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 Table below gives a summary of various isoforms of PPAR and their tissue distribution based on in situ hybridization of rate tissue.
Isoform
Liver
Kidney
Intestine
Spleen
Fat
Physiological control
α
β/δ
γ
++++
++
-
++
++
+/-
++++
+++
++
+
++
+++
-
-
++++
Lipid metabolism, regulation of inflammation.
Embryo implantation.
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
A/B
C
D
E/F
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
Endogenous ligands.
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. In a study carried out on rodents, it was shown that a high fat diet produced peroxisome proliferation as well as elevation of a number of enzymes e.g. peroxisomal acyl CoA oxidase, which is involved in metabolism of long chain fatty acids hence playing an important role in fatty acid metabolism.16
In another study, thiol-substituted fatty acid tetradecylthioacetic acid (TTA), was found to be a potent peroxisome proliferator-activated receptor activator and was shown to activate PPAR in a similar manner to potent hypolipidaemic drug WY - 14, 643, suggesting as well that the physiological role of PPAR is to regulate fatty acid metabolism by promoting the intracellular accumulation of fatty acids which in turn is thought to induce transcription activity of PPAR hence affecting a number of genes which are believed to be related to lipid metabolism resulting in peroxisomal proliferation.17
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.18
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.19
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.20 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.21
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. 22
PPAR ISOFORM
NATURAL LIGANDS
SYNTHETIC LIGANDS
PPAR α
Unsaturated fatty acids, Saturated fatty acids, Leukotriene B4, 8 - Hydroxyeicosatetraenoic acid.
WY 14,643, Clofibrate, Fenofibrate, Bezafibrate.
PPAR γ
Unsaturated fatty acids, 15d - PGJ2 , 15- Hydroxyeicosatetraenoic acid, Oxidized - LDL.
Rosiglitazone, Pioglitazone, Troglitazone, Ciglitazone.
PPAR β/δ
Unsaturated fatty acids, Saturated fatty acids, Prostacylin.
L1605041, GW0742X.
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 of 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.23
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. 24
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.25
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.26
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 δ agonists
So far to date, the exact role of PPAR δ still is not clear. However PPAR δ has shown to take part in a variety of physiological and pathophysiological processes shown in different animal studies. Several studies have shown that disruption of the PPAR δ gene causes death of embryos due to a defect caused in the placenta. This suggests that PPAR δ plays an important role in fertility and pregnancy since it is highly expressed in uterus. The knockout animals who survived during the studies were found to be much smaller and had skin defects, reduced fat mass and change in myelinisation suggesting that PPAR δ plays an important role in skin physiology .27
PPAR δ has been shown also to play an important role in the control of glucose levels and circulating lipids. In a study carried out on insulin - resistant obese rhesus monkeys, PPAR δ agonist GW501516 was given for 4 weeks. Results shown that fasting glucose and insulin normalized, there was an increase in HDL and a reduction in LDL. When the same agonist was given to genetically or dietary induced obese mice, insulin sensitivity was found to be improved. The studies suggested that like PPAR α and PPAR γ, PPAR δ plays an important role in controlling fatty acid catabolism in adipose and skeletal muscle.28
From the above information we can see that PPAR δ agonists may have a therapeutic benefit in metabolic syndrome by increasing the consumption of fatty acids in skeletal muscle and adipose tissues.
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 hypotheses 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 e.g. lipoprotein lipase (LPL), adipocyte fatty acid binding protein (A-FABP) and fatty acid transporter protein (FATP). Apart from their ability to increase glucose uptake in the adipose tissues, TZDs also increase the uptake of fatty acid and lipogenesis.29
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.30
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.29,31 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.32
ADIPOSE TISSUE
SKELETAL MUSCLE
LIVER
↑ Glucose uptake
↑ Fatty acid uptake
↑ Lipogenesis
↑ Glucose oxidation
↑ Glucose uptake
↑ Glycolysis
↑ Glycogenesis
↑ Glucose oxidation
↑ Gluconeogenesis
↑ Glycogenolysis
↑ Lipogenesis
↑ Glucose uptakeIn 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%).33 Below is a table which summaries the beneficial actions of TZDs on adipose tissue, skeletal muscles and liver.
