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Nuclear receptors are specific transcription factors which have common sequences and structures which are thought to bind as homodimers or heterodimers to specific consensus sequences of DNA referred to as response elements in the promoter region of certain gene targets. They either promote or repress transcription of these gene targets by binding to a variety of hydrophobic endogenous ligands which when bound to the receptor it leads to conformational changes in the receptor, allowing the recruitment or dissociation of protein partners generating a large protein complex. Nuclear receptors play an important role in terms of mammalian development, physiology and metabolism; however dysfunction of signalling controlled by these receptors can lead to reproductive metabolic and proliferative diseases, hence the ability of ligands binding to nuclear receptors makes them potential pharmaceutical targets.1
Peroxisome proliferator activated receptors (PPARs) are an example of a ligand activated transcription factor subfamily that belongs to this group of 48 member nuclear hormone receptor superfamily which also includes retinoic acid receptors (RARs), thyroid hormone receptors (TRs) and the steroid receptors.
To date PPARs exist in three different isoforms, each encoded by separate genes. PPAR α was the first isoform to be identified followed by PPAR β/δ and PPAR γ. It has been found that the different PPAR isoforms perform different physiological functions based on their differing patterns of tissue - specific expressions, different physiological outcomes when activated as well as their different ligand - binding specificities.2 PPAR α regulates fatty acid metabolism and is found to be highly expressed in liver, kidney and intestine. In different studies carried out, PPAR α has also shown to down regulate a number of inflammatory responses.3 PPAR β/δ is expressed in a variety of tissues, however its physiological function is not fully defined as yet.
Specific agonists of PPAR β/δ in mice however have shown to be involved in embryo implantation and decidualization (process involved in the adaptation of the uterus to enable implantation of the embryo). PPAR γ has been shown to exist in two different isoforms i.e. PPAR γ 1 and PPAR γ 2, both having different functions and tissue distributions. Expression of these isoforms however differ only in their N - terminal (PPAR γ 2 have 30 extra amino acids). PPAR γ 1 is mainly found in a broad range of tissues e.g. in the liver however to a lower extent in other tissues including adipose tissues. PPAR γ 2 on the other hand is restricted to the adipose tissues and is considered to be a potent regulator in adipoctye differentiation.4 the table below gives a summary of various isoforms of PPAR and their tissue distribution based on in situ hybridization of rate tissue.
Lipid metabolism, regulation of inflammation.
Adipocyte differentiation, regulation of inflammation.
Table 1. Properties of rodent isoforms of the peroxisome proliferator activated receptor based on tissue distribution on rat tissue.5
Structure and molecular signalling of PPARs.
In terms of understanding the structure of PPARs, it has been found that all three PPAR isoforms contain similar functional and structural features. They consist of five or six structural regions, in which four functional domains have been found known as A/B, C, D and E/F (Fig 1).
The N - terminal A/B domain contains a ligand - independent activation function (AF - 1) which is thought to be poorly conserved between the three different isotypes, however it is responsible for the phosphorylation of PPAR. The DNA binding domain (DBD) or C domain consists of two highly conserved zinc finger like structures which promotes the binding of PPAR to the peroxisome proliferator response element (PPRE) in the promoter regions of target genes.6 The C terminal, EF domain or ligand binding domain is thought to be responsible for ligand specificity and activation of PPAR binding to the PPRE of target genes.
The D site is involved as a docking domain for cofactors as well as linking the DNA binding domain (DBD) to the ligand binding domain (LBD). The ligand - dependent activation function
(AF - 2) carries out the function of recruiting PPAR co-factors which are used to assist gene transcription processes.7
DNA BINDING DOMAIN LIGAND BINDING DOMAIN
AF - 1
AF - 2
N - TERMINAL C - TERMINAL
Fig.1. Schematic representation of the four distinct functional domains of PPARs. A/B region located at the N terminal with AF - 1 responsible for phosphorylation, the C domain is implicated in DNA binding, domain D is the docking region for cofactors and domain E/F is the ligand specific domain, containing AF - 2, which promotes recruitment of cofactors required for gene transcription.
