Decaffeinated green and black tea polyphenols decrease weight gain and alter microbiome populations and function in diet-induced obese mice

Abstract

Purpose:
Decaffeinated green tea (GT) and black tea (BT) polyphenols inhibit weight gain in mice fed an obesogenic diet. Since the intestinal microflora is an important contributor to obesity, it was the objective of this study to determine whether the intestinal microflora plays a role in the anti-obesogenic effect of GT and BT.

Methods:
C57BL/6J mice were fed a high-fat/high-sucrose diet (HF/HS, 32% energy from fat; 25% energy from sucrose) or the same diet supplemented with 0.25% GTP or BTP or a low-fat/high-sucrose (LF/HS, 10.6% energy from fat, 25% energy from sucrose) diet for 4 weeks. Bacterial composition was assessed by MiSeq sequencing of the 16S rRNA gene.

Results:
GTP and BTP diets resulted in a decrease of cecum Firmicutes and increase in Bacteroidetes. The relative proportions of Blautia, Bryantella, Collinsella, Lactobacillus, Marvinbryantia, Turicibacter, Barnesiella, and Parabacteroides were significantly correlated with weight loss induced by tea extracts. BTP increased the relative proportion of Pseudobutyrivibrio and intestinal formation of short-chain fatty acids (SCFA) analyzed by gas chromatography. Cecum propionic acid content was significantly correlated with the relative proportion of Pseudobutyrivibrio. GTP and BTP induced a significant increase in hepatic 5'adenosylmonophosphate-activated protein kinase (AMPK) phosphorylation by 70 and 289%, respectively (P < 0.05) determined by Western blot.

Conclusion:
In summary, both BTP and GTP induced weight loss in association with alteration of the microbiota and increased hepatic AMPK phosphorylation. We hypothesize that BTP increased pAMPK through increased intestinal SCFA production, while GTPs increased hepatic AMPK through GTP present in the liver.

Full text

Decaffeinated green and black tea polyphenols decrease weight
gain and alter microbiome populations and function in diet-
induced obese mice
Susanne M. Henning1, Jieping Yang1, Mark Hsu1, Ru-Po Lee1, Emma M. Grojean1, Austin
Ly1, Chi-Hong Tseng2, David Heber1, Zhaoping Li1
1Center for Human Nutrition, David Geffen School of Medicine, University of California, Warren
Hall 14-166, 900 Veteran Avenue, Los Angeles, CA 90095, USA
2Department of Statistics Core, David Geffen School of Medicine, University of California, Los
Angeles, CA 90095, USA
Abstract
Purpose—Decaffeinated green tea (GT) and black tea (BT) polyphenols inhibit weight gain in
mice fed an obesogenic diet. Since the intestinal microflora is an important contributor to obesity,
it was the objective of this study to determine whether the intestinal microflora plays a role in the
anti-obesogenic effect of GT and BT.
Methods—C57BL/6J mice were fed a high-fat/high-sucrose diet (HF/HS, 32% energy from fat;
25% energy from sucrose) or the same diet supplemented with 0.25% GTP or BTP or a low-fat/
high-sucrose (LF/HS, 10.6% energy from fat, 25% energy from sucrose) diet for 4 weeks.
Bacterial composition was assessed by MiSeq sequencing of the 16S rRNA gene.
Results—GTP and BTP diets resulted in a decrease of cecum Firmicutes and increase in
Bacteroidetes. The relative pro-portions of
Blautia, Bryantella, Collinsella, Lactobacillus,
Marvinbryantia, Turicibacter, Barnesiella,
and
Parabacteroides
were significantly correlated with
weight loss induced by tea extracts. BTP increased the relative proportion of
Pseudobutyrivibrio
and intestinal formation of short-chain fatty acids (SCFA) analyzed by gas chromatography.
Cecum propionic acid content was significantly correlated with the relative proportion of
Pseudobutyrivibrio
. GTP and BTP induced a significant increase in hepatic
5′adenosylmonophosphate-activated protein kinase (AMPK) phosphorylation by 70 and 289%,
respectively (
P
< 0.05) determined by Western blot.
Conclusion—In summary, both BTP and GTP induced weight loss in association with alteration
of the microbiota and increased hepatic AMPK phosphorylation. We hypothesize that BTP
increased pAMPK through increased intestinal SCFA production, while GTPs increased hepatic
AMPK through GTP present in the liver.
Susanne M. Henning shenning@mednet.ucla.edu.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Electronic supplementary material The online version of this article (doi:10.1007/s00394-017-1542-8) contains supplementary
material, which is available to authorized users.
HHS Public Access
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Eur J Nutr
. Author manuscript; available in PMC 2020 July 17.
