Flindissone, a Limonoid Isolated from Trichilia prieuriana, Is an LXR Agonist

Abstract

In this study, the ability of six limonoids from Trichilia prieuriana (Meliaceae) to activate the liver X receptor (LXR) was assessed. One of these limonoids, flindissone, was shown to activate LXR by reporter-gene assays. Flindissone is a ring-intact limonoid, structurally similar to sterol-like LXR ligands. In endogenous cellular settings, flindissone showed an activity profile that is characteristic of LXR agonists. It induced cholesterol efflux in THP-1 macrophages by increasing the cholesterol transporter ABCA1 and ABCG1 gene expression. In HepG2 cells, flindissone induced the expression of IDOL, an LXR-target gene that is associated with the downregulation of the LDL receptor. However, unlike synthetic and similarly to sterol-based LXR agonists, flindissone did not induce the expression of the SREBP1c gene, a major transcription factor regulating de novo lipogenesis. Additionally, flindissone also appeared to be able to inhibit post-translational activation of SREBP1c. The results presented here reveal a natural product as a new LXR agonist and point to an additional property of T. prieuriana and other plant extracts containing flindissone.

Chart 1. Structures of the Compounds Explored in This Study

Figure 1

Concentration–response curves for flindissone (1) and 22R-hydroxycholesterol (22R-OHC) activating the LXRα (A) and LXRβ (B) receptors in luciferase assays employing full-length receptors. Data points show mean ± SEM, N = 4. EC₅₀ and E_max values were obtained by the nonlinear fitting of the log-transformed concentrations using the variable slope. Activation of LXRα-Gal4 (C) and LXRβ-Gal4 (D). Bars show mean, error bars SD, and data points individual values. N = 3. One-way ANOVA with Dunnett’s post hoc test. Significance indicated in comparison to the DMSO (0.1%) control: *: p = 0.05, **: p = 0.01, ***: p = 0.001, ****: p = 0.0001, no indication = not significant.

Figure 2

Depiction of the likely binding mode of (A) 22R-hydroxycholesterol (22R-OHC; green carbon atoms) and (B) flindissone (1; cyan carbon atoms) for the LXRβ ligand binding domain, derived by docking. 24(S),25-Epoxycholesterol (eCH), the ligand present in the cocrystal structure used for docking (PDB 1P8B), is depicted with gray carbon atoms. Magenta dashed lines indicate hydrogen bond interactions; the numbers indicate distances in Å. Amino acid residues forming hydrogen bonds with the ligands via H₂O molecules are marked by the thick tube representation.

Figure 3

Flindissone (1)-mediated stimulation of cholesterol efflux in THP-1 M0 macrophages (A). The gene expression (mRNA level) of the cholesterol transporter ABCA1, measured by RT-qPCR (B) and its protein quantification with the representative blot below (C). Upper panel: 250 kDa: ABCA1. Lower panel: 50 kDa: α/β-tubulin. For all graphs: bars show mean, error bars SD, and data points individual values. N = 3. One-way ANOVA with Dunnett’s post hoc test. Significance indicated in comparison to the DMSO (0.1%) control: *: p = 0.05, **: p = 0.01, ***: p = 0.001, ****: p = 0.0001, no indication = not significant.

Figure 4

Flindissone-mediated target-gene regulation in the HepG2 cell line. The mRNA levels of IDOL (A) and LDLR (B) were measured by RT-qPCR. N = 3. Bars show mean ± SD. One-way ANOVA with Dunnett’s post hoc test. Significance indicated in comparison to the DMSO (0.1%) control: *: p = 0.05, ****: p = 0.0001, ns = not significant.

Figure 5

Flindissone-mediated regulation of lipogenesis in HepG2 cells. Measurements of the cellular neutral lipid content by Oil Red O staining (A). N = 4. Representative images were taken at 40× magnification. Gene expression (mRNA) of SREBF1 (B), measured by RT-qPCR. N = 3. Quantification of the SREBP1c 130 kDa (C) and cleaved 60 kDa product relative to its 130 kDa precursor (D). Representative blots of the SREBP1c protein levels below. Upper panel: 130 kDa: SREBP1c precursor; 60 kDa: cleaved SREBP1c. Lower panel: 42 kDa: actin. For all graphs: Bars show mean, error bars SD, and data points individual values. One-way ANOVA with Dunnett’s post hoc test. Significance indicated in comparison to the DMSO (0.1%) control: *: p = 0.05, **, p = 0.01, no indication or ns = not significant.