Hypolipidemic agent Z-guggulsterone: metabolism interplays with induction of carboxylesterase and bile salt export pump

Abstract

Z-Guggulsterone is a major ingredient in the Indian traditional hypolipidemic remedy guggul. A study in mice has established that its hypolipidemic effect involves the farnesoid X receptor (FXR), presumably by acting as an antagonist of this receptor. It is generally assumed that the antagonism leads to induction of cytochrome P450 7A1 (CYP7A1), the rate-limiting enzyme converting free cholesterol to bile acids. In this study, we tested whether Z-guggulsterone indeed induces human CYP7A1. In addition, the expression of cholesteryl ester hydrolase CES1 and bile salt export pump (BSEP) was monitored. Contrary to the general assumption, Z-guggulsterone did not induce CYP7A1. Instead, this phytosterol significantly induced CES1 and BSEP through transactivation. Z-Guggulsterone underwent metabolism by CYP3A4, and the metabolites greatly increased the induction potency on BSEP but not on CES1. BSEP induction favors cholesterol elimination, whereas CES1 involves both elimination and retention (probably when excessively induced). Interestingly, clinical trials reported the hypolipidemic response rates from 18% to 80% and showed that higher dosages actually increased VLDL cholesterol. Our findings predict that better hypolipidemic outcomes likely occur in individuals who have a relatively higher capacity of metabolizing Z-guggulsterone with moderate CES1 induction, a scenario possibly achieved by lowering the dosing regimens.

Fig. 1.

Effect of Z-guggulsterone on the expression of CES1, CYP7A1, and BSEP. (A) Chemical structure of Z-guggulsterone. (B–D) Regulated expression in human primary hepatocytes. Hepatocytes (n = 4–8) were treated with Z-guggulsterone (10 μM), CDCA (10 μM), or both for 24 h. DMSO (0.1%) was used as the control. The mRNA levels of CES1, CYP7A1, BSEP, GAPDH, or polymerase II by Taqman probes. Columns labeled with a different letter were statistically significant (P < 0.05). To determine the changes in the protein level, lysates (0.5 µg for CES1 and 20 µg for CYP7A1 or BSEP) from pooled samples (n = 4) were resolved by 7.5% SDS-PAGE and transferred electrophoretically to nitrocellulose membranes. The blots were incubated with an antibody against CYP7A1, CES1, or BSEP and then developed with chemiluminescent substrate and reprobed by GAPDH antibody. The signal was captured by Carestream 2200 PRO Imager.

Fig. 2.

Stimulation of the CES1A1 promoter. (A) Concentration-dependent induction of CES1 and BSEP Huh7 cells were treated with Z-guggulsterone (Gugg) at various concentrations (0–20 µM) for 24 h, and then the levels of CES1 and BSEP mRNA were determined as described above. An asterisk indicates statistical significance of a treatment over solvent control, and an alpha indicates statistical significance between two columns linked by a line (P < 0.05). (B) Effect of actinomycin D (Act D) on induction. Huh7 cells were treated with Z-guggulsterone (5 μM) for 24 h in the absence or presence of actinomycin D (1 μM), and the levels of CES1 and BSEP mRNA were determined. An asterisk indicates statistical significance of a treatment over solvent control (P < 0.05). (C) Activation of CES1A1 reporters. Huh7 cells were seeded in 48-well plates. After an overnight incubation, the cells were transfected with a reporter (50 ng) along with 5 ng of the null-Renilla luciferase plasmid. The transfected cells were treated with Z-guggulsterone (10 μM) or the same volume of DMSO for 24 h. Luciferase activities were determined with a dual-luciferase reporter assay. Data in this figure are from three separate experiments.

Fig. 3.

Characterization of CES1A1 guggulsterone element. (A) Stimulation of the CES1A1 and BSEP element reporters. Reporter assays were performed as described above but with element reporters. An asterisk indicates statistical significance of a treatment over solvent control, and an alpha indicates statistical significance between two columns linked by a line (P < 0.05). (B) EMSA analysis. Nuclear extracts (5 μg) from Huh7 cells treated with Z-guggulsterone (10 μM) were incubated with a biotinylated probe (0.01 pmol) for 20 min. In the competition assay, nuclear extracts were preincubated with the unlabeled probe or mutant at 50× (+) or 100× (++) excess for 20 min, and then incubated with the biotinylated probe. The protein-DNA complexes were electrophoretically resolved and detected with streptavidin-conjugated horseradish peroxidase and chemiluminescent substrate.

Fig. 4.