Upon binding of endogenous ligands and synthetic ligands, PPARs, form heterodimers with the 9 - cis retinoic receptors (retinoid X receptor, RXR), a process which is thought to be facilitated by the ligand binding domain. The resultant heterodimer complex than undergoes a conformational change which allows the binding of the heterodimer to the peroxisome proliferator response element (PPRE) which consists of two hexonucleotides (5' - AGGTCA and AGGTCA - 3') located in the promoter region of the target gene.8 The PPAR / RXR heterodimer than binds to the PPRE, in which PPAR occupies the 5' end half site, whilst RXR occupies the 3' end site. The PPRE sequence (5' - AGGTCA n AGGTCA - 3') consists of a direct repeat pattern which fits two direct repeats spaced by one nucleotide and is thought to be specific for the PPAR/RXR heterodimer, hence making it different from the other nuclear receptor subtypes.9
PPARs as mentioned above undergo conformational stages, which lead to the enrolment of several proteins which act as co - activators and co - repressors which interact with the nuclear receptors in a ligand dependent manner either to initiate or suppress transcription process. In the unbound state (not in the presence of ligand), the PPAR/RXR associates with a number of co - repressors which contain histone deacetylase activity, such as silencing mediator for retinoid and thyroid receptor (SMRT) and nuclear receptor co - repressor (NCoR) which prevent gene transcription .10
However, once a ligand binds to the receptor, the histone acetylase activity which is essential for co - activators like steroid receptor co -activator (SRC)-1 and PPAR binding protein (PBP) initiate a sequence of events that lead to gene transcription.11
Studies have also suggested that transcription can also be modulated by phosphorylation of the A/B domains of PPAR α and PPAR γ through a mitogen - activated protein kinase dependent pathway (MAPK).12
Fig. 2. Diagram showing heterodimerization of PPARs with RXR to produce an active transcription complex which binds to PPRE. In the absence of ligand, heterodimer forms complexes with co - repressor proteins, such as (N - CoR), which prevents transcription activation by sequestration of the receptor complex from the promoter. In contrast in the presence of ligand, conformational change takes place; heterodimer gets activated and binds to PPRE. co - activators like PPAR γ co - activator 1 (PGC -1) promotes the assembly of an effective transcriptional complex which includes histone acetyltransferases (HATs) and steroid receptor co - activator -1 (SR-1).12
Natural ligands such as fatty acids and eicosanoids have been shown to bind and activate all three different isoforms of PPAR to a variable extent in terms of their chain length and degree of saturation. The ligand binding pocket accommodates different forms of saturated, monounsaturated and polyunsaturated fatty acids, however at only micro level concentrations. PPAR α has been shown to be the most common of the PPAR isoforms in terms of showing a strong binding affinity for both unsaturated and saturated fatty acids e.g. palmitic acid, oleic acid, linoleic acid and arachidonic acid.
PPAR δ also binds to a range of fatty acids more selectively however at a lower affinity than PPAR α. e.g. dihomo - γ - linolenic acid, arachidonic acid and palmitic acid and its metabolically stable analogue 2 - bromopalmitic acid. Primary polyunsaturated fatty acids including the essential fatty acids linoleic acid, linolenic acid, arachidonic acid and eicosapentaenoic acid have been shown to bind more selectively to PPAR γ.13 A prostaglandin derivative 15d - PGJ2 has been shown to be a relatively weak ( 2 - 5 μM) ligand for PPAR γ, however several studies have indicated that it exerts independent effects suggesting that it is not an endogenous ligand for PPAR γ receptors.14
In terms of considering fatty acids as a potential endogenous ligand for PPAR we must understand the mechanism in which these molecules become concentrated in the nucleus and activate PPAR. Studies have suggested that fatty acid mediated PPAR activation in the nucleus is via the activation of phospholipases and fatty acid transport.15
In vitro, the affinities of most of these fatty acids respective of their PPAR receptors they activate, are in the micromolar and submillimolar range indicating if they were true selective endogenous ligands their affinities should be within the nanomolar range at much lower concentrations.
Synthetic ligands (agonists and antagonists)
PPARs have the ability to be activated by a wide range of structurally diverse synthetic ligands, which vary between the 3 different isoforms due to their differences in heterogeneity of the ligand binding domain as well as the degree of ligand specificity. The synthetic ligands must have similar structural requirements for interacting and activating PPARs so that they are able to cause biological effects in humans. Most synthetic ligands are amphipathic molecules which contain a hydrophobic backbone (aliphatic or aromatic) linked to an acidic function, which is thought to be essential for ligand activity. They also consist of a carboxyl group which may be converted metabolically to a carboxyl group.16
Synthetic ligands fibrates (clofibrate, gemfibrozil, fenofibrate, bezafibrate and WY - 14,643), are examples of PPAR α agonists which have shown to preferentially activate PPAR α isoform which are commonly used to reduce plasma triglycerides. Clofibrate was developed before PPARs were identified which was later found to induce peroxisome proliferation in rodents. Studies have shown that clofibrate and fenofibrate activate PPAR α with tenfold selectivity over PPAR γ, however bezafibrate has shown to have a similar potency on all three different PPAR isoforms.17
Unsaturated fatty acids, Saturated fatty acids, Leukotriene B4, 8 - Hydroxyeicosatetraenoic acid.
