**3. The PPAR family**

PPARs are members of the nuclear receptor superfamily of ligand-activated transcription factors that regulate the expression of genes involved in fatty acid uptake and oxidation, lipid metabolism and inflammation (Kersten et al., 2000). To be transcriptionally active, PPARs need to heterodimerize with the 9-*cis* retinoic acid receptor (RXR) (NR2B) (Figure 1). PPAR-RXR heterodimers bind to DNA-specific sequences called peroxisome proliferatorresponse elements (PPREs), consisting of an imperfect direct repeat of the consensus binding site for nuclear hormone receptors (AGGTCA) separated by one nucleotide (DR-1). These

of multiple metabolic abnormalities, including abdominal obesity, dyslipidemia (high serum triglycerides and low serum HDL-C levels), glucose intolerance and hypertension (Eckel et

It is now recognized that the atherogenic dyslipidemia is mainly initiated by the hepatic overproduction of the plasma lipoproteins carrying triglycerides, the VLDL, which induce a sequence of lipoprotein changes leading to atherogenic lipid abnormalities in type 2 diabetes mellitus and metabolic syndrome (Adiels et al., 2008). Under these pathological conditions, the presence of insulin resistance at the level of adipose tissue leads to enhanced lypolisis and reduced free fatty acid (FFA) uptake and esterification which results in increased flux into the liver of FFA, which are either oxidized or esterified for triglyceride production, leading to hepatic steatosis and oversecretion into plasma of larger triglyceriderich VLDL particles (Chan & Watts, 2011). These particles compete with chylomicrons and its remnants for clearance pathways regulated by lipoprotein lipase, an endothelial-bound enzyme, and by hepatic receptors, thereby exacerbating postprandial dyslipidemia. In addition, insulin resistance increases hepatic secretion of apoC-III, which is attached to VLDL delaying the catabolism of TRLs by inhibiting lipoprotein lipase and binding of remnant TRLs to hepatic clearance receptors (Chan & Watts, 2011). Finally, expansion of the VLDL triglyceride pool leads to cholesterol depletion and triglyceride enrichment of LDL and HDL through cholesteryl ester transfer protein, which facilitates the movement of cholesterol esters to VLDL, intermediate-density lipoprotein (IDL) and LDL from cholesterol ester rich HDL, leading to the accumulation in plasma of small, dense LDLs and a reduction

Since residual risk remains even after achieving an optimal LDL-C concentration with statins (Barter et al., 2007), probably due to other risk factors, such as high triglycerides, low HDL-C levels, defective glucose metabolism and other non-lipid-related risk factors (Kannel, 1983; Castelli, 1992; Lorenzo et al., 2010; Cederberg et al., 2010), the development of new drugs aimed at improving risk reduction is necessary. Among the new drugs for the treatment of the risk factors leading to the residual risk, PPARβ/δ activators might have a promising future. Interestingly, PPARβ/δ agonists have been demonstrated to be effective raising HDL-C and lowering triglyceride concentrations (Kersten, 2008). In addition to their lipid-modifying properties, PPARβ/δ agonists improve insulin resistance, which may also confer protection against the development of dyslipidemia (Coll et al., 2010a). This review summarizes the effects of PPARβ/δ on dyslipidemia identified during the last few years.

PPARs are members of the nuclear receptor superfamily of ligand-activated transcription factors that regulate the expression of genes involved in fatty acid uptake and oxidation, lipid metabolism and inflammation (Kersten et al., 2000). To be transcriptionally active, PPARs need to heterodimerize with the 9-*cis* retinoic acid receptor (RXR) (NR2B) (Figure 1). PPAR-RXR heterodimers bind to DNA-specific sequences called peroxisome proliferatorresponse elements (PPREs), consisting of an imperfect direct repeat of the consensus binding site for nuclear hormone receptors (AGGTCA) separated by one nucleotide (DR-1). These

**2. Hypertriglyceridemia is crucial in the pathogenesis of atherogenic** 

al., 2005; Grundy et al., 2005).

in HDLs (Taskinen, 2003).