Published in final edited form as:
Eur J Nutr
. 2018 December ; 57(8): 2759–2769. doi:10.1007/s00394-017-1542-8.
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Keywords
Black tea; Green tea; Polyphenols; Microflora; Obesity; AMPK phosphorylation; Short-chain fatty
acids
Introduction
Consumption of green tea (GT) and black tea (BT) polyphenols provides distinct health
benefits due to differences in the chemical structure of tea polyphenols in GT and BT [1, 2].
BTPs such as theaflavins and thearubigins are of large molecular weight and are not
absorbed in the small intestine and transported to the colon, whereas GTP are rapidly
absorbed in the small intestine. Our previous mouse studies demonstrated that the
supplementation of a high-fat, high-sucrose (HF/HS), Western diet with decaffeinated GT
and BT polyphenols (P) for 20 weeks decreased body and adipose tissue weight [3].
According to a recent review, the two major mechanisms of the antiobesity effect of GTP are
(1) stimulation of fat oxidation and decrease in fatty acid synthesis through AMP-activated
protein kinase (AMPK) phosphorylation by tissue polyphenols (epigallocatechin gallate,
epicatechin, epigallocatechin, and epicatechin gallate) and (2) decrease in digestion and
absorption of lipids in the intestine [4–7]. Since BTP, however, are not absorbed into tissues,
it is more likely that their primary site of activity is in the intestine. Therefore, we speculate
that changes in the microbiota may represent an important mechanism for BTP and possibly
GTP to induce weight loss.
The gut microbiota is an important contributor to human health and has been implicated in
the development of obesity and obesity-related diseases such as diabetes [8, 9] and
cardiovascular disease [10]. The two most abundant bacterial phyla in humans and in mice
are Firmicutes (40–60%) and Bacteroidetes (20–40%) with lower abundance of
Actinobacteria, Fusobacteria, Proteobacteria, and Verrucomicrobia [11]. It has been reported
that consuming a Western-type diet high in fat and sucrose (HF/HS) alters cecum microflora
populations and produces a relative increase in Firmicutes, a relative decrease in
Bacteroidetes and a reduction in overall microbiota diversity [12, 13].
Recent studies have examined the effect of tea consumption on human intestinal bacteria
[14, 15]. However, these studies used in vitro batch fermentation of fecal material and little
is known about the effects of dietary intake of tea polyphenols on gut microflora
composition and metabolism in vivo [16]. One recent study determined the effect of brewed
GT, BT, and oolong tea provided as drinking fluid on the intestinal microbiota in mice fed a
high-fat diet [17]. Brewed tea contains tea polyphenols, alkaloids (caffeine), amino acids/
peptides, and carbohydrates [2]. Since our investigation was focused on the effect of tea
polyphenols on the microbiome, we used a 75% alcohol:water extract that is enriched for tea
polyphenols.
One potential link between changes in the microbiota and weight loss might be the
formation of short-chain fatty acids, which can be absorbed in the colon, transported to the
liver, and have been shown to induce changes via AMPK activation [18]. AMPK plays a key
role in regulating carbohydrate and fat metabolism, serving as a metabolic master switch in
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response to alterations in cellular energy charge [19]. Recently, den Besten et al.
demonstrated that short-chain fatty acid (SCFA) formation by the intestinal microflora
protected against the metabolic syndrome via a signaling cascade that involved AMP-
activated protein kinase (AMPK) activation [18].
To examine whether changes in the intestinal microbiota leading to an increase in intestinal
SCFA formation play a role in the anti-obesogenic effect of GT and BT polyphenols, mice
were fed a high-fat/high-sucrose Western diet supplemented with GT or BT polyphenols and
changes in relative proportion of intestinal microbiota, intestinal concentration of SCFAs,
and changes in hepatic AMPK phosphorylation were determined.
Methods
Tea polyphenol extracts
Chemical reagents and plant materials—All solvents were HPLC grade and
purchased from Fisher Scientific Co. (Tustin, CA, USA). Gallic acid (> 98%), catechins,
black tea extract (> 80% theaflavin), theanine, and caffeine standards were purchased from
Sigma-Aldrich Co. (St. Louis, MO, USA). GT and BT leaves were collected and purchased
in a selected location in Sichuan Province, China. The samples were kept in sealed bags at
room temperature before extraction.
Tea polyphenol extract preparation
500 g of tea leaves were extracted with 2 L of 75% ethanol in water at room temperature for
3 h. The leaves were separated and the procedure was repeated twice. The ethanol was
evaporated in a rotary evaporator under reduced pressure at 40 °C. The dried extract was
suspended in 500 mL of pure water and extracted with chloroform to remove caffeine. The
decaffeinated water solution of tea extract was subjected to an XAD-16 resin column, rinsed
with five bed volumes of water and eluted with pure methanol. The extract was dried using
the rotary evaporator.
