HS218 as an FXR antagonist suppresses gluconeogenesis by inhibiting FXR binding to PGC-1α promoter

Farnesoid X receptor (FXR) as a member of nuclear receptor is tightly associated with glucose metabolism. Accumulated evidence has addressed the potential of FXR antagonist in the treatment of type 2 diabetes mellitus (T2DM), although the related mechanisms remain unclear. Here, we determined a specific FXR antagonist HS218 (N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,4-dichlorobenzamide), which exhibited high activities in suppressing gluconeogenesis and ameliorating glucose homeostasis in db/db and HFD/STZ-induced T2DM mice. We would like to investigate the mechanisms underlying FXR antagonism in the regulation of gluconeogenesis by using HS218 as a probe.

HS218 was evaluated by glucose output assay. Binding affinity of HS218 to the ligand binding domain of FXR (FXR-LBD) was detected by Surface Plasmon Resonance (SPR) technology-based Biacore and fluorescence quenching assays. Mammalian one-hybrid and transactivation assays were carried out to detect the antagonistic effect of HS218 on FXR. Real-time PCR assay was performed to measure the expressions of FXR-target and gluconeogenic genes. Anti-diabetic efficiencies of HS218 were determined in db/db and HFD/STZ-induced T2DM mice. Assays by promoter 5'-deletion analysis and Chromatin immunoprecipitation (ChIP) were performed to detect the binding of FXR to peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) promoter. Western blot assay was used to determine the protein level in either cells or the liver tissues of mice.

We determined that HS218 as a new FXR specific antagonist could FXR-dependently suppress gluconeogenesis in mouse primary hepatocytes, and effectively improve glucose homeostasis in db/db and HFD/STZ-induced T2DM mice. HS218 decreased gluconeogenesis by inhibiting the FXR-induced increase in the promoter activity of the key gluconeogenic gene PGC-1α, leading to the repression of PGC-1α and its target gene peroxisome proliferator-activated receptor α (PPARα).

To our knowledge, our work might be the first report on the mechanism underlying FXR antagonist in the regulation of gluconeogenesis, and all results have also highlighted the potential of HS218 in the treatment of T2DM.