**3. The PPAR family** 

**dyslipidemia** 

sequences have been characterized within the promoter regions of PPAR target genes. The binding occurs in such a way that PPAR is always oriented to the DNA's 5'-end, while RXR is to the 3'-end. In the absence of ligand, high-affinity complexes are formed between PPAR-RXR heterodimers and nuclear receptor co-repressor proteins, which block transcriptional activation by sequestering the heterodimer from the promoter. Binding of the ligand to PPAR induces a conformational change resulting in dissociation of co-repressor proteins, so that the PPAR-RXR heterodimer can then bind to PPREs. Moreover, once activated by the ligand, the heterodimer recruits co-activator proteins that promote the initiation of transcription (Feige et al., 2006). As a consequence of these changes in transcriptional activity, binding of ligands to the receptor results in changes in the expression level of mRNAs encoded by PPAR target genes. In a specific cellular context, the activity of PPARs regulating the transcription of their target genes depends on many factors (relative expression of the PPARs, the promoter context of the target gene, the presence of coactivator and co-repressor proteins, etc.).

Thus, the transcriptional activity of PPARs is modulated by co-activators and co-repressors (Feige et al., 2006). One of the best described PPAR co-activators is PPARγ coactivator 1 α (PGC-1α). Silencing mediator for retinoic and thyroid hormone receptor (SMRT) and the nuclear receptor co-repressor (NCoR) are co-repressors that interact with the PPARs in the absence of ligands (Zamir et al., 1997). Receptor-interacting protein 140 (RIP140), an important metabolic regulator, is another ligand-dependent co-repressor which interacts with PPARs.

Finally, PPAR activity is also regulated at the post-transcriptional level by phosphorylation, ubiquitinylation, and sumoylation (for a detailed review see (Feige et al., 2006)).

However, the regulation of gene transcription by PPARs extends beyond their ability to trans-activate specific target genes in an agonist-dependent manner. PPARs also regulate gene expression independently of binding to PPREs. They cross-talk with other types of transcription factors and influence their function without binding to DNA, through a mechanism termed receptor-dependent trans-repression (Daynes & Jones, 2002). Most of the anti-inflammatory effects of PPARs are probably explained by this mechanism (Kamei et al., 1996; Li et al., 2000). Thus, through this DNA-binding independent mechanism, PPARs suppress the activities of several transcription factors, including nuclear factor κB (NF-κB), activator protein 1 (AP-1), signal transducers and activators of transcription (STATs) and nuclear factor of activated T cells (NFAT). There are three main trans-repression mechanisms by which ligand-activated PPAR-RXR complexes negatively regulate the activities of other transcription factors. First, trans-repression may result from competition for limiting amounts of shared co-activators. Under conditions in which the levels of specific co-activators are rate-limiting, activation of PPAR may suppress the activity of other transcription factors that use the same co-activators (Delerive et al., 1999; Delerive et al., 2002). In the second mechanism, activated PPAR-RXR heterodimers are believed to act through physical interaction with other transcription factors (for example AP-1, NF-κB, NFAT or STATs). This association prevents the transcription factor from binding to its response element and thereby inhibits its ability to induce gene transcription (Desreumaux et al., 2001). The third trans-repression mechanism relies on the ability of activated PPAR-RXR heterodimers to inhibit the phosphorylation and activation of certain members of the mitogen-activated protein kinase (MAPK) cascade (Johnson et al., 1997), preventing activation of downstream transcription factors.

Peroxisome Proliferator-Activated

**5. Role of PPARβ/δ in lipoprotein metabolism** 

al., 2003).

PPARβ/δ activation.

2003).

Receptor β/δ (PPARβ/δ) as a Potential Therapeutic Target for Dyslipidemia 219

length) fatty acids have affinities for PPARβ/δ in the low micromolar range (Xu et al., 1999; Forman et al., 1997; Yu et al., 1995; Krey et al., 1997). In addition, all-trans-retinoic acid (vitamin A) (Shaw et al., 2003) and fatty acids derived from VLDL (Chawla et al., 2003) can activate PPARβ/δFinally, the availability of three synthetic ligands (GW501516, GW0742 and L-165041) that activate PPARβ/δ at very low concentrations both in vivo and in vitro with high selectivity over other PPAR isotypes (Sznaidman et al., 2003) led to a huge increase in experimental studies on the role of PPARβ/δ in cellular processes. The EC50 for these compounds assessed with recombinant human PPARβ/δ were 1.0 nM for GW0742, 1.1 nM for GW501516 and 50 nM for L-165041 (Berger et al., 1999; Sznaidman et

