A tea catechin, epigallocatechin-3-gallate, is a unique modulator of the farnesoid X receptor

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Abstract

Farnesoid X receptor (FXR) is a ligand-activated nuclear receptor and serves as a key regulator to maintain health of the liver and intestine. Bile acids are endogenous ligands of FXR, and there are increasing efforts to identify FXR modulators to serve as biological probes and/or pharmaceutical agents. Natural FXR ligands isolated from plants may serve as models to synthesize novel FXR modulators. In this study, we demonstrated that epigallocatechin-3-gallate (EGCG), a major tea catechin, specifically and dose-dependently activates FXR. In addition, EGCG induced FXR target gene expression in vitro. Surprisingly, in a co-activator (SRC2) recruitment assay, we found that EGCG does not recruit SRC2 to FXR, but it dose-dependently inhibits recruitment of SRC2 to FXR (IC50, 1 μM) by GW6064, which is a potent FXR synthetic ligand. In addition, EGCG suppressed FXR target gene expression induced by either GW4064 or chenodeoxycholic acid in vitro. Furthermore, wild-type and FXR knockout mice treated with an acute dose of EGCG had induced mRNA expression in a subset of FXR target genes in the intestine but not in the liver. In conclusion, EGCG is a unique modulator of FXR in the intestine and may serve as an important model for future development of FXR modulators.

Highlights

► Epigallocatechin-3-gallate (EGCG) is a unique farnesoid X receptor (FXR) modulator. ► EGCG activates FXR by itself, but inhibits FXR transactivation by other agonists. ► Low concentration of EGCG activates FXR in mouse intestine but not liver. ► EGCG activates FXR to induce a subset of FXR target genes in mouse intestine.

Introduction

FXR (farnesoid X receptor, NR1H4) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. Bile acids are FXR's endogenous ligands. FXR regulates the biosynthesis and enterohepatic circulation of bile acids in addition to its regulation of triglyceride, cholesterol and glucose metabolism (Sinal et al., 2000, Cariou et al., 2005, Rizzo et al., 2005, Ma et al., 2006, Zhang et al., 2006). Recent evidence shows that FXR is also critical in maintaining innate immune responses in intestine (Vavassori et al., 2009). In addition, FXR deficiency in mice results in increased cholestasis, non-alcoholic fatty liver diseases, hepatocellular carcinoma and colon cancer (Gadaleta et al., 2010). This evidence suggests that FXR and FXR-mediated signaling pathways may be promising novel drug targets for the treatment of common metabolic diseases. However, use of synthetic FXR ligands in the clinic has not yet been approved.

The goal of the current study is to screen for FXR modulators in tea plant. Tea is a traditional medicinal plant and is also the most widely consumed beverage in the world, second only to water. The most commonly consumed teas are black, green, and oolong. These teas are all derived from the plant Camellia sinensis, which is a member of the Theaceae family. Epidemiologic and animal studies suggest that drinking green tea offers protection against cardiovascular diseases and a variety of cancers (oral cavity, esophagus, stomach, liver, small and large intestine, and mammary gland) (Setiawan et al., 2001, Zhang et al., 2002, Wu et al., 2003, Jian et al., 2004, Cabrera et al., 2006). Catechins are polyphenolic compounds and are one class of major chemicals found in tea. Examples of tea catechins include (−)-epigallocatechins-3-gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin-3-gallate (ECG), and (−)-epicatechin (EC) (Fig. 1). EGCG is the major constituent and the most biologically active catechin of green tea. Numerous studies report that EGCG can lower oxidative stress, atherosclerosis, inflammation, and cholesterol (Dona et al., 2003, Frei and Higdon, 2003, Raederstorff et al., 2003, Chyu et al., 2004). However, no effect of tea compounds on FXR activity has been reported. Therefore, we aimed to determine the effects of tea catechins, particularly EGCG, in modulating FXR activity in this study.

Section snippets

Chemicals

Thirty-four commercially available samples of green tea (5 g) were extracted with 10 ml of H2O (70 °C, for 10 min) to simulate conditions of regular infusion. The resulting aqueous extracts were concentrated in vacuo and dried overnight at 30 °C in a vacuum oven. All plant samples were prepared at the Department of Medicinal Chemistry, University of Kansas, Lawrence. GW4064 was synthesized by the Department of Medicinal Chemistry, University of Kansas. Chenodeoxycholic acid (CDCA), ECG, EGC, and

Statistical analysis

All data were presented as mean ± SD. All data were analyzed by one-way analysis of variance followed by the Student–Newman–Keuls test. P < 0.05 is considered statistically significant.

Screen of tea preparations for activation of FXR by luciferase assay

Crude extracts from various tea aqueous extracts were used to screen for FXR modulators by luciferase assay. Most of the crude tea extracts activated FXR, but FXR activation was weak (Fig. S1). The major active components in tea are polyphenols with EGCG as the major component followed by EGC, ECG and EC. However, great variations of polyphenols are observed among different types and sources of teas. Therefore, we tested two chemical preparations, A007 (ECG + EGC) and A009 (EGC + EGCG), to

Discussion

The current study identified unique FXR modulators from tea. FXR was activated by tea catechins EGCG, EGC and ECG in a dose-dependent manner as shown by luciferase assay. Activation of FXR by tea catechins induced FXR target gene expression in vitro. However, EGCG by itself did not recruit coactivators to FXR; it instead inhibited FXR agonist-mediated recruitment of coactivators and transactivation of FXR. Notably, activation of FXR by EGCG in vivo appears to be tissue-specific because only

Funding

This study was supported by the National Institutes of Health funding [Grants DK081343 (GLG), GM086415 (TC) and P20-RR021940 (GLG)]; and KUMC Endowment fund (GLG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest statement

The authors have declared that there are no conflicts of interest.

Acknowledgments

The authors would like to appreciate the support from Ms. Melinda Broward for her excellence in coordinating the studies. The authors would like to thank Ms. Noriko Esterly, and Drs. Gemma O'Donnell and Huaping Zhang, as well as Dr. Bruno Hagenbuch and Ms. Megan Ruth for their participation and discussion in the study. We would like to thank Satyanarayana R. Pondugula and Alexander A. Tong for technical assistance.

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