Black tea extract analysis—Theaflavin and thearubigin fractions were extracted
following a modification of the method by Xie et al. [20]. 100 mg of BT extract was
dissolved in 20 mL boiling water and shaken for 5 min. 10 mL of ethylacetate was added
and shaken for 5 min. The mixture was centrifuged at 2050
g
for 10 min and the ethylacetate
fraction (catechins and theaflavins) was transferred into fresh centrifuge vial. The procedure
was repeated two more times and all ethylacetate extracts were combined and evaporate to
dryness. The water fraction was further extracted with 10 mL butanol and shaken for 5 min.
After centrifugation for 10 min at 2050
g
, the butanol fraction (thearubigins) was transferred
into a fresh centrifuge vial. The procedure was repeated two more times and all butanol
extracts were combined and evaporate to dryness. The amount of dried residue was
determined by weight. The residues were reconstituted in 50% methanol:50% water and
analyzed using HPLC and LC–MS/MS.
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Liquid chromatography–mass spectrometry (LC–MS)
The HPLC with electrospray ionization mass spectrometry (ESI/MS) system consisted of an
LCQ Advantage Finnigan system (ThermoFinnigan, San Jose, CA, USA), equipped with a
Surveyor LC system consisting of an autosampler/injector, quaternary pump, column heater,
and diode array detector (DAD) with the Xcalibur 1.2 software (Finnigan Corp., San Jose,
CA, USA). The HPLC column was an Agilent Zorbax Sb-C18 3.5 um (150 × 2.1 mm). The
flow rate was 0.2 mL/min and gradient elution was used. The gradient started at 5% solution
A (acetonitrile) and 95% solution B (1% acetic acid/water) increasing over 40 min to 40%
solution A and 60% solution B. DAD range 210–600 nm, 440 nm as detection wavelength
for the theaflavin fraction, and 400 nm for the thearubigin fraction. MS parameters:
ionization mode ESI+ and ESI−; scan range 500–1000 amu; spray voltage, 5 kV; auxiliary
gas, 40; and capillary temperature, 275 °C. Peak identities were obtained by matching their
molecular M+ and M− with expected theoretical molecular weights from the literature [21].
Gallic acid equivalent (GAE)—The assays were performed as previously reported with
some modification using the Folin–Ciocalteau reagent [22]. The absorbance was read at 755
nm in a ThermoMax microplate reader (Molecular Devices, Sunnyvale, CA, USA) at room
temperature. The standard curves were used to convert the average absorbance of each
sample into mg/g gallic acid equivalent (GAE).
HPLC condition for analysis of GT and BT extracts—A Water Alliance 2695 HPLC
system coupled with a photodiode array detector and Empower 2 software was used to
analyze the catechins, gallic acid, theaflavins, and thearubigin fraction and caffeine. The
separation of compounds was conducted on an Agilent Zorbax SBC18 4.6 × 250 mm
column (Agilent) with a gradient of acetonitrile (solution A) and 0.1% phosphoric acid in
water (solution B). The gradient started at 5% solution A and 95% solution B, increasing
over 40 min to 40% solution A and 60% solution B. The detection wavelength was 280 nm
for catechins, 440 nm for theaflavin fraction, and 400 nm for thearubigin fraction. Theanine
was determined after reaction with AccQ Fluor reagent (Waters WAT-052880) and measured
by Waters 474 fluorescence detector.
Experimental animal and body composition studies—All mouse procedures were
approved by the UCLA Animal Research Committee in compliance with the Association for
Assessment and Accreditation of Laboratory Care (AAA-LAC) International and have been
performed in accordance with the ethical standards laid down in the 1964 Declaration of
Helsinki and its later amendments. 48 male C57BL/6J mice (strain JAX 000664) were
received from the Jackson Laboratory at 6–7 weeks of age (body weight: 16–18 g). After 1
week of acclimation, 28-day-old male C57BL/6J mice were assigned to four groups with
similar body weight distribution in each group and fed either a low-fat/high-sucrose diet
(LF/HS) (D12489B from Research Diets Inc., New Brunswick, NJ, USA), high-fat (HF)/HS
(D12266B from Research Diets Inc.) (Supplemental Table 1), or HF/HS diet supplemented
with GTP and BTP (Table 1) at 0.5 g/100 g of diet providing 0.25 g polyphenols/100 g diet
[3]. Based on the food intake, we calculated that mice fed the GTP diet (Fig. 1) in average
consumed 240 mg of GTP and 320 mg of BTP per kg body weight. Tea extracts were mixed
into the diet by Research Diets Inc. Body weights were recorded weekly and food
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consumption three times per week. Mice were euthanized after 4 weeks of dietary treatment.