Treatment of the atherogenic dyslipidemia associated with type 2 diabetes mellitus and metabolic syndrome requires lowering triglycerides, increasing HDL-C and increasing the size of the LDL-C particle. Studies using the PPARβ/δ agonist GW501516 have demonstrated that this drug increased HDL-C (79%), and decreased triglycerides (56%), LDL-C (29%) and fasting insulin levels (48%) in obese rhesus monkeys, a model for human obesity and its associated metabolic disorders (Oliver, Jr. et al., 2001). A decrease in small dense LDL was also observed in treated animals (Oliver, Jr. et al., 2001). It has been suggested that the increase in HDL-C levels after PPARβ/δ treatment is caused by enhanced cholesterol efflux stimulated by a higher expression of the reverse cholesterol transporter ATP-binding cassette A1 (ABCA1) in several tissues, including human and mouse macrophages and intestinal cells and fibroblasts (Leibowitz et al., 2000; van, V et al., 2005). Apart from these beneficial effects of PPARβ/δ activation on HDL levels, treatment with this compound also increased HDL particle size in primates (Wallace et al., 2005), an effect which is thought to be protective against the progression of coronary artery disease in humans (Rosenson et al., 2002). In addition, PPARβ/δ activation reduces cholesterol absorption through a mechanism that may involve, at least in part, reduced intestinal expression of Niemann-Pick C1-like 1 (Npc1l1), the proposed target for the inhibitor of cholesterol absorption ezetimibe (van, V et al., 2005). However, additional studies are necessary to clearly demonstrate that the effects of these drugs are mediated through

In obese and diabetic db/db mice, administration of a PPARβ/δ agonist modestly increased HDL particles, without affecting triglyceride levels (Leibowitz et al., 2000), whereas in a shorter treatment with GW501516 a reduction in plasma free fatty acids and triglyceride levels was observed in db/db mice, but not in mice exposed to a high fat diet (Tanaka et al.,

In mice, deletion of PPARβ/δ led to enhanced LDL and triglyceride levels (Akiyama et al., 2004). It has been proposed that the increase in triglycerides observed in these PPARβ/δnull mice is caused by a combination of increased VLDL production and decreased plasma triglyceride clearance, as demonstrated by a decrease in postheparin LPL activity and increased hepatic expression of the LPL inhibitors Angptl3 and 4 (Akiyama et al., 2004). Recent findings obtained by our laboratory indicate that additional mechanisms can also contribute to the hypotriglyceridemic effect of PPARβ/δ (Barroso et al., 2011). Interestingly, the main factor influencing hepatic triglyceride secretion is fatty acid availability (Lewis, 1997). In liver, fatty acids are either incorporated into triglycerides or oxidized by

The PPAR family consists of three members, PPARα (NR1C1 according to the unified nomenclature system for the nuclear receptor superfamily), PPARβ/δ (NR1C2) and PPARγ (NR1C3) (Auwerx et al., 1999). PPARα was the first PPAR to be identified and is the molecular target of the fibrate hypolipidemic class of drugs. This PPAR isotype is expressed primarily in tissues with a high level of fatty acid catabolism such as liver, brown fat, kidney, heart and skeletal muscle (Braissant et al., 1996). PPARγ has a restricted pattern of expression, mainly in white and brown adipose tissues and macrophages, whereas other tissues such as skeletal muscle and heart contain limited amounts. The γ isotype is the molecular target for the anti-diabetic drugs, thiazolidinediones. PPARβ/δ is ubiquitously expressed and, for this reason, was initially thought to be a "housekeeping gene" (Kliewer et al., 1994). However, studies with knockout mice (Barak et al., 2002; Peters et al., 2000; Tan et al., 2001) and the development of specific and high-affinity ligands for this receptor have shown that PPARβ/δ is a potential molecular target for prevention or treatment of several disorders. In this review we will highlight the role of PPARβ/δ in those metabolic processes with potential for treating dyslipidemia.

Fig. 1. Molecular mechanisms of Peroxisome Proliferator-Activated Receptors (PPARs). PPARs are ligand-activated transcription factors that regulate gene expression through two mechanisms: transactivation and transrepression. In transactivation PPAR-RXR heterodimers bind to DNA-specific sequences called peroxisome proliferator-response elements (PPREs), which are located in the promoter regions of genes involved in glucose and fatty acid metabolism. PPARs may also regulate gene expression through a DNAindependent mechanism called transrepression. Through this mechanism, PPARs inhibit the activity of several transcription factors such as NF-κB, which leads to anti-inflammatory effects. STAT denotes signal transducers and activators of transcription.
