Green tea and one of its constituents, Epigallocatechine-3-gallate, are potent inhibitors of human 11β-hydroxysteroid dehydrogenase type 1

Abstract

The microsomal enzyme 11β-hydroxysteroid deydrogenase type 1 (11β-HSD1) catalyzes the interconversion of glucocorticoid receptor-inert cortisone to receptor- active cortisol, thereby acting as an intracellular switch for regulating the access of glucocorticoid hormones to the glucocorticoid receptor. There is strong evidence for an important aetiological role of 11β-HSD1 in various metabolic disorders including insulin resistance, diabetes type 2, hypertension, dyslipidemia and obesity. Hence, modulation of 11β-HSD1 activity with selective inhibitors is being pursued as a new therapeutic approach for the treatment of the metabolic syndrome. Since tea has been associated with health benefits for thousands of years, we sought to elucidate the active principle in tea with regard to diabetes type 2 prevention. Several teas and tea specific polyphenolic compounds were tested for their possible inhibition of cortisone reduction with human liver microsomes and purified human 11β-HSD1. Indeed we found that tea extracts inhibited 11β-HSD1 mediated cortisone reduction, where green tea exhibited the highest inhibitory potency with an IC50 value of 3.749 mg dried tea leaves per ml. Consequently, major polyphenolic compounds from green tea, in particular catechins were tested with the same systems. (-)-Epigallocatechin gallate (EGCG) revealed the highest inhibition of 11β-HSD1 activity (reduction: IC50 = 57.99 µM; oxidation: IC50 = 131.2 µM). Detailed kinetic studies indicate a direct competition mode of EGCG, with substrate and/or cofactor binding. Inhibition constants of EGCG on cortisone reduction were Ki = 22.68 µM for microsomes and Ki = 18.74 µM for purified 11β-HSD1. In silicio docking studies support the view that EGCG binds directly to the active site of 11β-HSD1 by forming a hydrogen bond with Lys187 of the catalytic triade. Our study is the first to provide evidence that the health benefits of green tea and its polyphenolic compounds may be attributed to an inhibition of the cortisol producing enzyme 11β-HSD1.

Figure 1. Physiological role of the two isoenzymes 11β-HSD type 1 and 2.

Predominant reaction direction of the NADP(H)-dependent enzyme 11β-HSD1 by catalyzing the conversion of inactive cortisone to receptor-active cortisol. The reverse reaction is mediated by the unidirectional NAD-dependent 11β-HSD type 2.

Figure 2. Inhibition of cortisone reduction by three different types of tea (Green, Black and White Tea) in human liver microsomes.

Product yields were quantified by integration of cortisol peaks. The product yields of the controls without any type of tea were normalized to 100% enzyme activity and the residual activity expressed as percent of the control. Bars represent the mean ±SD of at least three repeat experiments.

Figure 3. Dose-dependent inhibition of cortisone reduction by green tea in human liver microsomes. Results are presented as means ±SD (n = 4).

Figure 4. Inhibitory effects of five tea catechins on cortisone reduction in human liver microsomes.

Bars represent the mean ±SD of at least three repeat experiments.

Figure 5. Chemical structures of five green tea catechins used in this study.

EGCG, (−)-epigallocatechin-3-gallate; EGC, (−)-epigallocatechin; EC, (−)-epicatechin; GC, (−)-gallocatechin; C, (−)-catechin.

Figure 6. Dose-dependent inhibition of cortisone reduction by EGCG in human liver microsomes.

Results are presented as means ±SD (n = 4).

Figure 7. Dose-dependent inhibition of cortisol oxidation by EGCG in human liver microsomes.

Results are presented as means ±SD (n = 4).

Figure 8. Dixon plot to characterize the inhibition type and to determine the Ki of EGCG on cortisone reduction in human liver microsomes.

The inset shows direct plotting of the same data.

Figure 9. Dixon plot to characterize the inhibition type and to determine the Ki of EGCG on cortisone reduction by purified 11β-HSD1.

The inset shows direct plotting of the same data.

Figure 10. Western Blot analysis of EGCG treated and EGCG untreated purified human liver 11β-HSD1.

Figure 11. Binding model of EGCG docked to the active site of human 11β-HSD1.

Shown is a binary complex of 11β-HSD1 (PDB: 2RBE) with EGCG (blue) and NADPH (green). K187 belongs to the catalytic triade (yellow) and forms a predicted hydrogen bond with EGCG (distance: 2.1 Å).