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Green Tea and One of Its Constituents, Epigallocatechine-3-gallate, Are Potent Inhibitors of Human 11β-hydroxysteroid Dehydrogenase Type 1

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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.
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Green Tea and One of Its Constituents,
Epigallocatechine-3-gallate, Are Potent Inhibitors of
Human 11b-hydroxysteroid Dehydrogenase Type 1
Jan Hintzpeter, Claudia Stapelfeld, Christine Loerz, Hans-Joerg Martin, Edmund Maser*
Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Germany
Abstract
The microsomal enzyme 11b-hydroxysteroid deydrogenase type 1 (11b-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 11b-HSD1 in various metabolic disorders including insulin resistance, diabetes type 2, hypertension, dyslipidemia and
obesity. Hence, modulation of 11b-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 11b-HSD1. Indeed we found that tea extracts inhibited 11b-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. (2)-
Epigallocatechin gallate (EGCG) revealed the highest inhibition of 11b-HSD1 activity (reduction: IC50 = 57.99 mM; oxidation:
IC50 = 131.2 mM). 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 mM for microsomes and Ki = 18.74 mM for
purified 11b-HSD1. In silicio docking studies support the view that EGCG binds directly to the active site of 11b-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
11b-HSD1.
Citation: Hintzpeter J, Stapelfeld C, Loerz C, Martin H-J, Maser E (2014) Green Tea and One of Its Constituents, Epigallocatechine-3-gallate, Are Potent Inhibitors
of Human 11b-hydroxysteroid Dehydrogenase Type 1. PLoS ONE 9(1): e84468. doi:10.1371/journal.pone.0084468
Editor: Kang Sun, Fudan University, China
Received August 13, 2013; Accepted November 21, 2013; Published January 3, 2014
Copyright: ß2014 Hintzpeter et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Work on 11b-HSD1 in our laboratory is funded by the German Federal Ministry of Education and Research (0315397A) and the Deutsche
Forschungsgemeinschaft (MA 1704/5-2). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: maser@toxi.uni-kiel.de
Introduction
Tea (Camellia sinensis) is the second most widely consumed
beverage in the world after water [1] and has been cultivated for
thousands of years due to its medicinal benefits and general health
promotion purposes.
The tea plant is naturally occurring in South China, but is
nowadays cultivated in many other regions of the major tea
producing countries in the world, like India, Japan, Sri Lanka,
Indonesia and Kenia. In general, tea can be divided into three
types (percentage of worlds tea production): non-fermented green
tea (20%), semi-fermented tea (e.g. oolong tea and white tea) (2%)
and fermented black tea (78%) [2]. Additionally, there are more
than 300 different kinds of tea that differ regarding the
manufacturing process.
The most popular form of tea consumed in the world is black
tea, whereas green tea is mainly consumed in China and Japan.
Recently, plenty of commercial beverages came to market that
contain tea extracts or catechins from tea. Nowadays, tea or
beverages containing tea extracts, if consumed daily, belong to a
life-style that might support healthiness and long life, which is
underpined by several laboratory, epidemiological and human
intervention studies [3–7].
In particular, consumption of green tea has been associated with
a reduction of the risk of cardiovascular disease, some forms of
cancer, as well as with the promotion of oral health and other
physiological functions such as antibacterial and antiviral activity,
neuroprotective properties, anti-hypertensive effects, body weight
control and diabetes type 2 prevention [8,9].
The latter diseases are risk factors of the metabolic syndrome
(obesity, insulin resistance, hypertension, diabetes type 2, dyslipi-
demia) against which the therapeutical potential of tea has been
shown in humans and model organisms in numerous studies [10–
14].
In most cases the beneficial effects have been attributed to the
polyphenolic compounds, especially catechins, although a large
number of potentially bioactive chemicals are present in tea as well
[15]. (2)-Epigallocatechin-3-gallate (EGCG) is the major compo-
nent among the tea catechins and is believed to have a
considerable therapeutical potential [16].
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Unlike semi-fermented and fermented teas (black and white
teas) unfermented green tea contains more catechins [17]. A
typical green tea infusion of 250 ml hot water with 2.5 g tea leaves
approximately contains 620–880 mg of water-extractable solid
compounds. About 30–42% of the dry weight of green tea consists
of phenolic compounds [18,19], from which EGCG is the most
abundant one (up to 50–80% of the total catechins [18]).
Other catechins are present in smaller amounts: (2)-epigallo-
catechin (EGC).(2)-epicatechin gallate (ECG).(2)-epicatechin
(EC).gallocatechin gallate (GCG).gallocatechin (GC).cate-
chin gallate (CG).catechin (C).epigallocatechin digallate .
epicatechin digallate [18].
Many other components have been identified in tea that might
have an effect on human health: theaflavins, thearubigins,
theasinesins, gallic acid, quinic acid, theogallin, coumaryl-quinic
acid, caffeine, theobromine and theophylline, L-theanine (unique
to tea), kaempferol, myricetin and quercetin [2,17].
It has been estimated that up to one-third of patients with
diabetes type 2, hypertension and dyslipidaemia consume some
form of complementary and alternative medicine, involving the
use of herbs and other dietary supplements, like plant infusions, as
alternatives to mainstream Western medical treatment [20]. For
this reason, our lab is in search of herbs that are traditionally used
for the treatment of symptoms of the metabolic syndrome in the
medicine of several indigenous populations as well as in traditional
Chinese medicine. In comparison with high-throughput screening
of large chemical libraries to discover new drugs, medical
treatments of chronic diseases by traditional Chinese medicine
and indigenous populations feature clear benefits based on their
thousand-year-old experience in dealing with some of the above
mentioned chronic diseases [21].