Tissues were collected, weighed, and stored at − 80 °C until analysis.
Cecum short-chain fatty acid concentration—Intestinal cecum content was diluted,
acidified, and filtered and SCFA (acetic, propionic, butyric, and valeric acids) were
quantified by gas chromatography flame ionization detection (Agilent 7890A) and RTX-
Stabilwax column (Restek corp. 30 m × 0.25 mm) [23]. SCFA standard mix was purchased
from Sigma-Aldrich (St Louis, MO, USA).
Sequencing of bacterial DNA—DNA from cecum content was extracted using QIAamp
Stool DNA Extraction Kit (Qiagen, Valencia, CA, USA). The quality of the DNA samples
was confirmed using a Nanodrop 1000 (Thermo Fisher Scientific, Wilmington, DE, USA).
The 16S rRNA gene V4 variable region PCR primers 530/960 with barcode on the forward
primer were used in a 30 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen,
USA) under the following conditions: 94 °C for 3 min, followed by 28 cycles of 94 °C for
30 s, 53 °C for 40 s, and 72 °C for 1 min, after which a final elongation step at 72 °C for 5
min was performed. After amplification, PCR products are checked in 2% agarose gel to
determine the success of amplification and the relative intensity of bands. Sequencing was
performed at MR DNA (http://www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq
following the manufacturer’s guidelines. Sequence data were processed using a proprietary
analysis pipeline (MR DNA, Shallowater, TX, USA). Operational taxonomic units (OTUs)
were defined by clustering at 3% divergence (97% similarity). Final OTUs were
taxonomically classified using BLASTn against a curated GreenGenes database [24]. β-
Diversity was measured by calculating the unweighted UniFrac distances [25] using
Quantitative Insights Into Microbial Ecology (QIIME) default scripts. In addition, UniFrac
PCoA biplot was visualized using EMPEROR. Statistical difference between different time
points was analyzed by PERMDISP.
Western blotting—30 mg of liver tissue was homogenized and lysed in cell lysis buffer
containing 20 mM Tris-HCl, 0.5 M NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA,
10 mM β-glycophosphate, 10 m MNaF, 300 μM Na3VO4, 1 mM benzamidine, 2 μM PMSF,
and 1 mM DTT. Protein concentrations were determined by a BCA protein assay kit
(Thermo Scientific, Rockford, IL, USA). Protein was separated by SDS-PAGE, transferred
to a nitrocellulose membrane, blocked in 5% BSA, and probed with the following primary
antibodies: phospho-AMPKα (T172), AMPKα, and GAPDH (Cell Signaling Technology,
Boston, MA, USA). The membranes were incubated with horseradish peroxidase (HRP)-
conjugated secondary antibodies (Sigma) and visualized with SuperSignal™ West Femto
Maximum Sensitivity Substrate (Thermo scientific, Rockford, IL, USA) according to the
manufacturer’s recommended protocol.
Statistical analysis—All statistical analyses were conducted using the IBM SPSS
Statistics version 21; mean values, standard deviation, and standard errors were calculated
using descriptive statistics. Body weight, % epididymal, % mesenteric, % subcutaneous fat/
body weight, cecum SCFA content, and hepatic AMPK protein phosphorylation were
analyzed with one-way ANOVA, with the factor being diet. Tukey–Kramer multiple
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comparison procedure was used for post-hoc comparisons. Wilcoxon rank sum test was
utilized to evaluate the differences in bacterial relative proportion between study groups. All
tests were two sided and all analyses of the microbiota were conducted using the SAS 9.3
(Statistical Analysis System, Cary, NC, USA, 2008) and R (http://www.r-project.org)
software.
P
values < 0.05 were considered statistically significant.
Results
Chemical composition of tea extracts
GTP and BTP extracts were analyzed by HPLC (Table 1). GTP contained tenfold higher
concentration of EGCG compared to BTP, while the total phenolic content (GAE) was
similar in GTP and BTP extracts. The BTP extract contained more gallic acid and theanine
compared to GTP (Table 1). The BTP theaflavin fraction was quantified as 1.49% and
thearubigin fraction 18% of solids. Theaflavin content in the ethylacetate fraction was
quantified based on the commercial BT extract standard. In the absence of standards,
thearubigins in the butanol fraction were quantified by weight. The theaflavin and
thearubigin fractions were further analyzed by LC–MS to identify molecular mass of
compounds (Supplemental Table 2, Supplemental Figs. 1, 2, 3). The theaflavin fraction
contained theaflavin, theaflavin-3′-gallate, theaflavin-3-gallate, and theaflavin-3,3′-digallate
(Supplemental Fig. 1). The following compounds were identified in the thearubigin fraction
with LC/MS positive mode: theasinensin-digallate, theasinensin, and theaflavin-gallate, and
in negative mode: theacitrin, chalcan-flavan dimer-gallate, and theacitrin-gallate based on
molecular weight (Supplemental Figs. 2, 3) compared to Kuhnert et al. [21].