WY 14,643, Clofibrate, Fenofibrate, Bezafibrate.
Unsaturated fatty acids, 15d - PGJ2 , 15- Hydroxyeicosatetraenoic acid, Oxidized - LDL.
Rosiglitazone, Pioglitazone, Troglitazone, Ciglitazone.
Unsaturated fatty acids, Saturated fatty acids, Prostacylin.
L1605041, GW0742X.Thiazolidinediones (TZDs) are the most common synthetic compounds that have PPAR γ activation properties which not only have been found to improve insulin resistance but also lower blood glucose levels in type II diabetes. TZDs (troglitazone, rosiglitazone, ciglitazone and pioglitazone) are examples of PPAR γ agonists which have shown to be more selective to PPAR γ compared to PPAR α and PPAR β/δ for e.g. rosiglitazone when compared to fibrates had a Kd of 43 nM as compared to micromolar affinity associated with fibrates.18 Partial PPAR γ agonists (CDDO) has been shown to have anti - inflammatory properties, and antagonists like bisphenol diglycidal ether (BADGE), T0070907 have been identified however they have less clinical significance and are normally used to understand the physiology of PPAR γ as well as being useful in the identification of new ligands.19
In addition to PPAR α and PPAR γ agonists, synthetic ligands for PPAR δ have also been developed. GW0742X and L165041 a phenoxyacetic acid derivative act specifically at PPAR δ and have shown beneficial effects in addition to its important role in fertility and cancer, on lipid and glucose metabolism. 20 1670 words
Table 2.showing a summary of the different natural and synthetic ligands for specific PPAR isoforms.
Clinical exploitation of PPAR agonists
PPAR α agonists
PPAR α controls the expression of a large number of proteins which are involved in both the β oxidation and transport of free fatty acids e.g. fatty acid transport protein which is thought to help in the uptake of long fatty acid chains across the plasma membrane and transport of key enzymes which are involved in catabolism in the cell. PPAR α has also shown to induce activation a number of other key enzymes like acyl -CoA oxidase, acyl- CoA dehydrogenase and thiolase which are important for the β oxidation of fatty acids within the mitochondria, microsomes and peroxisomes.21
An example of PPAR α agonists is fibrates (bezafibrate, gemfibrozil, ciprofibrate, clofibrate and fenofibrate), which are useful in the treatment of hypoalphalipoproteinemia (low plasma HDL) and hypertriglyceridemia (raised levels of triglycerides). Raised levels of triglycerides are often associated with low levels of HDL cholesterol and hence increasing the risk of coronary heart diseases, therefore fibrates can be beneficial in terms of reducing this risk. 22
In terms of the most prominent effects, fibrates have been shown to decrease plasma triglycerides rich lipoproteins (TRLs) as well as decrease LDL cholesterol and increasing HDL cholesterol concentrations. Studies have suggested that the effects of fibrates are caused through changes in transcription of genes that encode for proteins that control lipoprotein metabolism.23
Fibrates have shown to stimulate cellular fatty acid uptake by converting them to acyl CoA derivatives as well as catabolism by the β oxidation pathways, hence reducing fatty acid and triglyceride synthesis resulting in a reduced production of VLDL. Fibrates have also shown to have an effect on HDL cholesterol, they transcriptionally induce the synthesis of major HDL apoliproproteins, apoA-I and apoA-II as well as lowering hepatic apoC-III production which are markers for increased risk of atherogenesis.24
In general fibrates have been shown to be well tolerated and a very low percentage of people taking fibrates have shown to have serious side effects. However in combination with statins, may cause muscle pains (rhabdomyolysis), increasing the risk of bleeding when taken with warfarin.
PPAR γ agonists
PPAR γ agonists (thiazolidinediones) are a group of oral antidiabetic drugs which have been shown to improve metabolic control in patients with type II diabetes by lowering glucose levels by improving insulin sensitivity. They have a widespread action by also enabling to reduce insulin resistance in several tissues e.g. adipose tissue, muscle and liver.