Table 3.showing a summary of the widespread action of TZDs in different target tissues.30
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. 13 double blind studies showed 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.34
Thiazolidinediones has also been associated with weight gain as well as patients having fluid retention leading to peripheral oedema. 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.33 Oedema was found in 4 to 6 % of patients undergoing treatment with TZDs compared to those receiving a placebo. 35
The increase in body weight and oedema 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.36
Apart from its recognized role in controlling glucose and lipid metabolism, PPAR γ has been shown to be expressed in a range of other physiological roles. PPAR γ agonists are used in the management of type II diabetes, however they have shown to have a number of protective vascular effects through a large number of mechanisms therefore showing additional positive cardiovascular effects. These include in vivo evidence of reduction in blood pressure and increased NO availability in vitro, anti inflammatory action and atherosclerosis.37 In one of the studies, TZDs were found to lower the blood pressure in non diabetic rats.38
PPAR γ has been shown to be expressed in the vascular system including vascular smooth muscle cells, endothelial cells and monocytes/macrophages in which their mechanism of action which is still not very well understood may involve reduction in smooth muscle contraction, improvement of endothelial dysfunction and inhibition of inflammation and proliferation.39
The aim of this project was to compare the effects of different TZDs in different smooth muscle preparations, as well as find out what evidence suggests that TZDs causes vasodilation, and whether the effect was independent to PPAR γ or through gene transcription (dependent effect).
Methods
Tissue Preparation
Hearts were obtained from freshly slaughtered pigs which were collected from a local abattoir; and transported back to the laboratory in ice - cold Krebs - Henseleit buffer. Coronary arteries were dissected, washed with Krebs - Henseleit (K - H) solution composed in mM, of the following: NaCl, 128; KCl, 4.8; MgSO4, 1.1; NaHCO3, 25; KH2PO4, 1.2; glucose, 12; CaCl2, 1.25 and pre-gassed with O2-CO2 mixture (95:5). The dissected coronary arteries were then refrigerated overnight at 4°C in Krebs- Henseleit solution.
On the following day, the arteries were then cleaned of any fat or loose connective tissue and care was taken not to stretch or damage it. The artery was then cut into ring segments of about 2-3 mm long, and was attached to a wire connected to an isometric force transducer, with a silk thread. The transducer was then connected to a power lab data acquisition unit via an amplifier, responses were recorded on a computer using lab chart version 7 (AD Instruments, UK). The ring segments were suspended into 25 ml organ baths containing K-H solution which was maintained a constant temperature of 37°C, gassed with 95% O2 and 5% CO2 and at a pH of about 7.4. Depending on the size of the ring segments, 6 - 8 grams tension was applied to each of the segments following a 20 minute equilibration period which were then allowed to relax to a final resting tension of about 2-3 grams.
Experimental protocol
Following equilibration, the ring segments were then exposed twice to 60 mM KCl which was used to determine tissue viability and maximum contractile responses. In between times, rinsing twice with Krebs - Henseleit buffer following a 20 minute recovery period was done after each addition of KCl. The ring segments were then contracted with a thromboxane mimetic U46619 (2 to 13 nmol /L) which was used to achieve 40% - 80 % of the maximum contraction obtained with the maximum KCl contraction response.
i) Effects of the addition of different concentrations of troglitazone.
The first experiment carried out was to investigate the role of PPAR γ agonist troglitazone on coronary artery segments. Three different concentrations of troglitazone 3μM, 10μM and 30μM were compared to control dimethyl sulphoxide (0.1% DMSO), which were added following equilibration of U46619, responses were measured for 60 minutes.
ii) Effects of PPAR γ antagonist T0070907.
In a second experiment, investigation of the reversal effect of PPAR γ agonist was carried out by using a potent PPAR γ antagonist T0070907. Following equilibration, the ring segments were exposed twice to 60 mM KCl and before they were contracted with U46619, T0070907 1 μM was added to two of the organ baths containing the coronary ring segments. In the remaining two organ baths, (vehicle DMSO 0.01%) was added. Tissues were then contracted with U46619. Troglitazone 10μM was then added to one organ bath containing the antagonist T0070907 and one containing the vehicle DMSO 0.01%, in the remaining 2 organ baths control DMSO 0.1% was added. Table 1 below gives the summary of the experiment carried out.
CH1
CH2
CH3
CH4
Vehicle DMSO 0.01%
+
Varying concentrations of U46619.
Vehicle DMSO 0.01%
+
Varying concentrations of U46619.
Antagonist T0070907(1 μM )
+
Varying concentrations of U46619.
Antagonist T0070907(1 μM )
+
Varying concentrations of U46619.
45 minutes / 40% - 80 % of the maximum contraction achieved with U46619
Added
troglitazone 10μM
Added
DMSO 0.1%
Added
troglitazone 10μM
Added
DMSO 0.1%
AGONIST
CONTROL
ANTAGONIST
+
AGONIST
ANTAGONIST
+
CONTROL
iii) Effects of L-NAME on relaxatory response.
In a separate experiment, we also decided to explore the mechanism of troglitazone induced relaxation by using a nitric oxide synthase (NOS) inhibitor, NG- nitro- L-arginine methyl ester (L - NAME). Following equilibration, the ring segments were exposed twice to 60 mM KCl and before they were contracted with U46619, L - NAME (0.1 mM) was added to two organ baths containing coronary ring segments, whereas the other two organ baths were used as a control.