Body, liver, and fat depot weights in mice fed the HF/ HS diet supplemented with tea
extracts
The diet contained 0.25% of tea polyphenols. Based on the food intake, average
consumption of polyphenols was 240 and 320 mg per kg body weight for mice fed the GTP
and BTP diet, respectively. During the 4-week dietary intervention, the HF/HS-treated mice
had significantly higher body weight and subcutaneous and epididymal fat by weight (Fig.
1) compared to the LF/HS group. Supplementation of the HF/HS diet with GT and BT
polyphenols significantly decreased body weight, subcutaneous, mesenteric, and epididymal
fat normalized to body weight compared to HF/HS control equal to the body composition of
mice fed the LF/HS diet (Fig. 1). No change in liver weight normalized to body weight was
observed among the treatment groups (data not shown).
Cecum microbiota, SCFA concentration, cecum content weight, and total DNA in mice fed
the HF/HS diet supplemented with tea extracts
Cecum weight and DNA content normalized to body weight was significantly increased in
mice fed the HF/HS-GTP and BTP diets, while feeding the LF/HS diet had no effect
compared to the HF/HS diet (Supplemental Fig. 4). The unweighted UniFrac distance metric
was calculated and visualized via principle coordinate analyses (PCoA) (Fig. 2). For
unweighted UniFrac distance metrics, mice fed the GTP and BTP supplemented diet formed
clusters distinctly different from LF/HS and HF/HS microbial patterns (Fig. 2).
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Sequencing of the bacterial DNA demonstrated a significant increase in the relative
proportion of cecum phyla Bacteroidetes and decrease in Firmicutes and Actinobacter in
mice fed the HF/HS-GTP and BTP diet compared to HF/HS and LF/HS diets (Fig. 3). We
observed a significant correlation between Firmicutes and body weight and inverse
correlation between Bacteroidetes and body weight (Fig. 4). On the genus level, we found
that the relative proportion of
Blautia, Bryantella, Collinsella, Lactobacillus,
Marvinbryantia, and Turicibacter
were positively correlated with body weight (Table 2),
while
Barnesiella
and
Parabacteroides
were negatively correlated with body weight (Table
2). On the genus level feeding, the HF/HS-GTP and BTP diets were associated with a
significant increase in relative proportion of
Parabacteroides, Bacteroides,
and
Prevotella
and a significant decrease in several genera from the phylum Firmicutes such as
Roseburia
,
Lactobacillus, Blautia, Anaerostipes, Shuttleworthia, Bryantella
,
Lactococcus,
and
Acetitomaculum
as well as a significant decrease in
Collinsella
from the Actinobacter
phylum (Fig. 5). Changes only induced by GTP but not BTP consumption included an
increase in the genus
Clostridium
and
Coprococcus
, and decrease in
Turicibacter
and
Marvinbryantia
. Changes only induced by BTP and not GTP consumption were increase in
Oscillibacter
,
Anaerotruncus,
and
Pseudobutyrivibrio
(Fig. 5). In comparison with the
HF/HS diet mice fed the LF/HS diet showed an increase in
Bacteroides
and decrease in
Blautia
and
Bryantella
(Fig. 5c).
The consumption of HF/HS-BTP diet was associated with a significant increase in
concentration of the fecal SCFA propionic acid, i-butyric acid, and a trend to increase in
butyric acid (
P
= 0.07) with the sum increased by 24% compared to the HF/HS diet (Table
3). The cecum concentration of propionic and i-butyric acids was significantly correlated
with the relative proportion of
Pseudobutyrivibrio
(Table 4). Propionic acid formation was
correlated with
Oscillibacter
and
Maryell
(Table 4). To confirm the sequencing results, we
also performed pPCR using duodenum bacterial DNA (Supplemental Fig. 5). We confirmed
the significant increase in the abundance of Bacteroidetes and decrease in Firmicutes.
Hepatic AMPK phosphorylation in mice fed the HF/HS diet supplemented with tea extracts
Feeding the LF/HS, HF/HS-GTP and HF/HS-BTP diets increased AMPK phosphorylation
compared to the HF/HS diet (Fig. 6). Increased pAMPK/tAMPK ratio was significantly
correlated with the relative proportion of
Anaerococcus
(
P
= 0.0054).
Discussion
Our data demonstrate the basic differences in the mechanism of the anti-obesogenic activity
between GT and BT polyphenols that involve complex changes in the intestinal microbiota.