In humans high levels of intracellular cortisol can increase
glucose output in the liver, induce fat accumulation and weaken
insulin sensitivity in adipose tissues, resulting in an increased risk
for the metabolic syndrome [22]. Cortisol-levels are regulated by
two known isoforms of 11b-hydroxysteroid dehydrogenase (see
Figure 1): a microsomal NADPH-dependent 11b-hydroxysteroid
dehydrogenase type 1 (11b-HSD1) primarily acting as a reductase
in liver, fat tissues, lung and macrophages and a NAD dependent
11b-hydroxysteroid dehydrogenase type 2 (11b-HSD2) that is
expressed in kidney, salivary glands, colon and placenta. 11b-
HSD2 is a unidirectional oxidase that deactivates glucocorticoid-
receptor active cortisol to cortisone [23–26].
Multiple evidence argues for an important aetiological role of
11b-HSD1 in various metabolic disorders including insulin
resistance or rather diabetes type 2, hypertension, dyslipidaemia
and obesity [22,27–31]. 11b-HSD1-deficient mice show enhanced
glucose tolerance, improved hepatic insulin resistance, attenuated
gluconeogenesis, and an improved lipid and lipoprotein profile
[32–34]. Moreover, overexpression of 11b-HSD1 selectively in
adipose tissue causes visceral obesity, insulin resistance, type 2
diabetes, dyslipidemia, and hypertension in mice [22].
Collectively, these findings emphasize the potential benefits of a
specific inhibitor of human 11b-HSD1 in the treatment of obesity
and type 2 diabetes. In the present investigation, we tested several
teas and some major tea-specific polyphenolic compounds,
catechins, for their capability to inhibit the cortisol producing
enzyme 11b-HSD1. Inhibition of 11b-HSD1 would shed light on
– at least some of – the mechanisms by which green tea performs
its health effect.
Materials and Methods
Ethics Statement
Work on pluripotent carbonyl reductases/hydroxysteroid de-
hydrogenases, including 11b-HSD1, was approved by the ethics
commission of the Medical Faculty, University of Kiel. Human
liver tissue was obtained with written informed consent and
approval by ethics commission of the Medical Faculty, University
of Kiel. All experimental studies were performed in accordance
with German legislation.
Chemicals
Cortisol, cortisone and EGCG were purchased from Sigma-
Aldrich (St. Louis, MO). The following chemicals were procured
from Carl Roth GmbH +Co. KG (Karlsruhe, Germany):
Ethylacetate, NADP, NADPH, non-fat dry milkpowder, mono-
sodium phosphate and potassium chloride, HPLC column
LiChrospherH100 RP-18, methanol, sodium chloride and
monopotassium phosphate were obtained from Merck (Darmstadt,
Germany). (2)-Epicatechin gallate, (2)-catechin and (2)-galloca-
techin were purchased from Biomol GmbH (Hamburg, Germany),
Cayman Chemical (Ann Arbor, MI) and LKT Laboratories Inc.
(St. Paul, MN), respectively. The different types of commercially
available teas consisting of green, oolong and black teas were
bought from supermarkets in Kiel, Germany. The anti-HSD11B1
antibody (ab83522) was obtained from Abcam (Cambridge, UK).
Preparation of tea extracts
Dried tea leaves were reduced to small pieces and 1 g of the
crushed material was suspended in 10 mL water without stirring
for 90 min at room temperature. Insoluble material was removed
by centrifugation (13,000 RPM in a microcentrifuge) and the
resulting supernatant was used in enzyme inhibition experiments.
Preparation of 11b-HSD1-containing microsomes and
purification of human 11b-HSD1
Human liver microsomes and purified human 11b-HSD1 were
prepared as described previously [35,24].
Enzyme assay
In terms of 11b-HSD1 reductase activity, inhibition analysis was
performed as follows: 50 ml of tea extract were added to a mixture
containing 400 mM cortisone, 0.8 mM NADPH solution in
100 mM phosphate buffer pH 7.4. The reaction was started by
adding 5 ml of human liver microsomes (218 mg protein) and the
mixture was incubated for 3 h at 37uC. The total assay volume
was 250 ml.
The reaction was stopped by adding 250 ml ice-cold ethylacetate
and vortexing, followed by a 30 sec centrifugation at 13,000 RPM
in a microcentrifuge. The organic phase was separated and the
aqueous phase extracted two more times with 250 ml ethylacetate.
The ethylacetate phases were combined and the solvent was
removed in a SpeedVac (ThermoSavant). The resulting residue
was dissolved in 150 ml methanol-water (58:42, v/v) and used for
metabolite quantification by HPLC.
To analyze inhibition of 11b-HSD1 dehydrogenase activity, the
oxidation of cortisol to cortisone was measured essentially as
described above, except for the substitution of NADPH with
0.8 mM NADP, and the use of 400 mM cortisol instead of
cortisone. Here, the incubation time was 60 min at 37uCina
100 mM phosphate buffer pH 9.0.
11b-HSD1 Inhibition by Green Tea and EGCG
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Metabolite quantification by HPLC
After enzymatic conversion the glucocorticoids were resolved as
published previously [36]. Product yield was quantified by peak
integration of the cortisone and cortisol peaks, respectively.
Determination of the mode of 11b-HSD1 inhibition by
EGCG and of its inhibition constant Ki
The type of inhibition by EGCG (dissolved in buffer solution)
was determined with four inhibitor (0, 20, 50, 75, 100 mM EGCG)
and substrate (50, 100, 200, 400 mM cortisone) concentrations.
The data obtained were plotted as substrate-velocity curves and
analyzed by nonlinear-regression. The type of inhibition was
tested with both human liver microsomes (218 mg) and purified
11b-HSD1 (16.4 mg).
Western Blot analysis after EGCG incubation with
microsomal and purified 11b-HSD1
To exclude EGCG-induced 11b-HSD1 aggregate formation,
purified 11b-HSD1 (2.14 mg) was preincubated with 25–100 mM
EGCG (3 h at 37uC) and then prepared for sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) according to the
manufacturer’s protocol. As a control, the same volume of
untreated purified protein was loaded onto the parallel lane.
