oxidation has been associated with an
increased production of reactive oxygen species (ROSs) as long-chain fatty
acids provide reducing equivalents that fuel the electron transport chain.While the role of PPARα in skeletal and cardiac muscle
degeneration has been established in rodents, the effect of PPARδ activation on these parameters is less
clear. It has been reported that PPARδ activates distinct metabolic programs
in the mouse heart as compared to PPARα, leading to cardioprotection in the
setting of myocardial ischemia/reperfusion injury [77]. The exact mechanism for the
cardioprotection is currently unknown, but in contrast to PPARα, PPARδ overexpression induced cardiac glucose
oxidation as opposed to fatty acid oxidation [77]. Normal hearts exhibit
substrate flexibility by switching between lipid and glucose to match the
metabolic state. However, diabetic hearts mostly rely on fatty acids, leading
to excessive rates of myocardial fatty acid oxidation concomitant with reduced
glucose oxidation. In the heart of cardiac-specific PPARα transgenic mice, genes involved in
fatty acid uptake, lipogenesis, and triglyceride synthesis (fatty acid
transport protein (FATP), CD36, glycerol-3-phosphate acyltransferase (GPAT),
acyl-CoA synthetase (ACS), fatty acid synthase (FAS), microsomal triglyceride
transfer protein (MTP)) were significantly elevated while genes involved in
glucose metabolism (glucose transporter 4 (GLUT4), phosphofructokinase (PFK))
were not. In contrast, the expression of fatty acid transport and
esterification genes was unchanged in cardiac-specific PPARδ transgenic mice while genes regulating
glucose metabolism were significantly elevated. These results further
contribute to the hypothesis suggesting that increased and sustained rate of
fatty acid oxidation could be detrimental for cardiac functions and suggest
that this mechanism is PPARα-specific. Furthermore, it has been
reported that PPARδ activation is associated with elevated
expression of catalase [78], an enzyme responsible for
hydrogen peroxide degradation potentially providing an additional protective
effect.Recently, the concept of incomplete fatty acid oxidation
has emerged as another potential explanation for fatty acid oxidation-induced
metabolic disturbances. It was proposed that obesity results in an excessive
fatty acid load on mitochondria causing accumulation of incompletely oxidized
intermediates, including acylcarnitine esters, while decreasing levels of
metabolites of the tricarboxylic acid (TCA) cycle [79]. The exact mechanism leading
to metabolic disturbances is still unknown and whether acylcarnitine esters
directly induce insulin resistance and metabolic disorders remains to be
determined.


## 3.4. Safety Study Requirements Unique to PPAR Biology
and Drug Development

PPAR gamma (TZDs) and alpha (fibrates) agonists have been
used for many years to treat type 2 diabetic and dyslipidemic patients,
respectively. TZDs (Avandia and Actos) are very well characterized in rodents
and humans and result in significant improvements in insulin sensitivity and
glycemic control but are associated with increases in body weight and fluid
retention that can exacerbate congestive heart failure. The liabilities
associated with PPARγ agonists are observed in a small but
significant number of patients and are very closely related to their efficacy,
as reduction in HbA1c is directly correlated to body weight gain [80]. PPARγ-associated weight gain and fluid
retention liabilities are well understood and can be reversed by discontinuing
drug administration or by treating with diuretics.Recently, the PPARγ activator rosiglitazone has been
suggested to directly affect cardiac function independent from its effects on
fluid retention. A number of meta-analyses concluded that rosiglitazone
significantly increased the rates of myocardial infarction and death by
cardiovascular causes [81, 82]. However, controversies exist
as several recent studies refuting those results have been published [83, 84]. Interestingly, a significant
lower risk of death, myocardial infarction and stroke was observed with
pioglitazone (Actos) [85], suggesting a difference
between rosiglitazone and pioglitazone in relation to cardiovascular effects.
Pioglitazone has been previously shown to improve lipid profile (triglyceride,
LDL and HDL cholesterol) by weakly activating PPARα, while rosiglitazone seems to worsen
these parameters which could contribute to the deleterious effects on cardiac
functions [86–88]. The findings from these
meta-analyses will have to be confirmed with cardiovascular outcome studies, as
the clinical trials included in these analyses were not originally designed to
evaluate cardiovascular endpoints. The RECORD study is a clinical trial
evaluating the effects of rosiglitazone in approximately 2000 patients to
specifically address cardiovascular effects as primary outcomes. The interim
analysis indicated no significant increased myocardial infarction and death
with rosiglitazone, however, the authors admitted that their study might be
underpowered due to lower cardiovascular events in this patient population and
that the analysis was performed before completion of the study [89–92].Whether the cardiovascular effects of rosiglitazone and
pioglitazone are confirmed or not with clinical outcome studies, these recent
findings have changed the way regulatory agencies review and approve drugs and
protein therapeutics for the treatment of T2D. Earlier this year, the FDA
published their draft guidance for the development of new diabetes
therapeutics. The guidance affirms that in the absence of cardiovascular
signal, long-term cardiovascular studies could be conducted postapproval in a
reasonable timeframe [93]. However, large outcome
trials should be conducted prior to submission of regulatory dossiers for drugs
that show nonclinical or clinical evidence of increasing cardiovascular risk [93].In addition to the cardiovascular effects, multiple PPAR
modulators have been discontinued over the last several years due to
carcinogenicity findings in rodents. Because of the prevalence of positive
carcinogenicity findings with PPAR agonists and the lack of complete
understanding of PPAR-induced tumor development, 2-year carcinogenicity studies
in mice and rats are now required before clinical trials longer than 6 months
in duration can be initiated [93, 94]. Moreover, PPARγ activation has recently been linked to
an increased risk of fracture in humans [95–97]. It has been proposed that PPARγ activators could have a direct effect
on osteoblastogenesis [98, 99] and osteoclastogenesis [100] in rodents, but further
clinical studies will be necessary to understand if these mechanisms are also
involved in TZDs-induced bone loss in humans. Despite an increased risk of
fracture with PPARγ activators in humans, the role of other
PPAR isoforms on bone formation and resorption is less clear. The presence of
PPARδ both at protein and mRNA levels has
been reported in rat bone tissue sections, preosteoblasts, rodent and human
osteoblastic cell lines as well as rabbit osteoclasts [101–104], while PPARα has been found in preosteoblasts,
chondrocytes, and human peripheral blood mononuclear cell (PBMC)-derived
osteoclasts [101]. The role of PPARα and PPARδ in bone formation and resorption needs
to be further determined.


## 4. Summary and Perspectives

Obesity and associated disorders are serious diseases
affecting millions of people worldwide. The therapeutic options currently
offered are providing limited efficacy and are coupled with several serious
side effects. Despite significant challenges associated with their development,
PPAR modulators are potential new obesity therapies that could offer not only
weight management opportunities but also amelioration of the associated
disorders by correcting the causes of insulin resistance and dyslipidemia.
Molecules targeting PPARδ, alone or in combination with other
PPARs, offer significant advantages as PPARδ seems to have beneficial
cardioprotective effects in rodents by modulating fuel utilization in addition
to its anti-inflammatory and lipid effects. Whether or not such
weight-modulating therapies will be relevant to humans and ultimately
approvable by regulatory agencies remain to be determined.



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