Principal coordinates analysis of unweighted UniFrac distances revealed a distinct
separation of the gut microbial communities between GTP, BTP, and HF/HS-LF/HS diet
treatments. Although both teas induced similar changes on the phylum level of the intestinal
microbiota, on the genus level, we observed strong differences between GT and BT
polyphenol supplementation.
In the present study, the addition of GTP and BTP to the HF/HS diet significantly decreased
the ratio of Firmicutes to Bacteroidetes. These changes were significantly correlated with
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body weight. In support of our findings the study by Seo et al. also found that oral gavage of
an extract from fermented GT reversed the obesogenic effect of a high-fat diet (HFD)
associated with a decrease in the ratio of Firmicutes/Bacteroidetes ratio compared to HFD
fed control mice as determined by RT-PCR [26]. The study by Seo et al. was different in that
it used GT produced from dried green tea leaves fermented by Bacillus subtilis, a
microorganism used to produce fermented soy-bean products [26]. Another study by Liu et
al. did not find an effect on Firmicutes to Bacteroidetes ratio in mice fed an HFD and
provided with brewed GT, BT, or oolong tea as drinking fluid [17].
In the presented study, the largest change in relative proportion in the cecal microbiota after
GTP and BTP intervention was observed for
Parabacteroides
, a bile-resistant Gram-negative
anaerobic bacteria, which in addition to other Bacteroidetes has been associated with the
formation of the SCFA propionic acid in in vitro fermentation of different fiber sources with
human fecal microbiota [27]. Fermentation of polysaccharides to SCFA is one of the
potential mechanisms through which changes in the microbiota can contribute to inhibition
of weight gain. For example, the study by Ridaura et al. demonstrated that transplanted
microbiomes from lean co-twins to mice exhibited higher expression of genes involved in
polysaccharide fermentation [28]. The microbiome transplant from lean co-twins also
showed higher abundance of members of the
Bacteroides
and
Parabacteroides
genus and
resulted in increased formation of the fecal short-chain fatty acids propionate and butyrate
[28]. In the present study, however, we observed that cecum content of SCFA was only
increased significantly in mice fed the BTP diet. In difference to the GTP intervention,
feeding the HF/HS-BTP diet increased the relative proportion of
Pseudobutyrivibrio.
The
relative proportion of
Pseudobutyrivibrio
was significantly correlated with the formation of
propionic and i-butyric acids. Members of the
Pseudobutyrivibrio
genus have fructanolytic
and saccharolytic enzyme activity [29], which may have contributed to the formation of
SCFA.
Other bacteria significantly increased only in mice fed the GTP diet belong to the genus
Clostridium
and
Coprococcus.
Similar results were found in batch culture studies by
Tzounis et al. showing that the addition of GT polyphenols epicatechin and catechin
promoted the growth of members from the
Clostridium
genus, which have the ability to
generate SCFA by saccharolytic metabolism [14]. In addition, a rat study by Zhong et al.
linked the abundance of
Coprococcus
to significant increase in fecal SCFA concentration in
rats fed a high-fat diet supplemented with barley [30]. In the presented study, however,
SCFA were not increased in mice fed the GTP diet, which may depend on the interaction
between multiple bacteria present in the intestinal microbiota [30, 31].
SCFAs such as acetate, propionate, and butyrate are the main products of intestinal bacterial
fermentation of dietary fiber and complex carbohydrates [27]. GT and BT polyphenols have
been shown to inhibit α-amylase and α-glucosidase in saliva and small intestine, which may
lead to residual carbohydrate in the large intestine providing substrate for the SCFA
generation [32, 33]. SCFA serve as energy source for colonic epithelium (butyrate) and
peripheral tissues (acetate and propionate) [28]. SCFA are also absorbed into the
bloodstream and travel to the liver, where they play a role in energy metabolism [30]. The
addition of SCFAs to the diet has been demonstrated to be efficacious in protection against
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high-fat diet-induced metabolic syndrome [18]. Potential mechanisms are activation of
AMPK phosphorylation and inhibition of PPARγ. For example, Den Besten et al.
demonstrated that fiber (guar gum)-derived SCFA protected against metabolic syndrome by
AMPK activation and PPARγ inhibition in mice fed a high-fat diet for 12 weeks [34].
AMPK phosphorylation in turn can lead to stimulation of FA β-oxidation and inhibition of
gluconeogenesis [35].