The resolved proteins were transferred to nitrocellulose
membranes. After blocking the membrane for 1 h at RT with
5% non-fat dry milkpowder in PBS-buffer, the blocking solution
was replaced by the primary antibody at a 1:2,000 dilution and the
membrane was incubated at 4uC for about 16 h. Primary and
secondary antibodies were diluted in PBS containing 2.5% non-fat
dry milkpowder. The membrane was washed three times and then
the corresponding secondary antibody was added at a dilution of
1:10,000. After a 1.5 h incubation at RT the membrane was
washed three times and the specific Western blot signals of 11b-
HSD1 were detected by the ECL Western Blotting System
according to the manufacturer’s instructions.
Quantification of hydrogen peroxide by xylenol orange
According to Halliwell [37] and Lay et al. [38], EGCG induces
hydrogen peroxide production in various buffer systems. For this
reason, we determined the hydrogen peroxide concentration
produced by EGCG (concentration 200 mM) after 3 h incubation
at 37uC either in the buffer used or in our complete assay systems
[39]. Further, to exclude any inhibitory effects of hydrogen peroxide
on cortisone reduction, we tested the influence of hydrogen peroxide
on cortisol production by HPLC (as described above) with both
human liver microsomes and purified 11b-HSD1, respectively.
Figure 1. Physiological role of the two isoenzymes 11b-HSD type 1 and 2. Predominant reaction direction of the NADP(H)-dependent
enzyme 11b-HSD1 by catalyzing the conversion of inactive cortisone to receptor-active cortisol. The reverse reaction is mediated by the unidirectional
NAD-dependent 11b-HSD type 2.
doi:10.1371/journal.pone.0084468.g001
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 6SD of at least three
repeat experiments.
doi:10.1371/journal.pone.0084468.g002
Figure 3. Dose-dependent inhibition of cortisone reduction by
green tea in human liver microsomes. Results are presented as
means ±SD (n = 4).
doi:10.1371/journal.pone.0084468.g003
11b-HSD1 Inhibition by Green Tea and EGCG
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Results
Concentration-dependent inhibition of cortisol
production by three types of tea in human liver
microsomes
Inhibition of 11b-HSD1 activity was assessed by determining
cortisol yields after 3 h incubation at 37uC with equal volumes of
green, black and white tea infusions (100 mg tea per ml; prepared
as described above). Quantification was performed by integration
of the well-resolved product peaks in the above discribed HPLC
assay. Figure 2 shows the inhibitory effects of three commercially
available teas on human microsomal 11b-HSD1 reductase
activity. Conversion of cortisone to cortisol was inhibited by all
three types of tea in a concentration-dependent manner, with
green tea exhibiting the highest inhibition.
Figure 5. Chemical structures of five green tea catechins used in this study. EGCG, (2)-epigallocatechin-3-gallate; EGC, (2)-
epigallocatechin; EC, (2)-epicatechin; GC, (2)-gallocatechin; C, (2)-catechin.
doi:10.1371/journal.pone.0084468.g005
Figure 4. Inhibitory effects of five tea catechins on cortisone reduction in human liver microsomes. Bars represent the mean 6SD of at
least three repeat experiments.
doi:10.1371/journal.pone.0084468.g004
11b-HSD1 Inhibition by Green Tea and EGCG
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Concentration-dependent inhibition of cortisol
production by green tea in human liver microsomes
As shown in Figure 2, green tea extract displayed the highest
inhibitory potency of the three types of tea tested. With human
liver microsomes and different amounts of green tea extract a
dose-response inhibition curve of microsomal cortisone reduction
was observed. An IC50-value of 3.75 mg crushed dried tea leaves
per ml (this corresponds to a value of 0.75 vol% of green tea
extract in the reaction mixture) was determined (Fig. 3).
Inhibition of cortisol production by several constituents
of green tea in human liver microsomes
Inhibition of cortisone reduction in human liver microsomes
was assessed with several green tea constituents. Figure 4 shows the
residual enzymatic activity after incubation with five catechins
found in green tea (see Fig. 5) measured at three concentrations.
Highest inhibition was achieved with EGCG, followed by GC
(gallocatechin). The half maximal inhibitory concentration (IC50)
determined for EGCG on 11b-HSD1 activity in microsomes was
57.99 mM (Figure 6). It should be noted that an aqueous solution
of EGCG, when stored at 220uC for one week, resulted in an
IC50-value about half as high (26.88 mM) as that determined with
freshly prepared EGCG solutions.
To determine possible differences in the potential of EGCG to
inhibit reductase and dehydrogenase activity of 11b-HSD1, the
influence of EGCG on the oxidation of cortisol to cortisone was
monitored. Figure 7 shows the EGCG concentration-dependent
decrease of 11b-HSD1 cortisol dehydrogenase activity in micro-
somes. From these data an IC50-value of 131.20 mM was
calculated.
Determination of the type of inhibition by EGCG and of
its dissociation constant Ki with human liver microsomes
and purified 11b-HSD1
To determine the mechanism of 11b-HSD1 inhibition by
EGCG various inhibitor and substrate concentrations were used.
Non-linear regression analysis yielded Ki -values of
Ki = 22.68610.10 mM and Ki = 18.7468.00 mM for microsomes
and the purified enzyme, respectively (Insets Figs. 8,9 and Table 1).
The data were best fitted using a model of mixed inhibition. In line
with this calculation were the results of the Dixon-plots
(Figures 8,9). The regression lines converge above the abscissa
indicating a competetive or mixed mode of inhibition and Ki -
values similar to those obtained by non-linear regression (Table 1).
Western Blot analysis after EGCG incubation with purified
11b-HSD1
As shown in Figure 10, EGCG preincubation (3 h at 37uC) with
purified 11b-HSD1 did not induce protein aggregate formation.
