PPARα (EC50 = 6 nM); Chk2 (IC50 = 697.4 nM); PPARγ (EC50 = 1.1 μM); PPARδ (EC50 = 6.2 μM)

GW7647 (1 μM) causes both in the presence and absence of IL-1β a significant increase in PDZK1 protein expression in Caco2BBE cells, reaching 129.7 ± 6.5% of vehicle treated control[1]. GW7647 also attenuates the PDZK1 expression decrease mediated by IL-1β.
GW7647(50 nM) increases the amounts of NO released by stimulating the phosphorylation of PI3K and then Akt (Ser473) in the stripped antral mucosa. In antral mucous cells, GW7647 (50 nM) amplifies the first stage of Ca2+-regulated exocytotic events triggered by ACh, but GW7647 by itself does not cause any exocytotic events. In antral mucous cells, GW7647 plus ACh increases the effects of wortmannin (50 nM) and AKT-inh (100 nM) on exocytotic events[2].
GW7647 (100 nM) decreases the AQP9 protein abundance by 43%; however, at 10 and 1,000 nM, it has no discernible effec in WIF-B9 hepatocytes. HepG2 cells exposed to GW7647 (100 nM) exhibit a 24% decrease in AQP9 protein abundance; however, L-FABP protein abundance in HepG2 hepatocytes is not significantly increased[3].

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Effect of the PPARα agonist GW7647 on PDZK1 expression [1]
The transcription factor peroxisome proliferator-activated receptor alpha (PPARα) has been identified as a strong activator of the PDZK1 promoter (Tachibana et al., 2008). The peroxisome proliferator response element, to which the heterodimer PPARα and RXRα binds, is not located in the 5′ end of the PDZK1 promoter, but close to the start of transcription site at 98–86 bp, thus, not in the region sensitive to IL-1ß. Interestingly, IL-1β incubation also decreased PPARα expression in Caco2BBE cells to 65.5 ± 9.6% at 24 h incubation (Supplementary Figure 4), whereas a significant effect on PDZK1 mRNA expression was seen earlier (Figure 3A). It is nevertheless feasible that a PPARα agonist is able to restore PDZK1 expression in pro-inflammatory settings, offering a window for pharmacological intervention. We therefore incubated Caco2BBE cells with the PPARα agonist GW7647, in the absence and presence of IL-1β. At 24 h after GW7647 application, resulted in a significant increase of PDZK1 protein expression to 129.7 ± 6.5% of vehicle treated control (Figures 8A,B), and GW7647 was able to attenuate the IL-1β-mediated decrease in PDZK1 expression (61.1 ± 3.4% in the absence and 33.9 ± 2.3% in the presence of GW7647).

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A PPARα (peroxisome proliferation activation receptor α) agonist (GW7647) activates nitric oxide synthase 1 (NOS1) to produce NO leading to cGMP accumulation in antral mucous cells. In this study, we examined how PPARα activates NOS1. The NO production stimulated by GW7647 was suppressed by inhibitors of PI3K (wortmannin) and Akt (AKT 1/2 Kinase Inhibitor, AKT-inh), although it was also suppressed by the inhibitors of PPARα (GW6471) and NOS1 (N-PLA). GW7647 enhanced the ACh (acetylcholine)-stimulated exocytosis (Ca(2+)-regulated exocytosis) mediated via NO, which was abolished by GW6471, N-PLA, wortmannin, and AKT-inh. The Western blotting revealed that GW7647 phosphorylates NOS1 via phosphorylation of PI3K/Akt in antral mucous cells. The immunofluorescence examinations demonstrated that PPARα existing with NOS1 co-localizes with PI3K and Akt in the cytoplasm of antral mucous cells. ACh alone and AACOCF3, an analogue of arachidonic acid (AA), induced the NOS1 phosphorylation via PI3K/Akt to produce NO, which was inhibited by GW6471. Since AA is a natural ligand for PPARα, ACh stimulates PPARα probably via AA. In conclusion, PPARα activates NOS1 via PI3K/Akt phosphorylation to produce NO in antral mucous cells during ACh stimulation. [2]

GW7647 (3 mg/kg per day) inhibits the decrease in left ventricular ejection fraction but does not stop the development of cardiac hypertrophy in vivo[4].