Both teas induced hepatic AMPK phosphorylation but most likely through different
mechanisms based on the difference in chemical composition. In our previous study, using
the same GTP and BTP supplemented diets, we found 540 pmol of polyphenols (EGCG, 4″-
meEGCG, EGC, and ECG) per g liver in mice fed the HF/HS-GTP diet, whereas only 20
pmol/g liver of ECG was found in mice fed the HF/HS-BTP diet [3]. We, therefore, suggest
that GTP intervention induced weight loss in part by increased hepatic AMPK
phosphorylation through polyphenols present in the tissue. The formation of phenolic acids
as microbial metabolites by catabolism of tea polyphenols provides another potential
mechanism. In one of our previous human tea intervention studies and in a recent study by
Pereira–Caro, it was demonstrated that GT and BT polyphenols are metabolized to smaller
phenolic acids and are present in serum and urine after the consumption of GT and BT [36,
37]. It is possible that some phenolic acids contribute to the effect on AMPK
phosphorylation. In addition, the formation of SCFA may contribute to the increase in
AMPK phosphorylation. SCFAs in turn can be absorbed and have been shown to induce
hepatic AMPK phosphorylation [34]. Increased AMPK phosphorylation has also been
observed by Rocha et al. in obese rats gavaged with GT extract dissolved in water [38] and
in obese mice gavaged with EGCG enriched GT extract [39]. In the presented study, a low
fiber chemically defined rodent diet was used, and therefore, SCFA formation was relatively
low. Future studies using a combination of tea extracts and fiber may enhance the formation
of SCFA and increase the effect on AMPK phosphorylation.
In addition, based on our findings that eight bacterial genera (Blautia, Bryantella,
Collinsella, Lactobacillus, Marvinbryantia and Turicibacter, Barnesiella, and
Parabacteroides) were significantly correlated with body weight, we conclude that additional
mechanisms related to the intestinal microbiota are involved in the anti-obesogenic activity
of both teas.
In summary, we demonstrated that both GTP and BTP administration were associated with
significant changes in the cecum microflora. Based on our results and published data, we
conclude that the obesogenic effect of tea polyphenols involves multiple mechanisms such
as changes in the composition of the intestinal microbiota, changes in microbial metabolite
formation, and increase in residual complex carbohydrate through inhibition of α-amylase
and α-glucosidase leading to an increase in SCFA formation. In the present study, only
changes in microbiota induced by the BTP diet were associated with a significant increase in
cecum concentration of SCFA. Future studies using high-fat diets containing fermentable
fiber together with GT and BT polyphenols may shed more light on the role of microflora
and SCFA formation in inhibiting weight gain when consuming an HF/HS diet.
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Up to date, green tea polyphenols are recognized as more effective agents that offer more
health benefits than black tea based on the bioavailability of green tea polyphenols.
However, our novel findings that feeding a diet containing black tea polyphenols was
associated with an increase in cecum SCFA formation and increased hepatic AMPK
phosphorylation provides novel insights in the health benefits of black tea.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
This work was supported by the National Institute of Health (R03CA171583 and P50CA092131) and departmental
funds of the Center for Human Nutrition, Department of Medicine, David Geffen School of Medicine, University of
California, Los Angeles.
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Fig. 1.
Effects of tea extracts on (a) body weight, (b) % subcutaneous fat, (c) % mesenteric fat, (d)
% epididymal fat normalized to body weight, (e) food intake, and (f) energy intake in male
C57BL/6J mice fed an HF/HS, LF/HS, HF/HS-GTP, or HF/HS-BTP diet for 4 weeks. Data
are mean ± SEM (
n
= 11–12). Labeled means of dietary interventions without a common
letter differ by diet;
P
< 0.05
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Fig. 2.
Beta-diversity analysis of the microbiome of cecum content from mice fed the HF/HS,
LF/HS, HF/HS-GTP, or HF/HS-BTP diet for 4 weeks. Three-dimensional principal
coordinate analyses (PCoA) based on the unweighted UniFrac distance between samples
were performed using QIIME (
n
= 9)
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Fig. 3.
Effects of tea extracts on cecum bacteria relative proportion in male C57BL/6J mice fed an
HF/HS, LF/HS, HF/HS-GTP, HF/HSOTP, or HF/HS-BTP diet for 4 weeks. Data are means
(
n
= 9). Wilcoxon rank sum test was utilized to evaluate the differences in bacterial relative
proportion comparing each tea and LF/HS intervention with HF/HS control diet. Labeled
means are different from the HF/HS group with *
P
≤ 0.05, **
P
≤ 0.01, ***
P
≤ 0.001
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Fig. 4.
Correlation of body weight and bacteria relative proportion of a Bacteroidetes and b
Firmicutes phyla including data from all intervention groups. The correlation was evaluated
using the GraphPad Prism6 software (San Diego, CA, USA)
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Fig. 5.
Effects of tea extracts on microbiome on the genus level in cecum content from male
C57BL/6J mice fed an HF/HS, LF/HS, HF/HS-GTP, or HF/HS-BTP diet for 4 weeks.