No differences in the treated (lane 2–4) and untreated samples
(lane 1) of purified 11b-HSD1 were observed, even at EGCG
concentrations up to 100 mM.
Quantification of EGCG generated hydrogen peroxide by
xylenol orange
When 200 mM EGCG was incubated for 3 h at 37uC in buffer
only (same buffer as used in our assays above), the formation of
220 mM hydrogen peroxide was determined by the xylenol orange
assay. However, when human liver microsomes, NADPH and
cortisone was added (in the same concentrations as in the enzyme
assays), only 12 mM hydrogen peroxide could be detected.
Further, we tested the influence of up to 300 mM hydrogen
peroxide on cortisol production with both human liver micro-
somes and purified human 11b-HSD1, respectively. No 11b-
HSD1 inhibition by hydrogen peroxide could be detected (data
not shown).
Discusssion
Tea infusions have not only been a popular beverage for
thousands of years but also have been traditionally used for the
treatment of the metabolic syndrome and other chronic diseases.
The metabolic syndrome is a complex metabolic disorder that may
lead to diabetes type 2 and is one of the fastest growing diseases
within the western civilisation. The role of glucocorticoid
hormones in maintaining the blood glucose level by mobilization
of energy sources is essential for homeostasis: Cortisol deficiency
results in low blood glucose levels, dizziness, generalized weakness
and stress sensitivity while cortisol overproduction, hypercortisol-
ism, leads to high blood pressure, increased abdominal fat
deposition, insulin resistance etc. Due to its pivotal role in cortisol
production, inhibition of 11b-HSD1 maybe of therapeutic value in
the treatment of the metabolic syndrome and diabetes.
For these reasons our laboratory has investigated the inhibitory
effects of tea, in particular green tea and some of its constitutents,
on microsomal and purified 11b-HSD1 mediated cortisone
reductase (and cortisol dehydrogenase) activity. The present study
demonstrates that tea and green tea, respectively, and some of
their polyphenolic constituents, EGCG and GC, have the
capability to inhibit human 11b-HSD1.
Figure 6. Dose-dependent inhibition of cortisone reduction by
EGCG in human liver microsomes. Results are presented as means
6SD (n = 4).
doi:10.1371/journal.pone.0084468.g006
Figure 7. Dose-dependent inhibition of cortisol oxidation by
EGCG in human liver microsomes. Results are presented as means
6SD (n = 4).
doi:10.1371/journal.pone.0084468.g007
11b-HSD1 Inhibition by Green Tea and EGCG
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Initially, we could show that non-fermented green tea, semi-
fermented tea (e.g. oolong tea and white tea) and fermented black
tea have different potencies to inhibit human 11b-HSD1 mediated
cortisone reduction. Non-fermented green tea showed the highest
inhibitory potential (IC50 of 3.75 mg crushed dried tea leaves per
ml, which corresponds to a value of 0.75 vol% of green tea
extract), whereas a weaker inhibition potency was observed by
semi-fermented white and fermented black tea.
A reason for higher 11b-HSD1 inhibition by green tea
compared to other teas might be a higher concentration of
phenolic compounds in the former. During the fermentation
process phenolic compounds, like catechins, are enzymatically
oxidized. To protect the polyphenolic compounds from enzymatic
oxidation, tea leaves that are destined to become green tea are
withered by air drying prior to heat inactivation of polyphenol
oxidases.
Consequently, we tested several polyphenolic compounds
known from green tea, in particular catechins, for their inhibition
of 11b-HSD1 cortisone reduction. Interestingly, we observed big
differences in the inhibition potency among the structurally related
catechins (see Figure 5). For example, (2)-epigallocatechin, (2)-
epicatechin and (2)-catechin showed almost no or very little
inhibition ((2)-epigallocatechin ca. 25% inhibition at 200 mM).
However, when (2)-epigallocatechin gallate (ca. 75% inhibition at
100 mM) and (2)-gallocatechin (ca. 53% inhibition at 100 mM)
were used as inhibitors, a much larger effect could be detected. A
possible explanation might be that all other catechins tested in this
study are non-gallic acid esterified compounds (Fig. 5). (2)-
Epigallocatechin and (2)-gallocatechin are diastereoisomers (Fig. 5)
from which (2)-gallocatechin showed a greater inhibition (Fig. 4).
The gallic acid esterified form of (2)-epigallocatechin, EGCG, is
the most potent substance tested so far, which may lead to the
assumption that the gallic acid ester form of (2)-gallocatechin, (2)-
gallocatechin gallate may exhibit an even greater inhibitory
potential than EGCG. Nevertheless, EGCG showed the highest
inhibition in this study and was an obvious choice for further
investigation.
From cell culture experiments it is known that polyphenols like
EGCG and EGC trigger a rapid hydrogen peroxide generation in
various buffer systems. Concentrations up to 220 mM of hydrogen
peroxide generated by 200 mM EGCG (incubated for 3 h at 37uC)
in the buffer systems used could be detected. In contrast, addition
of human liver microsomes, NADPH and cortisone to the reaction
mixture resulted in about 12 mM hydrogen peroxide. A possible
explanation for this hydrogen peroxide preventing effect might be
the presence of peroxisomes in the microsomal fraction which
contain several peroxidases known to catalyze the reduction of
hydrogen peroxide to water. Further, due to the possibility that
hydrogen peroxide generated by catechins could be responsible for
the 11b-HSD1 inhibition observed in our study, we tested the
inhibitory effects of up to 300 mM hydrogen peroxide on cortisone
reduction. However, no inhibition of cortisol formation by both
human liver microsomes and purified human 11b-HSD1,
respectively, could be detected.