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Volume-overload cardiac hypertrophy was produced in 7-day-old rabbits via an aorto-caval shunt, after which, the rabbits were treated with or without GW7647 (3 mg/kg per day) for 14 days. Biventricular working hearts were subjected to 35 minutes of aerobic perfusion, 25 minutes of global no-flow ischemia, and 30 minutes of aerobic reperfusion. GW7647 treatment did not prevent the development of cardiac hypertrophy, but did prevent the decline in left ventricular ejection fraction in vivo. GW7647 treatment increased cardiac fatty acid β-oxidation rates before and after ischemia, which resulted in a significant increase in overall ATP production and an improved in vitro post-ischemic functional recovery. A decrease in post-ischemic proton production and endoplasmic reticulum stress, as well as an activation of sarcoplasmic reticulum calcium ATPase isoform 2 and citrate synthase, was evident in GW7647-treated hearts. Conclusions: Stimulating fatty acid β-oxidation in neonatal hearts may present a novel cardioprotective intervention to limit post-ischemic contractile dysfunction. [4]

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GW7647 Treatment Improves In Vivo and In Vitro Contractile Function in Hypertrophied Neonatal Rabbit Hearts. GW7647 Treatment Prevents the Hypertrophy-Induced Shift in Myocardial Energy Metabolism. GW7647 Treatment Enhances Rates of ATP Production and Tricarboxylic Acid Cycle Activity in Hypertrophied Hearts. GW7647 Treatment Increases Mitochondrial Biogenesis and Fatty Acid Oxidative Capacity. GW7647 Treatment Reduces Myocardial Triacylglycerol Content by Enhancing Its Turnover. GW7647 Treatment Activates Calcium-Handling Proteins, Reduces Ceramide Synthesis, Endoplasmic Reticulum Stress, and Reduces Inflammation. [4]

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Administration of GW7647 (3 mg/kg po bid) to cholesterol/cholic acid-fed rats5 for 4 days resulted in a 60% increase in HDL-cholesterol, a 60% decrease in triglycerides, and a 40% decrease in serum apolipoprotein CIII [5].

Cell culture, seeding density, and cytokine treatment [1]
Caco-2BBE cells were grown at 37°C in a humidified atmosphere containing 5% CO2 and 95% O2, in Dulbecco's Modified Eagle Medium containing 4.5 g·L−1 D-glucose and sodium pyruvate supplemented with 10% FCS, 50 units penicillin/streptomycin, and 1% non-essential amino acids. Huh-7 cells were grown in Dulbecco's Modified Eagle Medium containing 4.5 g·L−1 D-glucose and sodium pyruvate supplemented with 10% FCS, 50 units penicillin/ streptomycin. 0.25 × 106 cells were plated in 6 well-plates and grown for 24 h, serum starved (0.5% FCS in medium) for 24 h and then treated with IL-1β (0.1, 1, or 10 ng·mL−1), TNF-α (up to 20 ng·mL−1) and IFN-γ (up to 30 ng·mL−1) for the times indicated in the respective figures. Concentration response curves for these cytokines in Caco-2BBE cells had been tested in a previous study (Yeruva et al., 2008), and were applied accordingly in this study. For each experiment, two (qPCR) or three (Western Analysis) individual dishes were studied for each experimental condition, and the results pooled for n = 1. Each experiment was repeated three times in different cell passages. n indicates the number of repeats in different cell passages. Due to the homogeneity of a cell line, and the duplicate or triplicate measurements within the same experiment, we believe that three repeats are acceptable for a statistically valid conclusion.

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Transient transfections and luciferase assays [1]
3 × 104 cells were seeded in 24 wells and grown overnight. Two hundred and fifty nanograms of plasmid per well was mixed with 10 ng of renilla luciferase plasmid and transfections were done using a Jet prime® polyplus transfection reagent from Peqlab according manufacturer's protocol. Cells were serum starved overnight before adding cytokines and then treated with cytokines for the time periods indicated in the text. After the treatment cells were lysed in 1X passive lysis buffer by shaking at room temperature for 15 min. Luciferase assay was performed as described previously.

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Patients selection [1]
The details of the patients who provided the biopsies of UC patients are given in detail in a previous report (Yeruva et al., 2015).

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Inhibition of NF-κB and MAPKs pathways [1]
For NF-κB and MAPK pathway inhibition experiments, Caco-2BBE cells were pretreated with a NF-κB inhibitor BAY11-7082 (10 μM), a p38MAPK inhibitor BIRB-796 (μM), a JNK inhibitor SP600125 (25 μM), MEK1/2 inhibitor PD98059 (30 μM), and U0126 (10 μM) for 1 h, followed by exposure to IL-1β (10 ng·mL−1) for 48 h. Cells were lysed for Western blot analysis as described below.