Agonist troglitazone 10 μM was then added to all the organ baths after equilibration with U46619.
Data analysis
The computer program Prism (GraphPad software) was used to analyze the data. Graphs were plotted of time in minutes against the percentage degree of relaxation measured as a fraction achieved by U46619 expressed as a percentage of U46619 induced tone.
All results are expressed as Mean ± SEM, with n indicating the number of experiments conducted. Two-way ANOVA with Bonferroni post test was used to determine both the concentration dependent effects of Troglitazone against control and the effects of antagonist T0070907 against control. 2 tailed Students paired t -test was used to determine the statistical significance for the effect of L-NAME against control. A value of P < 0.05 was considered statistically significant.
Nomenclature
TROGLITAZONE T0070907 ANTAGONIST
.HCl
L - NAME DIMETHYL SULPHOXIDE (DMSO)
Materials
The following compounds were used: Krebs reagents (Fisher Scientific, UK); troglitazone (Tocris Bioscience, Bristol, UK); DMSO (Sigma, Poole, UK); T0070907 Antagonist (Sigma, Poole, UK); L - NAME (Sigma, Poole, UK); (5Z, 9α, 11α, 13E, 15(S))-15-hydroxy-9 (11) methanoepoxyprosta- 5,13-dien-1 oic acid (U46619).
Results
Relaxant effect of different concentrations of PPAR γ agonist troglitazone.
Fig. 1. Effects of different concentrations of PPAR γ agonist Troglitazone compared to control (0.1% DMSO). Time against relaxation in response to the different agonist concentrations as expressed as a percentage decrease from the U46619 induced contraction was plotted. Data given as means with error bars representing S.E.M. *, P < 0.05; ** P < 0.01 denotes significant difference between Troglitazone 30μM and Control treated segments of artery ( 2- way Anova test with bonferroni post test ).
From the graph it can be seen that all concentrations of troglitazone induced a relaxation greater than the control (DMSO 0.1%). The maximum relaxation at 60 minutes in the segments exposed to 3μM, 10μM and 30μM troglitazone were 18 ± 12% (n=4), 44 ± 18% (n=4) & 62 ± 12% (n=4) respectively. The two-way ANOVA with Bonferroni post test showed no difference when control was compared to both 3μM and10μM troglitazone, however it showed a significant relaxation where (p < 0.01) after 20 minutes with 30μM troglitazone.
Effect of using a potent PPAR γ antagonist T0070907
Fig. 2. Reversing effect of PPAR γ agonist by using a potent PPAR γ antagonist T0070907 (1mM), compared to a control DMSO 100% and PPAR γ agonist Troglitazone 10μM. Graph plotted of time against relaxation expressed as a percentage decrease from the U46619 induced contraction. Data given as means with error bars representing S.E.M. *, P < 0.05; ** P < 0.01; *** P < 0.001 denotes significant difference between both PPAR γ agonist troglitazone 10μM and PPAR γ agonist troglitazone 10μΜ + PPAR γ antagonist T0070907 (1mM) compared to control treated segments of artery ( 2- way Anova test).
In the second experiment, the maximum relaxation at 90 minutes in the segments exposed to the Control and the antagonist (T0070907) alone were 5 ± 9% (n=4) and 5± 13% (n=4) respectively. The antagonist (T0070907) + agonist (10μM troglitazone) and vehicle (DMSO 0.01%) + agonist (10μM troglitazone) combinations both induced relaxations of 86 ± 9% (n=4), & 89 ± 4% (n=4) respectively. The two-way ANOVA with Bonferroni post test showed a statistically significant relaxation when compared with control to both the antagonist (T0070907) + agonist (10μM troglitazone) and vehicle (DMSO 0.01%) + agonist (10μM troglitazone) combination, p < 0.001 after 30 minutes.
Effect of L-NAME on the relaxatory response.
Fig. 3. Effect of L-NAME (0.1 mM) compared to PPAR γ agonist Troglitazone 10μM. Time in minutes against relaxation expressed as a percentage decrease from the U46619 induced contraction was plotted. Data given as means with error bars representing S.E.M. *, P < 0.05; ** P < 0.01 denotes significant difference between L - NAME ( 0.1 mM ) compared to control treated segments of artery containing PPAR γ agonist Troglitazone 10μΜ (2 tailed paired Student's t test ).
In the final experiment, 10μM troglitazone induced relaxation was significantly inhibited by the treatment of L-NAME, maximum relaxation at 90 minutes in the segment exposed to 10μM troglitazone 84 ± 10% (n=4) in the absence of L-NAME and in the presence of L-NAME 38 ± 17% (n=4), p < 0.05 (two - tailed Student's paired t - test).