Metabolism of Z-guggulsterone. (A) Chromatograms generated by pooled human liver microsomes. The incubations were performed at 37°C in a total volume of 100 μl containing pooled human liver microsomes (20 μg) and Z-guggulsterone (10 μM) in the presence of NRS. The incubations lasted for 90 min. The elution trace was generated at 240 nm by injecting 20 μl supernatants. (B) Inhibited metabolism of Z-guggulsterone. Z-guggulsterone (10 μM) was metabolized by microsomes (20 μg) in the presence or absence of a CYP inhibitor: 50 μM furafylline, 100 μM pilocarpine, 75 μM thio tepa A, 10 μM quercetin, 20 μM sulfaphenazole, 1 μM ticlopidine, 10 μM quinidine, 100 μM chlormethiazole, or 1 μM ketoconazole. The reactions lasted for 40 min. The disappearance of Z-guggulsterone was monitored. The percentage of inhibition was calculated. (C) Metabolism by recombinant CYPs. The incubations were performed with a recombinant CYP (1 pmol) and Z-guggulsterone (10 μl) for 40 min. All experiments were conducted twice with each in triplicate.

Fig. 5.

Analyses of metabolites by HPLC and LC-MS. (A) Time-course metabolism by CYP3A4. The incubations were performed at 37°C with 1 pmol CYP3A4 and 10 μM Z-guggulsterone for 0–120 min. The HPLC elution trace was generated at 240 nm. (B–E) HPLC chromatogram and full scan of metabolites M1, M2, M3, and M4, respectively.

Fig. 6.

Functional characterization of the metabolites. (A) Differential induction of CES1 and BSEP Huh7 cells were treated with Z-guggulsterone (2 μM) or a metabolite (2 μM) for 24 h. The mRNA levels of CES1 and BSEP were determined. An asterisk indicates statistical significance of a metabolite over Z-guggulsterone, and an alpha indicates statistical significance between two columns linked by a line (P < 0.05). (B) Differential activation of CES1 and BSEP element reporters. Huh7 cells were seeded in 48-well plates. After overnight incubation, the cells were transfected with a reporter (50 ng) and 5 ng of the null-Renilla luciferase plasmid. The transfected cells were treated with Z-guggulsterone (2 μM), a metabolite (2 μM), or the same volume of DMSO for 24 h. The reporter luciferase activities (firefly) were normalized according to the null-Renilla luciferase activities. An asterisk indicates statistical significance of a metabolite over Z-guggulsterone, and an alpha indicates statistical significance between two columns linked by a line (P < 0.05). (C) Effect of CYP3A4 on the induction of CES1 and BSEP. Huh7 cells were seeded in 24-well plates at a density of 1.5 × 10⁵ and cultured overnight. The cells were transfected with a CYP3A4 expression construct or the corresponding vector (0.5 µg). The transfected cells were cultured for 24 h and then treated with Z-guggulsterone at 5 μM for 24 h. Total RNA was isolated and analyzed for the levels of CES1 and BSEP mRNA. The data were collected from three separate experiments. *P < 0.05.

Fig. 7.

Effect of Z-guggulsterone, CDCA, and both on the expression of mouse bsep, mouse cyp7a1, and esterase activity staining. (A) Expression of mouse bsep and cyp7a1. Mouse primary hepatocytes (n = 3) were treated with Z-guggulsterone (10 μM), CDCA (10 μM), or both for 24 h. DMSO (0.1%) was used as the control. Lysates (25 µg) were resolved by 7.5% SDS-PAGE and transferred electrophoretically to nitrocellulose membranes. The blots were incubated with an antibody against bsep or cyp7a1, developed with chemiluminescent substrate, and reprobed by GAPDH antibody. The signal was captured by Carestream 2200 PRO Imager. (B) Nondenaturing electrophoresis stained for hydrolytic activity. Mouse primary hepatocytes and human HepG2 cells were treated as described above. Lysates from mouse hepatocytes (5 µg) or HepG2 cells (20 µg) were subjected to native gel electrophoresis and stained for esterase activity with 1-naphythalacetate as described in Experimental Procedures. The staining intensity was captured by Carestream 2200 PRO Imager. Note the glycosylation variants of CES.

Fig. 8.

Response elements and interplays among CES1, BSEP, and CYP7A1. (A) CES1A1 and BSEP guggulsterone response elements. The antioxidant response elements (ARE) are italicized, whereas the activator protein-1 sites (AP-1) are bolded. (B) Multiple interplays among metabolism, CES1/BSEP induction, lipid retention, and bile acid synthesis The arrows point to the targets that an enzyme or compound acts on or is converted into. When two arrows points to the same target, the solid line denotes a major effect and the dotted line denotes a minor effect. The double-lined arrow denotes an increased role when the induction of CES1 exceeds the capacity of bile acid synthesis.