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9-cis retinoic acid treatment experiments [1]
For PDZK1 mRNA measurements, Caco-2BBE cells were pretreated for 30 mins with 9-cis retinoic acid (RA) or vehicle at a concentration of 1 μM and then treated with IL-1β for 3, 6, 12, and 24 h. For PDZK1 protein assessment, the cells were pretreated with 1 μM RA or vehicle for 30 min before addition of IL-1β (10 ng·mL−1) and the cells were harvested after 48 h.

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RNA isolation and real-time PCRs [1]
RNA isolation from cells was done using Qiagen RNA isolation kit and real time PCRs were performed as explained previously (Yeruva et al., 2015).

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Immunoblot analysis [1]
After the treatment, cells were lysed in lysis buffer and protein concentration was estimated with Bio-rad Bradford assay. Twenty to forty micrograms of total cellular proteins were separated on 8–10% SDS-poly acryl amide gels and transferred to polyvinylene difluoride membranes. Antibodies were diluted in TBST containing 5% non-fat dry milk and blots were incubated overnight at 4°C, washed with TBST and incubated with secondary antibodies conjugated to horseradish peroxidase, washed with TBST and then developed using enhanced chemiluminescence kit from GE health sciences.

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WST-1 cell viability assay [1]
The reagent WST-1 was used to determine cell viability according to the manufacturer's instructions. In brief, Caco-2BBE cells were seeded at a density of 1 × 104 cells in each well of a 96 well-plate and were grown and treated as stated in the section “Cell culture, seeding density, and cytokine treatment.” Cells were incubated with respective cytokines for 24 h. At the end of treatment, 10 μl WST-1 were added to each well and 1 h later absorbance was measured at 450 and 630 nm using the BioTek® Epoch Reader. No decrease in viability was detected during exposure of any of the tested cytokines or their combination (Supplementary Figure 5).

New Zealand newborn Seven-day-old white rabbits weighing between 90 and 200 grams are anesthetized with 2% isofluorane inhaled, and they undergo an aorto-caval shunt to cause volume-overload cardiac hypertrophy. When using a color flow doppler on postoperative days 7 and 13, it is possible to see a physical shunt between the inferior vena cava and the abdominal aorta in both the axial and transverse planes, indicating the presence of a successful fistula. The enlarged inferior vena cava serves as additional confirmation of this. Following validation, animals in the shunt group are randomized to receive either GW7647 (3 mg/kg per day; EC50=6 nM for PPARα) or the vehicle (dimethyl sulfoxide, the solvent of GW7647) twice daily for 14 days via intraperitoneal injection. Animals that have surgery to create a shunt are not allowed to continue in the study if the shunt closes or does not exhibit. Transthoracic echocardiography is used to measure the left ventricular ejection fraction (%) and other cardiac parameters on postoperative days 7 and 13. All animals are put to sleep with Na⁺ pentobarbital at age 21 (14 days after surgery), and their hearts are removed for isolated biventricular working heart perfusions.[4]

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Assessment of Myocardial Function [4]
Newborn New Zealand White rabbits of either sex (7 days old, 90–200 g) were anesthetized with inhaled isofluorane (2%), and were subjected to an aorto-caval shunt to induce volume-overload cardiac hypertrophy as described previously.18 The presence of a successful fistula was verified at postsurgical days 7 and 13 by color flow doppler that visualizes a physical shunt between the abdominal aorta and the inferior vena cava in both an axial and transverse plane. This is further validated by an enlarged inferior vena cava (an expanded Methods section is available in the Online Data Supplement). After validation, the animals in shunt group were randomly assigned to receive an intraperitoneal injection of vehicle (dimethyl sulfoxide, the solvent of GW7647) or GW7647 (3 mg/kg per day; EC50=6 nmol/L for PPARα) twice a day for 14 days. Animals that underwent surgery to create shunt, but consequently the shunt either did not exhibit or closed, were excluded from the study. Left ventricular ejection fraction (%) and other cardiac parameters were assessed by transthoracic echocardiography at postsurgical days 7 and 13 as described previously.18 At 21 days of age (14 days post surgery), all animals were euthanized with Na+ pentobarbital, and hearts were removed for isolated biventricular working heart perfusions.22

[1]. IL-1β-Induced Downregulation of the Multifunctional PDZ Adaptor PDZK1 Is Attenuated by ERK Inhibition, RXRα, or PPARα Stimulation in Enterocytes. Front Physiol. 2017 Feb 7;8:61.

