PPARa Rat Agonist Graph
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Rat PPARa Reporter Assay Kit

SIZE SKU PRICE
1 x-96 well format assays—
3 x-32 assays in-96 well format—
SIZE SKU
1 x-96 well format assays
3 x-32 assays in-96 well format
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Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Rat Peroxisome Proliferator-Activated Receptor Alpha. INDIGO’s rPPAR alpha reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the rPPAR alpha. In addition to rPPAR alpha Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against rPPAR alpha. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.

Features

  • Clear, Reproducible Results

  • All-Inclusive Assay Systems
  • Exceptional Cell Viability Post-Thaw
  • Consistent Results Lot to Lot

Product Specifications

Target TypeNuclear Hormone Receptor, Nuclear Receptor Orthologs
SpeciesRat
Receptor FormHybrid
Assay ModeAgonist, Antagonist
Kit Components
  • Rat PPARa Reporter Cells
  • Cell Recovery Medium (CRM)
  • Compound Screening Medium (CSM)
  • GW7647, (ref. agonist; in DMSO)
  • Detection Substrate
  • Detection Buffer
  • White, sterile, cell-culture ready assay plate
Shelf Life6 months
Shipping RequirementsDry Ice
Storage temperature-80C

Data

Dose-response analyses of rPPAR Reporter Cells were performed using the reference agonists GW590735 (provided), GW7647 (Tocris) and WY14643 (Tocris). Final assay concentrations were prepared by serial dilution in 4-fold decrements, ranging between 12 M and 183 pM. The highest assay concentration of residual DMSO was 0.12%, which has no effect on assay performance. To assess the level of background signal contributed by non-specific factors that cause gratuitous activation of the luciferase reporter gene, “mock” reporter cells were identically treated with GW590735 (mock reporter cells, which contain only the luciferase reporter vector, are not provided with assay kits). Treatment media were removed after 24 hr and LDR was applied. Luminescence was quantified using a GloMax-Multi+ luminometer (Promega). Average relative light units (RLU) and corresponding standard deviation (SD) values were determined for each treatment concentration (n ≥ 6). Signal-to-background (S/B; fold-activation) and Z’ values were calculated as described by Zhang, et al. (1999). Non-linear regression and EC50 analyses were performed using GraphPad Prism software. RESULTS: The high S/B and Z’ values, low background and %CV values, and the absence of non-specific reporter activity confirm the robust performance of this rPPARa Reporter Assay.
Antagonist dose-response assays were performed using GW6471 (Tocris) and MK886 (Tocris). CSM was first used to prepare serial 4-fold dilutions of each antagonist to generate the desired range of 2x-concentration treatment media. Next, frozen Rat PPARa Reporter Cells were thawed in CRM, supplemented with a '2x EC75 concentration' of GW590735, and 100 l of this treated cell suspension was dispensed into each well of the assay plate. 100 l of the prepared series of 2x-concentration treatment media were then dispensed per well, combining with the reporter cells. Final assay concentrations of the respective antagonists ranged between 40 M and 2.44 nM, including a 'no antagonist' control (n ≥ 6 per treatment; highest [DMSO] ≤ 0.12% f.c.). Each assay treatment contained ~ 1.3 M (approximating EC75) GW590735 as challenge agonist. Assay plates were incubated for 22-24 hrs, then further processed to quantify rPPARa activity for each treatment condition.

Target Background

INDIGO’s Rat PPAR alpha (nr1c1; pparA; rPPARα) Assay utilizes proprietary rodent cells engineered to provide high-level expression of rPPARα. Reporter Cells also incorporate a responsive luciferase reporter gene, therefore, quantifying expressed luciferase activity provides a sensitive surrogate measure of rPPARα activity in the treated cells.

The principle application of this reporter assay system is in the screening of test samples to quantify functional activity, either agonist or antagonist, that they may exert against rat PPARα.

Citations

KHK, PNPLA3 and PPAR as Novel Targets for the Anti-Steatotic Action of Bempedoic Acid

Bempedoic acid (BemA) is an ATP-citrate lyase (ACLY) inhibitor used to treat hypercholesterolemia. We studied the anti-steatotic effect of BemA, and the mechanisms involved, in a model of fatty liver in female rats obtained through the administration of a high-fat diet supplemented with liquid fructose (HFHFr) for three months. In the third month, a group of rats was treated with BemA (30 mg/kg/day) by gavage. Plasma analytes, liver histology, adiposity, and the expression of key genes controlling fatty acid metabolism were determined, and PPAR agonism was explored by using luciferase reporter assays. Our results showed that, compared to HFHFr, BemA-treated rats exhibited lower body weight, higher liver/body weight, and reduced hepatic steatosis. In addition to ACLY inhibition, we found three novel mechanisms that could account for the anti-steatotic effect: (1) reduction of liver ketohexokinase, leading to lower fructose intake and reduced de novo lipogenesis; (2) increased expression of patatin-like phospholipase domain-containing protein 3, a protein related to the export of liver triglycerides to blood; and (3) PPARα agonist activity, leading to increased hepatic fatty acid β-oxidation. In conclusion, BemA may represent a novel approach to treat hepatic steatosis, and therefore to avoid progression to advanced stages of non-alcoholic fatty liver disease.
2022-06-27

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Peroxisome Proliferator-Activated Receptor Alpha (PPARa, NR1C1)

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