Abstract Body: Background: Stevioside is used as a sweetener in food additives and pharmaceutical products. There is a growing concern about the adverse effects posed by stevioside usage. Stevioside is hydrolyzed into the aglycone steviol that is absorbed into the body. Recent studies suggest that steviol, but not stevioside, activates Pregnane X receptor (PXR) in human hepatic cells. In addition to the role in xenobiotic metabolism, the atherogenic and dyslipidemic effects of PXR have been revealed. Controversially, steviol not only increases the mRNA expression of PXR direct downstream gene CYP3A4, but also inhibits the enzymatic activities of CYP3A4. Thus, it is urgent to elucidate the molecular mechanisms by which steviol activates PXR signaling and to assess the possible adverse effects of steviol on pro-atherosclerotic events, such as dyslipidemia.

Objective: Our study aims to explore the molecular mechanisms by which exposure to steviol activates human PXR and increases the risk of dyslipidemia.

Methods: Both human hepatic and intestinal cells were used to test if steviol was a PXR agonist via transfection assay. The key residues within PXR’s ligand-binding pocket that steviol interacted with were investigated using a computational docking study with site-directed mutagenesis assay. Human intestinal cells were treated with steviol and/or PXR antagonist resveratrol (RES) to estimate the function of steviol in cholesterol uptake. Individual pairwise comparisons and other comparisons were analyzed with Student’s t-test and two-way ANOVA, respectively.

Results: Steviol was a more potent agonist of human PXR than mouse PXR. Steviol promoted PXR to dissociate from its nuclear co-repressors. The key amino acids that were essential for the agonistic effects of steviol within PXR’s ligand binding pocket were established. Mechanistically, steviol induced gene expression of key intestinal cholesterol transporters, which led to increased cholesterol uptake by intestinal cells. Future studies in mice are needed to explore if exposure to steviol alters in vivo lipid profiles via PXR signaling. Our study provides potential evidence for the future risk assessment of steviol on cardiovascular disease.

Keywords:
Steviol, PXR, cholesterol uptake, dyslipidemia