[2]. PPARα induced NOS1 phosphorylation via PI3K/Akt in guinea pig antral mucous cells: NO-enhancement in Ca(2+)-regulated exocytosis. Biomed Res. 2016;37(3):167-78.

[3]. Hepatic AQP9 expression in male rats is reduced in response to PPARα agonist treatment. Am J Physiol Gastrointest Liver Physiol. 2015 Feb 1;308(3):G198-205.

[4]. Activating PPARα prevents post-ischemic contractile dysfunction in hypertrophied neonatal hearts. Circ Res. 2015 Jun 19;117(1):41-51.

[5]. Identification of a subtype selective human PPARalpha agonist through parallel-array synthesis. Bioorg Med Chem Lett. 2001 May 7;11(9):1225-7.

GW 7647 is a monocarboxylic acid that is 2-(phenylsulfanyl)isobutyric acid in which the phenyl group is substituted at the para- position by a 3-aza-7-cyclohexylhept-1-yl group in which the nitrogen is acylated by a (cyclohexylamino)carbonyl group. It has a role as a PPARalpha agonist. It is a member of ureas, an aryl sulfide and a monocarboxylic acid.

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Background: The PDZ adaptor protein PDZK1 modulates the membrane expression and function of a variety of intestinal receptors and ion/nutrient transporters. Its expression is strongly decreased in inflamed intestinal mucosa of mice and IBD patients. Aim and Methods: We investigated whether the inflammation-associated PDZK1 downregulation is a direct consequence of proinflammatory cytokine release by treating intestinal Caco-2BBE cells with TNF-α, IFN-γ, and IL-1β, and analysing PDZK1 promotor activity, mRNA and protein expression. Results: IL-1β was found to significantly decrease PDZK1 promoter activity, mRNA and protein expression in Caco-2BBE cells. A distal region of the hPDZK1 promoter was identified to be important for basal expression and IL-1β-responsiveness. This region harbors the retinoid acid response element RARE as well as binding sites for transcription factors involved in IL-β downstream signaling. ERK1/2 inhibition by the specific MEK1/2 inhibitors PD98059/U0126 significantly attenuated the IL-1β mediated downregulation of PDZK1, while NF-κB, p38 MAPK, and JNK inhibition did not. Expression of the nuclear receptors RXRα and PPARα was decreased in inflamed colonic-mucosa of ulcerative colitis patients and in IL-1β-treated Caco2-BBE cells. Moreover, the RAR/RXR ligand 9-cis retinoic acid and the PPARα-agonist GW7647 stimulated PDZK1 mRNA and protein expression and attenuated IL-1β-mediated inhibition. Conclusions: The strong decrease in PDZK1 expression during intestinal inflammation may be in part a consequence of IL-1β-mediated RXRα and PPARα repression and can be attenuated by agonists for either nuclear receptor, or by ERK1/2 inhibition. The negative consequences of inflammation-induced PDZK1 downregulation on epithelial transport-function may thus be amenable to pharmacological therapy. [1]

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The peroxisome proliferator receptor α (PPARα) is a key regulator of the hepatic response to fasting with effects on both lipid and carbohydrate metabolism. A role in hepatic glycerol metabolism has also been found; however, the results are somewhat contradictive. Aquaporin 9 (AQP9) is a pore-forming transmembrane protein that facilitates hepatic uptake of glycerol. Its expression is inversely regulated by insulin in male rodents, with increased expression during fasting. Previous results indicate that PPARα plays a crucial role in the induction of AQP9 mRNA during fasting. In the present study, we use PPARα agonists to explore the effect of PPARα activation on hepatic AQP9 expression and on the abundance of enzymes involved in glycerol metabolism using both in vivo and in vitro systems. In male rats with free access to food, treatment with the PPARα agonist WY 14643 (3 mg·kg(-1)·day(-1)) caused a 50% reduction in hepatic AQP9 abundance with the effect being restricted to AQP9 expressed in periportal hepatocytes. The pharmacological activation of PPARα had no effect on the abundance of GlyK, whereas it caused an increased expression of hepatic GPD1, GPAT1, and L-FABP protein. In WIF-B9 and HepG2 hepatocytes, both WY 14643 and another PPARα agonist GW 7647 reduced the abundance of AQP9 protein. In conclusion, pharmacological PPARα activation results in a marked reduction in the abundance of AQP9 in periportal hepatocytes. Together with the effect on the enzymatic apparatus for glycerol metabolism, our results suggest that PPARα activation in the fed state directs glycerol into glycerolipid synthesis rather than into de novo synthesis of glucose. [3]