Comparison of relative proportion of bacteria between a HF/HS to HF/HS-GTP, b HF/HS to
HF/HS-BTP, and c HF/HS to LF/HS fed mice. Data are mean ± SD;
N
= 9. The difference in
relative proportion compared to mice fed the HF/HS control diet was significant for all
bacteria included in the figure (
P
< 0.05)
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Fig. 6.
Effects of tea extracts on protein expression of hepatic AMPK phosphorylation in liver from
male C57BL/6J mice fed an HF/HS, LF/HS, HF/HS-GTP, or HF/HS-BTP diet for 4 weeks.
Data are mean ± SEM (
n
= 5). Data were analyzed by one-way ANOVA, followed by
Tukey–Kramer multiple comparison procedure. Labeled means without a common letter
differ,
P
< 0.05
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Author Manuscript Author Manuscript Author Manuscript Author Manuscript
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Table 1
Gallic acid equivalent, and chemical composition of GTP and BTP
GAE (mg/g) EGCG (mg/g) ECG (mg/g) EGC (mg/g) EC (mg/g) GA (mg/g) Theanine (mg/g) Caffeine (mg/g)
GTP 565 ± 24 214 ± 4.5 48.7 ± 2.4 24.5 ± 2.0 21.4 ± 1.6 1.7 ± 0.6 0.7 ± 0.3 0.5 ± 0.1
BTP 532 ± 25 20 ± 0.8 7.1 ± 0.4 7.1 ± 0.4 9.3 ± 0.3 4.3 ± 0.2 3.1 ± 0.2 1.4 ± 0.1
Values are means ± SDs. Total phenolic content was expressed as GAE
BTP
black tea polyphenol,
GTP
green tea polyphenol,
GAE
gallic acid equivalent,
EGCG
epigallocatechin gallate,
ECG
epicatechin gallate,
EGC
epigallocatechin,
EC
epicatechin,
GA
gallic acid
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Table 2
Correlation between body weight and relative proportion of bacteria on the genus level
Pearson R P value Phylum
Barnesiella − 0.62 < 0.0001 Bacteroidetes
Parabacteroides − 0.44 0.0087 Bacteroidetes
Blautia 0.39 < 0.0001 Firmicutes
Bryantella 0.37 0.029 Firmicutes
Lactobacillus 0.48 0.0037 Firmicutes
Marvinbryantia 0.57 0.0004 Firmicutes
Turicibacter 0.46 0.0053 Firmicutes
Collinsella 0.39 0.0225 Actinobacter
Pearson correlation coefficient and
P
value were calculated using the Prism GraphPad software
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Table 3
Cecum short-chain fatty acid content
Acetic acid (μmol/g) Propionic acid (μmol/g) i-Butyric acid (μmol/g) n-Butyric acid (μmol/g)* i-Valeric acid (μmol/g) n-Valeric acid (μmol/g) Sum (μmol/g)
HF/HS 3.46 ± 0.53 0.34 ± 0.05b0.06 ± 0.01b0.62 ± 0.06a0.16 ± 0.02ab 0.12 ± 0.02b4.75 ± 0.67
HF/HS-GTE 2.97 ± 0.41 0.42 ± 0.07b0.06 ± 0.01b0.65 ± 0.12a0.15 ± 0.01ab 0.09 ± 0.03b4.35 ± 0.61
HS/HS-BTE 3.91 ± 0.57 0.59 ± 0.07a0.14 ± 0.02a0.86 ± 0.09a0.21 ± 0.03a0.18 ± 0.03a5.88 ± 0.78
LF/HS 3.55 ± 0.19 0.40 ± 0.04b0.05 ± 0.01b0.68 ± 0.08a0.10 ± 0.01c0.16 ± 0.01a4.94 ± 0.29
Data were analyzed by one-way ANOVA, followed by Tukey–Kramer multiple comparison procedure. Labeled means without a common letter differ,
P
< 0.05.
n
-Butyric acid: comparison between HF/HS
and HF/HS-BTP diets showed trend (
P
= 0.071). Values are means ± SEMs (
n
= 6)
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Table 4
Correlation between cecum concentration of short-chain fatty acids and relative proportion of bacteria in the cecum
Propionic acid Iso-butyric acid Phylum
Pearson R P value Pearson R P value
Bacteroidetes 0.190 0.374 0.38 0.067 Bacteroidetes
Parabacteroides 0.136 0.525 0.41 0.045 Bacteroidetes
Moryella 0.435 0.034 0.04 0.85 Firmicutes
Oscillibacter 0.566 0.004 0.34 0.101 Firmicutes
Pseudobutyrivibrio 0.556 0.005 0.67 0.0003 Firmicutes
Pearson correlation coefficient and
p
value were calculated using the Prism GraphPad software
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