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.
doi:10.1371/journal.pone.0084468.g008
11b-HSD1 Inhibition by Green Tea and EGCG
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Next, we determined the IC50-values of EGCG to inhibit the
conversion of cortisone to cortisol and vice versa by human liver
microsomes. The results indicate that the dehydrogenase activity
seems to be less affected by EGCG than the reducing activity
(reduction: IC50 = 57.99 mM; oxidation: IC50 = 131.20 mM; see
Figs. 6, 7).
As mentioned before, 11b-HSD1 reductase and dehydrogenase
activities are varying from tissue to tissue [25]. Moreover, recent
reports indicate the capability of cortisol oxidation by 11b-HSD1
in subcutaneous adipose tissues [40]. Hence, a shift in the
equilibrium towards cortisone may occur in vivo. In the past,
selectivity of inhibitors for 11b-HSD1 over 11b-HSD2 has
received most attention. In addition, effectiveness of inhibitors
should also consider a selectivity for cortisone reduction by 11b-
HSD1 over its cortisol to cortisone dehydrogenase reaction. This
assumption may be underpined by the fact that many selective
11b-HSD1 inhibitors failed in clinical trials [41].
Figure 9. Dixon plot to characterize the inhibition type and to determine the Ki of EGCG on cortisone reduction by purified 11b-
HSD1. The inset shows direct plotting of the same data.
doi:10.1371/journal.pone.0084468.g009
Table 1. Inhibition constants of EGCG on cortisone reduction with human liver microsomes and purified human 11b-HSD1.
human liver microsomes purified human 11b-HSD1
Ki (non-linear regression) 22.68 mM 18.74 mM
Ki (estimated by Dixon-plot) 17.80 mM 20.93 mM
Data are derived from non-linear regression curves and Dixon-plots (Figs. 8–11).
doi:10.1371/journal.pone.0084468.t001
11b-HSD1 Inhibition by Green Tea and EGCG
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A recently published paper about the inhibition of cortisol
production by EGCG in rat liver microsomes proposes a redox
shift in the lumen of the endoplasmic reticulum (ER) being
responsible for the EGCG dependent effect [42]. However, in our
present study we used purified 11b-HSD1 from human liver for
EGCG inhibition studies. Detailed kinetic analysis yielded a mixed
mode of EGCG on 11b-HSD1 inhibition with respect to cortisone.
Moreover, we were able to determine a Ki for EGCG of
18.74 mM using non-linear regression analysis, which is similar to
the Ki determined by using human liver microsomes
(Ki = 17.8 mM). As a consequence, our results indicate a direct
effect of EGCG to both purified and microsomal 11b-HSD1
rather than a EGCG-induced shift of NADPH to NADP in the ER
lumen. To strengthen our findings, EGCG was docked into the X-
ray crystal structure of human 11b-HSD1 (PDB: 2RBE) using
SwissDock [43]. The result of EGCG positioned into the active
site of 11b-HSD1 (Figure 11) support the direct binding of EGCG
into the substrate-binding pocket. EGCG forms a hydrogen bond
with Lys187 that is part of the catalytic triade of short-chain
dehydrogenases, suggesting a direct competition with binding of
the substrate and/or cofactor, respectively.
As reported by others [44], many microsomal (drug metabo-
lizing) enzymes (glyceraldehyde-3-phosphate dehydrogenase,
CYP1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A, catechol-O-
methyltransferase and microsomal glutathione transferase 1) are
inhibited by EGCG. In these cases, the underlying mechanism of
inhibition was suspected to be protein aggregate formation in
EGCG-treated microsomes. To exclude protein aggregate forma-
tion by EGCG in our studies, 11b-HSD1 was incubated with
increasing EGCG concentrations of up to 100 mM for 3 h at 37uC
and then analyzed by Western Blot. From our results any
crosslinking of purified 11b-HSD1 by EGCG could be ruled
out, as no multimeric forms at higher molecular weight regions
were observed (Figure 10).
In conclusion, we provide evidence that aqueous extracts of tea
(Camellia sinensis) are able to inhibit cortisol formation by the
enzyme 11b-HSD1. From several abundant constitutents of tea,
the major phenolic compound EGCG could be identified as the
most potent inhibitor of human 11b-HSD1, with inhibition
constants of Ki = 22.68 mM in microsomes and Ki = 18.74 mM
Figure 10. Western Blot analysis of EGCG treated and EGCG
untreated purified human liver 11b-HSD1.
doi:10.1371/journal.pone.0084468.g010
Figure 11. Binding model of EGCG docked to the active site of human 11b-HSD1. Shown is a binary complex of 11b-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 A
˚).
doi:10.1371/journal.pone.0084468.g011
11b-HSD1 Inhibition by Green Tea and EGCG
PLOS ONE | www.plosone.org 8 January 2014 | Volume 9 | Issue 1 | e84468
for the purified enzyme, respectively. The mechanism of EGCG
inhibition is most likely a direct binding to the active site of 11b-
HSD1, which is supported by enzyme kinetic studies and a
computer aided docking model. Our results decipher the
mechanism by which catechins such as EGCG, or green tea in
general, have been successfully consumed for thousand of years for
general health benefits. These polyphenolic compounds may serve
as model structures for the development of novel agents to treat
the metabolic syndrome and related diseases.
Author Contributions
Conceived and designed the experiments: JH CS CL HJM. Performed the
experiments: CS CL. Analyzed the data: JH CS CL HJM. Contributed
reagents/materials/analysis tools: EM. Wrote the paper: JH HJM. Added
valuable points of discussion: JH CL CS HJM EM.
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11b-HSD1 Inhibition by Green Tea and EGCG
PLOS ONE | www.plosone.org 9 January 2014 | Volume 9 | Issue 1 | e84468
... Une étude récente a montré que des souris KO pour cette enzyme présentait une résistance aux effets cognitifs et émotionnels du stress chronique et ce à long terme, suggérant que les effets neurobiologiques du stress dépendent en grande partie des GC générés localement par la 11β-HSD1 dans différentes structures cérébrales (Wheelan et al., 2018a). La 11β-HSD2, quant à elle, possède exclusivement une activité déshydrogénase (Hintzpeter et al., 2014), c'est-à-dire qu'elle inactive le cortisol (ou la corticostérone) en cortisone (figure 4 ...