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Rationale: Post-ischemic contractile dysfunction is a contributor to morbidity and mortality after the surgical correction of congenital heart defects in neonatal patients. Pre-existing hypertrophy in the newborn heart can exacerbate these ischemic injuries, which may partly be due to a decreased energy supply to the heart resulting from low fatty acid β-oxidation rates. Objective: We determined whether stimulating fatty acid β-oxidation with GW7647, a peroxisome proliferator-activated receptor-α (PPARα) activator, would improve cardiac energy production and post-ischemic functional recovery in neonatal rabbit hearts subjected to volume overload-induced cardiac hypertrophy. Methods and results: Volume-overload cardiac hypertrophy was produced in 7-day-old rabbits via an aorto-caval shunt, after which, the rabbits were treated with or without GW7647 (3 mg/kg per day) for 14 days. Biventricular working hearts were subjected to 35 minutes of aerobic perfusion, 25 minutes of global no-flow ischemia, and 30 minutes of aerobic reperfusion. GW7647 treatment did not prevent the development of cardiac hypertrophy, but did prevent the decline in left ventricular ejection fraction in vivo. GW7647 treatment increased cardiac fatty acid β-oxidation rates before and after ischemia, which resulted in a significant increase in overall ATP production and an improved in vitro post-ischemic functional recovery. A decrease in post-ischemic proton production and endoplasmic reticulum stress, as well as an activation of sarcoplasmic reticulum calcium ATPase isoform 2 and citrate synthase, was evident in GW7647-treated hearts. Conclusions: Stimulating fatty acid β-oxidation in neonatal hearts may present a novel cardioprotective intervention to limit post-ischemic contractile dysfunction. [4]

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Using solid-phase, parallel-array synthesis, a series of urea-substituted thioisobutyric acids was synthesized and assayed for activity on the human PPAR subtypes. GW7647 (3) was identified as a potent human PPARalpha agonist with approximately 200-fold selectivity over PPARgamma and PPARdelta, and potent lipid-lowering activity in animal models of dyslipidemia. GW7647 (3) will be a valuable chemical tool for studying the biology of PPARalpha in human cells and animal models of disease.
GW7647 (3) is a potent human PPARα agonist with ∼200-fold selectivity over the other subtypes and in vivo lipid-lowering activity. To further characterize this compound, it was assayed against the three murine PPAR subtypes. Compound 3 showed EC50=0.001, 1.3, and 2.9 μM on murine PPARα, PPARγ, and PPARδ, respectively. [5]

C29H46N2O3S

502.75214

502.322

C, 69.28; H, 9.22; N, 5.57; O, 9.55; S, 6.38

265129-71-3

265129-71-3

3392731

White to off-white solid powder

1.1±0.1 g/cm3

693.9±55.0 °C at 760 mmHg

373.5±31.5 °C

0.0±2.3 mmHg at 25°C

1.569

8.59

2

4

12

35

636

0

CC(C)(SC1=CC=C(CCN(C(NC2CCCCC2)=O)CCCCC3CCCCC3)C=C1)C(O)=O

PKNYXWMTHFMHKD-UHFFFAOYSA-N

InChI=1S/C29H46N2O3S/c1-29(2,27(32)33)35-26-18-16-24(17-19-26)20-22-31(28(34)30-25-14-7-4-8-15-25)21-10-9-13-23-11-5-3-6-12-23/h16-19,23,25H,3-15,20-22H2,1-2H3,(H,30,34)(H,32,33)

2-[4-[2-[4-cyclohexylbutyl(cyclohexylcarbamoyl)amino]ethyl]phenyl]sulfanyl-2-methylpropanoic acid

GW 7647; GW-7647; 265129-71-3; 2-[(4-{2-[(4-cyclohexylbutyl)(cyclohexylcarbamoyl)amino]ethyl}phenyl)sulfanyl]-2-methylpropanoic acid; 2-[[4-[2-[[(cyclohexylamino)carbonyl](4-cyclohexylbutyl)amino]ethyl]phenyl]thio]-2-methylpropanoic acid; MFCD06798379; NTL2A9CAZ7; GW7647

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~198.9 mM)
Ethanol: ~25 mg/mL (~49.7 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.97 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.97 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)