GW1929 

Ligand binding to bacterially expressed ligand binding domain (LBD) of hPPAR-γ is determined by scintillation proximity assay (SPA). The assay measures the ability of putative ligands to displace receptor bound [³H]BRL 49653. Assays are conducted in 96-well plates. Wells contained varying concentrations of GW1929 or troglitazone; streptavidin-modified SPA beads to which biotinylates PPAR-γ LBD is prebound; and 10 nM of the specific radioligand [³H]BRL 49653 in a volume of 100 μL. The amount of nonspecific binding, as assessed by control wells that contained 50 μM of the corresponding unlabeled ligand, is subtracted from each data point. For each compound tested, plots of ligand concentration versus counts/min of radioligand bound are constructed, and apparent K_i values are estimated from a nonlinear least squares fit of the data, assuming simple competitive binding. The results are expressed as pK_i, where pK_i = -log₁₀(K_I)^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

For the experiments, the cells are plated in 96-well plates at a density 2 × 10⁵ cells per cm² and cultured in the presence of TBBPA, in a concentrations range from 1 nM to 100 μM TBBPA. TBBPA is dissolved in DMSO, resulting in a final vehicle concentration of 0.1 % (v/v). Control (no vehicle) and DMSO-treated wells are included in the experimental design to determine the effect of DMSO. To study whether PPAR-γ is involved in the neurotoxic effect of TBBPA, cells are co-treated with 10 μM TBBPA and 10 μM GW1929 or GW9662. After 6 or 24 h of culture, 100 μL medium is collected for the LDH analysis, and the cells are collected and frozen at −70°C for the caspase-3 activity measurements^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Animals are housed at 72°F and 50% relative humidity with a 12-h light and dark cycle, and fed Formulab Diet 5008. Age- (60-day) and glucose-matched male Zucker diabetic fatty rats are gavaged twice daily for 14 days with vehicle (0.05 M N-methylglucamine), GW1929 (0.5, 1.0, or 5.0 mg/kg), or troglitazone (as the milled extrudate, in a suspension in methylcellulose, 50, 150, and 500 mg/kg). Another group of animals receives a mixture of Humulin N and Humulin R by subcutaneous injection twice daily. On days 7 and 14 of dosing, nonfasted measurements of glucose, lactate, insuline, total cholesterol, TGs, F FAs, and hematocrit are obtained. On day 14 of dosing, samples for serum drug levels (2-h postdose) and glycosylated hemoglobin measurements are also collected. In addition, once weekly, three animals from each group are placed in metabolic chambers for 48 h for quantitation of 24-h food and water consumption. Body weights are recorded throughout the study. At the conclusion of the study, perfused pancreas experiments are performed on 12 animals (n = 4 per group) that have received either GW1929 (1 and 5 mg/kg) or vehicle, to directly evaluate the effects of treatment on basal and glucose-stimulated insuline secretion. The remaining animals are killed, and their pancreases are processed for immunocytochemistry^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Brown KK, et al. A novel N-aryl tyrosine activator of peroxisome proliferator-activated receptor-gamma reverses the diabetic phenotype of the Zucker diabetic fatty rat. Diabetes. 1999 Jul;48(7):1415-24.  [Content Brief]

  [2]. Wojtowicz AK, et al. PPAR-γ agonist GW1929 but not antagonist GW9662 reduces TBBPA-induced neurotoxicity in primary neocortical cells. Neurotox Res. 2014 Apr;25(3):311-22.  [Content Brief]