... Une partie de ces glucocorticoïdes passe la barrière placentaire et atteignent le foetus, ce qui peut engendrer des perturbations de l'activité de son axe HPA en cours de développement (Koehl et al., 1999). En condition physiologique le foetus est protégé contre les GC maternels par l'enzyme 11βHSD2 qui dégrade la corticostérone (ou le cortisol chez l'Homme) en un dérivé inactif (Hintzpeter et al., 2014). En revanche, un stress prénatal (stress de contention infligé à la mère par exemple) entraîne une réduction de l'expression de la 11βHSD2 et de son activité dans le placenta , ce qui participe à la hausse de GC chez le foetus et à la réactivité accrue de son axe HPA, qui se maintiennent à la naissance (Maccari et al., 1995). ...
... Figure 4 : Mécanisme d'activation et d'inactivation du cortisolLa 11β-HSD active la cortisone en cortisol, tandis que la 11β-HSD2 inactive le cortisol en cortisone (d'aprèsHintzpeter et al., 2014).MR concerne majoritairement le système limbique notamment l'HPC, l'amygdale, le septum et l'hypothalamus. Par contre, la distribution des GR est ubiquitaire avec une forte densité dans l'HPC et le PVN de l'hypothalamus (figure 5). ...
Thesis
Le stress chronique est un phénomène universel qui concerne toutes les strates de la société, quels que soient l’âge, le sexe, le milieu social, les origines culturelles et géographiques. Néanmoins la vulnérabilité est variable selon l’âge d’exposition. Il est aujourd’hui avéré qu’une exposition chronique à un stress ou bien une exposition à un stress aigu intense chez l’enfant, voire même pendant la période périnatale, peut avoir des conséquences dramatiques sur le développement cérébral et peut engendrer un certain nombre de pathologies neuropsychiatriques plus tard au cours de la vie. Néanmoins, une plus forte résistance et une capacité de résilience apparaît à l’âge adulte se caractérisant notamment par une adaptabilité et la possibilité de retourner rapidement à un état initial après une exposition au stress. Toutefois, des travaux récents suggèrent qu’une seconde phase de vulnérabilité apparait en milieu de vie, période charnière durant laquelle certaines structures du système limbique commencent peu à peu à décliner. L’objectif de cette thèse a été ainsi dans un premier temps de mieux caractériser la nature des perturbations somatiques à court terme et des déficits comportementaux à long terme qui apparaissent suite à un stress chronique durant cette période charnière au cours de laquelle s’installe une vulnérabilité accrue aux stress. Pour répondre à cette première problématique, le modèle de stress chronique léger et imprédictible mis au point chez la souris a été utilisé. Les impacts à court terme ont été déterminés en analysant les troubles somatiques et endocriniens, puis ceux à long-terme (chez le sujet âgé), ont été déterminés en analysant les troubles comportementaux anxieux, dépressifs et cognitifs, caractéristiques du syndrome de stress. D’autre part, il s’agissait d’identifier les réorganisations épigénétiques associés au vieillissement et au stress chronique ayant eu lieu en milieu de vie. Nous nous sommes concentrés sur les perturbations de la marque permissive H3K9ac et la marque répressive H3K27me3 en fonction du vieillissement et du stress chronique à long terme. Enfin, une approche pharmacologique a été utilisée pour évaluer l’impact d’un traitement ciblant ces mécanismes épigénétiques sur les déficits comportementaux. Un traitement chronique de 3 semaines à base de butyrate de sodium (NaB) qui est un iHDAC, a été administré aux animaux après la phase de stress chronique, puis les évaluations comportementales ont été réalisé à long terme, chez les sujets âgés.
... μM (Feng et al. 2011). A catechin derivative, EGCG (35), from green tea showed moderate inhibitory effect against h-11β-HSD1 with an IC 50 of 57.99 μM (Hintzpeter et al. 2014). A cyclic tetrapeptide, penicopeptide A (268) isolated from the culture of endophytic fungus Penicillium commune of grape plant, Vitis vinifera, in rice, inhibited the activity of h-11β-HSD1 with an IC 50 of 9.07 μM ). ...
... Accumulating evidence strongly demonstrates that the enzyme 11β-HSD1 is expressed in high concentrations in subcutaneous adipose tissues of obese and type 2 diabetic patients, and is the marker of insulin resistance and abdominal fat deposition. EGCG strongly inhibited the activity of human 11β-HSD1 activity with an IC 50 value of 57.99 μM (reduction) and inhibition constant Ki of 22.68 and 18.74 μM for human liver microsomes and purified human 11β-HSD1, respectively (Hintzpeter et al. 2014). In diabetic vascular tissues, the elevated expression of vasoconstrictor factor, endothelin-1 (ET-1), causes restriction of vasorelaxation process of vascular endothelial cells in arteries by decreasing nitric oxide production from eNOS and leads to cardiovascular diseases. ...