The mechanism of action of TZDs has been shown to be related to their ability of increasing insulin sensitivity by increasing peripheral glucose utilization; however the exact mechanism is not completely understood. However one of the hypothesis in regards to its ability of increasing insulin sensitivity has been suggested that TZDs are able to bind and activate nuclear PPAR γ receptors which are abundantly found in adipocytes, stimulating the expression of a number of genes which encode proteins involved in the metabolism of glucose and lipids. Apart from their ability to increase glucose uptake in the adipose tissues, TZDs also increase the uptake of fatty acid and lipogenesis.25
Currently, there are only two TZD drugs on the market, rosiglitazone and pioglitazone. A third TZD, troglitazone was withdrawn from the market due to its affects on the liver leading to hepatotoxicity. Experimental agents include rivoglitazone and the early non marketed TZD ciglitazone. TZDs have shown to vary in potency (rosiglitazone > pioglitazone > troglitazone and ciglitazone), however all of them have shown to have generally similar effects on carbohydrate and lipid metabolism.26
In a study carried out on insulin - resistant animal models for obesity/type II diabetes, ciglitazone was shown to decrease levels of hyperglycaemia and hyperinsulinaemia, as well as increased insulin sensitivity in adipose tissue, skeletal muscles and liver.25,27 The improved insulin sensitivity seen with TZDs has been suggested in animal models with hyperinsulinaemia, by increasing peripheral glucose disposal and reducing hepatic glucose production. However, in a study carried out on nonobese diabetic rats which were not hyperinsulinaemic, only reduced hepatic glucose production was found with troglitazone.28
In terms of hypoglycaemic effects of TZDs, placebo controlled studies have shown that both pioglitazone and rosiglitazone have found to be effective in achieving glycemic control. At maximal doses (8mg rosiglitazone and 30 to 45mg pioglitazone), both drugs have shown to decrease glycosylated haemoglobin values by 1 to 1.5 percent which in a type II diabetic patient the glycosylated haemoglobin to decrease from 8.5 % to around 7% (normal range 4 to 6%).29 Below is a table which summaries the beneficial actions of TZDs on adipose tissue, skeletal muscles and liver.
↑ Glucose uptake
↑ Fatty acid uptake
↑ Glucose oxidation
↑ Glucose uptake
↑ Glucose oxidation
↑ Glucose uptake
Table 3.showing a summary of the widespread action of TZDs in different target tissues.26
In terms of side effects and risks of TZDs, they have been found to be well tolerated and are associated with few side effects. As mentioned above hepatotoxicity was observed with troglitazone and was withdrawn from the market in the year 2000. in 13 double blind studies, it was found that 1.91% of 2,510 patients, 0.26% of 1,526 patients, and 0.17% percent of 3,503 patients receiving troglitazone, pioglitazone and rosiglitazone had alanine aminotransferase levels three times more than the upper limit reference range. Although the percentage of raised alanine aminotransferase is low in pioglitazone and rosiglitazone, FDA recommends that liver enzyme monitoring is essential and should be checked regularly when these drugs are prescribed.30
Thiazolidinediones has also been associated with weight gain as well as patients having fluid retention leading to peripheral edema. In a clinical study carried out, weight gain was found reported from 2 to 6 kg during the first 6 months to 1 year treatment with TZDs.29 Edema was found in 4 to 6 % of patients undergoing treatment with TZDs compared to those receiving a placebo. 31
The increase in body weight and edema can cause cardiovascular risks in which evidence from a recent study referred to as the RECORD study was carried out to compare the cardiovascular safety outcomes in patients with type II diabetes taking rosiglitazone plus other antidiabetic medication (metformin or a sulfonylurea) compared to patients taking metformin and a sulfonylurea.
The study was carried out for almost 6 years in which patients were monitored for occurrence of primary endpoints i.e. cardiovascular death and . cardiovascular hospitalizations. Secondary endpoints included cardiovascular death, heart attack or stroke. The study was found to show no difference in primary endpoints in rosiglitazone group [hazard ratio = 0.99 (95% Confidence Interval of 0.85 to 1.16)] compared to the combined use of metformin and a sulfonylurea. However a significant difference was found in secondary endpoints in increasing heart failure which is one of the side effects of rosiglitazone as well as pioglitazone. The FDA has asked patients to report any side effects and new observational studies are being carried out in regards to the safety of rosiglitazone.32