**1. Introduction**

Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that is activated by ligand binding as well as via ligand-independent activation. PPARγ plays an active role in regulation of glucose and lipid metabolism but more recently has been examined for its role in numerous disease states including: diabetes, cardiovascular disease, cancer, inflammation, angiogenesis and metastasis [1–6]. Some research also suggests it provides potential for anti-aging activity [7]. Prior to the discovery of the thiazolidinedione (TZD) drugs used for treatment of diabetes, the general consensus was that PPARγ was subject only to ligand-independent activation [8]. It was the discovery of these drugs that suggested that PPARγ was also sensitive to ligand-dependent activation [8–11]. The goal of the first generation TZDs was to target insulin sensitivity and although the drugs were successful with this they were not without toxicity concerns. The focus more recently has looked to natural methods to accomplish therapeutic level results and PPARγ provides a viable target with multiple mechanisms available for activation and a variety of functional food sources for PPARγ ligands.

PPARγ is a member of the nuclear receptors gene subfamily and is found on chromosome 3. Other members of the gene family include PPARα and PPARβ/δ. In addition due to alternative splicing, there are multiple isoforms of PPARγ, known as PPARγ1 and PPARγ2 [12, 13]. PPARγ1 is expressed at high levels in white and brown adipose tissue, however, lower levels of expression of PPARγ1 are found in all tissues as well as the immune cells. PPARγ2 is expressed only in white and brown adipose

tissue [14]. Although disease states are sometimes related to changes in expression of these isoforms and inefficient signaling associated with the pathway, some research has demonstrated that specific variants have been linked to early onset type 2 diabetes. Nearly half of these patients had a variant in PPARγ2 resulting in an amino acid substitution of tyrosine to cysteine in the activation function domain 1 (AF1). Of note is also that of these patients, 83% also had diabetic kidney disease [15]. Another study demonstrated that loss of function mutation of arginine 288 to histidine in the ligand-binding domain (LBD) resulted in significant conformational changes in the protein that lead to blunting of the activation via prostaglandins [16].

Mouse studies have shown that knockout of PPARγ2 results in insulin resistance [13]. In contrast, it was demonstrated that activation of PPARγ in mouse adipocytes improves insulin sensitivity [17]. Together these studies support the effects of PPARγ activity on insulin resistance and type 2 diabetes in addition to its known role in adipocyte differentiation [12, 18]. PPARγ also controls the expression of the skeletal muscle and adipose glucose transporter (GLUT4), and adipose tissue based hormones adiponectin, resistin and leptin [19–21]. It is clear from these studies that modulation of PPARγ activity plays a significant role in patient wellness considering the effects of activation on glucose homeostasis, lipid metabolism and adipocyte differentiation. These effects strongly suggest that activation of PPARγ in a controlled fashion provide strong potential for improvement of patient quality of life. Nutraceuticals offer this type of low level controlled activation that may benefit the patient even beyond the potential for the synthetic drugs targeting PPARγ activation.

The structure of PPARγ contains multiple protein domains of importance as shown in **Figure 1**. The individual domains are separated based on function and can themselves be modified in most cases. The activation function domain 1 (AF1) is on the N-terminus and is subject to phosphorylation, and small ubiquitin like modifiers (SUMOylation). In general SUMOylation leads to suppression of transcriptional activity while ubiquitination increase transcriptional activity. Phosphorylation can increase or decrease transcriptional activity depending on the site of phosphorylation and the enzyme catalyzing the phosphorylation event. In general deacetylation by Sirt-1, the histone deacetylase leads to dissociation of the nuclear receptor corepressor (NCoR) and increased transcriptional activity [22]. The next domain is the DNA binding domain (DBD) responsible for interacting with the DNA in conjunction with the retinoid c receptor (RXR). RXR houses a binding site for 9-cis-retinoic acid, which when bound allows RXR to complex with the PPARγ complex and interaction with DNA [23]. The HD domain is a regulatory domain named due to the histidine

#### **Figure 1.**

*The structure of PPARγ. AF-1 is the activated function 1 domain. A/B houses a SUMOylation (S) and phosphorylation (P) site. DBD is the DNA binding domain. HD is the conserved protein domain with histidine and aspartate that interacts with the coactivator PGC-1α. The ligand binding domain (LBD) is attached via a hinge region and houses a SUMOylation, phosphorylation, ubiquitination (Ub) and two acetylation (Ac) sites. AF-2 is the activated function.*

(H)-aspartate (D) amino acids conserved in the region. The HD domain interacts with either the coactivator, peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) or the corepressors NCoR and silencing mediator of retinoid and thyroid hormone receptors (SMRT) [24]. On the C-terminal end is the ligand binding domain (LBD) that when ligand bound by an activator, stimulates transcription. When no ligand is bound, and the corepressor is bound, transcription is limited. The LBD is also subject to phosphorylation, ubiquitination, SUMOylation and acetylation. Each of these affect the likelihood of ligand activation. Of interest are the histone deacetylase enzyme (HDAC) which deacetylate the ligand binding domain. Inhibition of this HDAC, limits deacetylation of the LBD and leads towards PPARγ activation via the ligand independent pathway [25].

The PPARγ pathway involves several overlapping cellular functions. Activation of PPARγ, either ligand-independent or ligand dependent, leads to change in the immune system, metabolic organs including skeletal muscle and adipose tissue [26–28]. For example in the immune system macrophages and regular T cells, activation of PPARγ in general will decrease inflammation. Inflammation is decreased in the heart and brain but lipid storage is increased in the heart as well as growth. In white adipose tissue lipid metabolism and glucose homeostasis is improved with activation. Of particular interest is the fact that activation of PPARγ increases remodeling and browning of white adipose tissue, a benefit that will likely lead to better ability for patients to manage weight (ref). It is clear that there are strong benefits to PPARγ activation, but potential side effects such as lipid storage in the heart, sodium and fluid retention and increased desire for food can also result in unwanted effects [22].

It is important to consider how PPARγ activation can be achieved while minimizing the potentially harmful side effects. A benefit also exists to activate PPARγ in a ligand dependent fashion versus the ligand independent activation. Endogenous ligands to PPARγ are typically fairly weak agonists while the TZDs are strong agonists [8, 29]. The first generation TZD troglitazone was pulled from the market in the US approximately 3 years after first becoming available because it resulted in severe or fatal hepatotoxicity in numerous cases [30–34]. Other TZDs were taken off the European market and restricted in the US markets due to potentially dangerous side effects including myocardial infarction, heart failure, hepatic failure [35–38]. It is still currently believed that these side effects resulted in part to the full PPARγ activation rather than the moderate activation offered by the weaker ligand agonists [29]. Gene expression, especially expression of genes involved in metabolic process that are sensitive to endogenous ligands as well, should be tightly controlled with an effective regulatory method that allows frequent adjustment of expression levels in response to the environment. Altering expression of genes fully by fully activating PPARγ poses several concerns, especially since there is so much overlap within the PPARγ activation pathway. Thus, an increased emphasis on natural and less specific activators is warranted. Natural ligands are particularly useful in this situation because the full and permanent activation is not desired. Given the range of disease state linked to PPARγ signaling, exploring the potential natural food based ligands is an essential tool in moving patient wellness forward.