Chapter
Pre-diabetes is a stage that usually precedes the onset of type 2 diabetes with a slight increase in fasting glucose, between 5.6 and 6.9 mmol/L; decreased glucose tolerance, between 7.8 and 11 mmol/L; and glycated hemoglobin, between 5.5 and 6.4% (ADA 2020). These biochemical abnormalities are usually caused by defects in insulin secretion from pancreatic cells, low insulin activity, or both (Unwin et al. 2002). Pre-diabetes can eventually lead to type 2 diabetes, which is characterized by hyperglycemia due to the inability of cells to respond fully to insulin, or to be resistant to insulin. Insulin resistance is characterized by insulin inefficiency in promoting glucose uptake by tissues and low intracellular glucose concentration signals for increased insulin production by the pancreas. Over time, depleted pancreatic beta cells reduce or stop insulin production, further elevating blood glucose levels (>6.9 mmol/L), characteristic of the diabetes condition. According to epidemiological data, type 2 diabetes is common in the elderly, but its frequency has increased in children and young adults due to high rates of obesity, sedentary lifestyle, and unbalanced diet, with excess fat and sugar. All of these factors indicate that both type 1 and type 2 diabetes result from a combination of genetic predisposition and environmental triggers (IDF 2020). Type 2 diabetes symptoms are similar to those of type 1 diabetes, but they are difficult to be identified in the early stages due to the long pre-diagnosis process, and, therefore, up to one third of the population may not be diagnosed early (Bansal 2015; Kaur 2014; Buchanan et al. 2002). This can be detrimental for a favorable prognosis after a long period of latent disease, and complications such as retinopathy or ulcers in the lower limbs that do not heal may occur (Chiasson et al. 2002). Furthermore,visceral obesity common in overweight and obese patients is related to the local and systemic inflammatory process, closely linked to the development of these comorbidities (ADA 2020). Regarding the healthy population, individuals with pre-diabetes and diabetes have a higher risk over the time of developing cardiovascular disease, metabolic syndrome, and polycystic ovaries, in addition to higher morbidity and mortality rates (Bansal 2015; Kaur 2014). Considering the close relationship between pre-diabetes, obesity, and diet, it is important to examine the influence of intestinal microbiota in this context. Intestinal microbiota is characterized by a diverse community of bacteria responsible for influencing nutrient metabolism, immune responses, and resistance to infectious pathogens (Nicholson et al. 2012; Belkaid and Hand 2014; van Nood et al. 2013). In the diabetic population, the intestinal microbiota presents a pattern of dysbiosis, which can be the starting point for the evolution of pre-diabetes to type 2 diabetes and other chronic diseases (Pratley 2013; Ziemer et al. 2008; Tsui et al. 2008; Stefanakia et al. 2018). Current scientific evidence has shown that probiotics or prebiotics, including phenolic compounds, can play a widely recognized role in the regulation of the intestinal microbiota, altering microbial composition and the metabolism of the bacteria and host (Tsai et al. 2019; Wang et al. 2020). Based on these fundamentals, this chapter will address the intrinsic relationships between the consumption of probiotics and prebiotics in individuals with pre-diabetes and type 2 diabetes.
... μM (Feng et al. 2011). A catechin derivative, EGCG (35), from green tea showed moderate inhibitory effect against h-11β-HSD1 with an IC 50 of 57.99 μM (Hintzpeter et al. 2014). A cyclic tetrapeptide, penicopeptide A (268) isolated from the culture of endophytic fungus Penicillium commune of grape plant, Vitis vinifera, in rice, inhibited the activity of h-11β-HSD1 with an IC 50 of 9.07 μM ). ...
... Accumulating evidence strongly demonstrates that the enzyme 11β-HSD1 is expressed in high concentrations in subcutaneous adipose tissues of obese and type 2 diabetic patients, and is the marker of insulin resistance and abdominal fat deposition. EGCG strongly inhibited the activity of human 11β-HSD1 activity with an IC 50 value of 57.99 μM (reduction) and inhibition constant Ki of 22.68 and 18.74 μM for human liver microsomes and purified human 11β-HSD1, respectively (Hintzpeter et al. 2014). In diabetic vascular tissues, the elevated expression of vasoconstrictor factor, endothelin-1 (ET-1), causes restriction of vasorelaxation process of vascular endothelial cells in arteries by decreasing nitric oxide production from eNOS and leads to cardiovascular diseases. ...
Chapter
The increased worldwide prevalence of pre-diabetes and diabetes, especially type 2 diabetes, requiring different strategies for their prevention and management. A new focus is the reversal of diabetes dysbiosis, a disruption of gut microbiota homeostasis, which is closely related to elevated blood glucose levels and altered metabolic parameters. In this sense, a balanced diet plays a key role, and, particularly, probiotic and prebiotic have shown a promising role. This chapter explored current knowledge on the potential of probiotic and prebiotic to modulate glucose homeostasis. We showed that the consumption of probiotics and prebiotics is a promising strategy with a beneficial impact on gut microbiota and glycemic control. Furthermore, specific probiotic strains, such as L. acidophilus, L. casei strain Shirota, and B. lactis Bb12, have demonstrated the ability to improve parameters related to pre-diabetes and type 2 diabetes. In addition, polyphenols are emerging as a new alternative in glycemic control through the production of short chain fat acids (SCFA) and decreased lipopolysaccharides (LPS) translocation that leads to metabolic endotoxemia. Finally, the ingestion of beneficial bacteria, and foods rich in fiber and polyphenols, or a combination of them, is a good strategy for the control of pre-diabetes and type 2 diabetes, but more studies are still needed, mainly clinical trials, for these strategies are improved and widely used.
... Significant changes in subjects treated with placebo were not observed. The interconversion of the inert cortisone to active cortisol is catalyzed by the microsomal enzyme 11β-HSD1 and thereby acts as a key for regulating the access of glucocorticoid hormones to the glucocorticoid receptor [41]. There were significant reductions of salivary cortisol/cortisone ratios which indicate 11-β HSD1 activity in the PE group only from day 0 to day 28 at all-time points: morning from a mean of 1.12 ± 0.31 to 0.81 ± 0.33 (p = 0.003), noon from 1.31 ± 0.25 to 1.02 ± 0.38 (p = 0.047) and evening from 1.39 ± 0.41 to 1.06 ± 0.46 (p = 0.045). ...
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Pomegranates are known to possess anti-hypertensive, anti-atherogenic and cardioprotective effects mainly due to their pleiotropic effects on various cellular pathways, especially those triggered by oxidative stress. The aim of this study was to investigate the effect of natural standardized pomegranate (PE) extract on cardiovascular risk factors in 24 healthy volunteers who participated in a randomized, single-blind placebo-controlled study. There were 12 subjects in the PE group and 12 in the placebo group. Variables were measured at baseline and after 14 and 28 days of supplementation are anthropometry, BP, pulse wave velocity, fat and lean body mass, salivary and urinary cortisol, and cortisone, total phenolics, antioxidant capacity and lipid peroxidation. Urinary total phenolics excretion and antioxidant capacity were significantly increased after 14 and 28 days of PE intake. At day 28, there were also statistically significant decreases in systolic and diastolic blood pressure (BP), pulse wave velocity, body fat and fat mass, as well as an increase in lean body mass. Significant changes in the placebo group were not found. Glucocorticoid levels showed a significant decrease in saliva cortisol at day 28 (morning) in the PE group, and cortisol/cortisone ratio was significantly decreased following 28 days of PE intake at morning, noon, and evening. Urine free cortisol was significantly reduced at day 14. These findings suggest that pomegranate extract intake may improve antioxidant and oxidative stress status and play a beneficial role in the attenuation of some cardiovascular risk factors. Future studies should concentrate on overweight and older people.
... 11β-HSD1, by converting cortisone (inactive) to cortisol (active), contributes to the differentiation of adipocytes [176,180]. The polyphenolic compounds including flavonoids such as apigenin obtained from Matricaria chamomilla and quercetin from Brassica oleracea, isoflavonoid such as genistein isolated from Glycine max and catechins such as epigallocatechin-3-gallate (EGCG) extracted from Camellia sinensis have the crucial role in the inhibition of 11β-HSD1 expression, especially in the adipose tissues and inactivation of cortisol in the liver leading to improved glucose metabolism and insulin signaling ( Table 1) [48,181]. It has been revealed that 11β-HSD1 is centrally involved in cortisol metabolism, thus inhibition of 11β-HSD1 particularly in the adipose tissues may provide a therapeutic strategy for the management of diabesity (Fig. 2) [170]. ...
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Obesity-associated diabetes mellitus, a chronic metabolic affliction accounting for 90% of all diabetic patients, has been affecting humanity extremely badly and escalating the risk of developing other serious disorders. It is observed that 0.4 billion people globally have diabetes, whose major cause is obesity. Currently, innumerable synthetic drugs like alogliptin and rosiglitazone are being used to get through diabetes, but they have certain complications, restrictions with severe side effects, and toxicity issues. Recently, the frequency of plant-derived phytochemicals as advantageous substitutes against diabesity is increasing progressively due to their unparalleled benefit of producing less side effects and toxicity. Of these phytochemicals, dietary polyphenols have been accepted as potent agents against the dual sword “diabesity”. These polyphenols target certain genes and molecular pathways through dual mechanisms such as adiponectin upregulation, cannabinoid receptor antagonism, free fatty acid oxidation, ghrelin antagonism, glucocorticoid inhibition, sodium-glucose cotransporter inhibition, oxidative stress and inflammation inhibition etc. which sequentially help to combat both diabetes and obesity. In this review, we have summarized the most beneficial natural polyphenols along with their complex molecular pathways during diabesity
... Hepatic CCAAT-enhancer binding proteinα (C/EBP-α) is the main transcription factor required for 11β-HSD1 mRNA expression found to increase by supplementation of TFAs and SFAs in the rat. The consumption of medicinal plants such as tea is reported to reduce 11β-HSD1 activity (Hintzpeter, Stapelfeld, Loerz, Martin, & Maser, 2014). About five compounds isolated from tea showed slight inhibitory effects on both human and mouse 11β-HSD1 activity (G. ...
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... Various teas (Camellia sinensis (L)Kuntze ), tea particular polyphenolic agents were investigated for human liver microsomes and human purified 11 β-HSD1 for their probable antidiabetic action through reduction of cortisone by hampering action on 11 β-HSD1. 67 The polyphenol(-_ epigallocatechin gallate(EGCG) demonstrated, maximum robust hampering action on 11 β-HSD1((IC 50 value =57. 99 μMfor reduction; IC 50 value =131. 2 μMfor Oxidation). ...
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Epigallocatechin gallate (EGCG) inhibits drug metabolizing enzymes by unknown mechanisms. Here we examined if the inhibition is due to covalent-binding of EGCG to the enzymes or formation of protein aggregates. EGCG was incubated with rat liver microsomes at 1-100μM for 30min. The EGCG-binding proteins were affinity purified using m-aminophenylboronic acid agarose and probed with antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, cytochrome P450 (CYP) 1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A, catechol-O-methyltransferase (COMT) and microsomal glutathione transferase 1 (MGST1). All but actin and soluble COMT were positively detected at ≥1μM EGCG, indicating EGCG selectively bound to a subset of proteins including membrane-bound COMT. The binding correlated well with inhibition of CYP activities, except for CYP2E1 whose activity was unaffected despite evident binding. The antioxidant enzyme MGST1, but not cytosolic GSTs, was remarkably inhibited, providing novel evidence supporting the pro-oxidative effects of EGCG. When microsomes incubated with EGCG were probed on Western blots, all but the actin and CYP2E1 antibodies showed a significant reduction in binding at ≥1μM EGCG, suggesting that a fraction of the indicated proteins formed aggregates that likely contributed to the inhibitory effects of EGCG but were not recognizable by antibodies against the intact proteins. This raised the possibility that previous reports on EGCG regulating protein expression using GAPDH as a reference should be revisited for accuracy. Remarkable protein aggregate formation in EGCG-treated microsomes was also observed by analyzing Coomassie Blue-stained SDS-PAGE gels. EGCG effects were partially abolished in the presence of 1mM glutathione, suggesting they are particularly relevant to the in vivo conditions when glutathione is depleted by toxicant insults.
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