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Journal of Nutritional Biochemistry 26 (2015) 893 – 902
RESEARCH ARTICLES
Decaffeinated green tea extract rich in epigallocatechin-3-gallate improves insulin
resistance and metabolic profiles in normolipidic diet—but not high-fat diet-fed mice
Aline Santana a , Aline Santamarina a , Gabriel Souza b , Laís Mennitti c , Marcos Okuda a , Daniel Venancio d ,
Marilia Seelaender e, Claudia Oller do Nascimento a , Eliane Ribeiro a , Fabio Lira f, Lila Oyama a,⁎
a
Departamento de Fisiologia, Universidade Federal de São Paulo, Sao Paulo, Brazil
Laboratório de Movimento Humano, Universidade São Judas Tadeu, Sao Paulo, Brazil
c
Programa de Pós-Graduação Interdisciplinar em Ciências da Saúde, Universidade Federal de São Paulo, Sao Paulo, Brazil
d
Departamento de Psicobiologia, Universidade Federal de São Paulo, Sao Paulo, Brazil
e
Cancer Metabolism Research Group, Institute of Biomedical Sciences, University of São Paulo, Sao Paulo, Brazil
f
Exercise and Immunometabolism Research Group, Department of Physical Education, Universidade Estadual Paulista, UNESP, Presidente Prudente, SP, Brazil
b
Received 25 August 2014; received in revised form 12 February 2015; accepted 2 March 2015
Abstract
Supplementation with epigallocatechin-3-gallate (EGCG), which restores metabolic profiles, has been proposed as an option for preventing and treating
obesity. We investigated whether decaffeinated green tea extract rich in EGCG, attenuates high-fat diet (HFD)-induced metabolic alterations in Swiss mice. The
mice were maintained on either a control diet (CD) or HFD for 8 weeks and supplemented with either a placebo or EGCG (50 mg/kg/day). Body weight, serum
lipid profiles, cytokine protein expression, and content in epididymal (EPI) and retroperitoneal (RET) adipose tissues, and adipocyte area were measured. The
body weights of HFD + placebo-fed mice were increased compared with those of HFD + EGCG-fed mice (28 and 21%, respectively), whereas the body weights of
CD + EGCG-fed mice were decreased 16% compared with those of the CD + placebo group. Serum triglyceride levels were decreased 32% in the CD + EGCG
group compared with the CD + placebo group. Compared with the CD + placebo group, increased phosphorylation of AMPK and hormone-sensitive lipase in EPI
and RET, respectively, was found in the CD + EGCG group. Increased acetyl-CoA carboxylase phosphorylation was observed in both adipose tissues. In addition,
TNF-α and IL-10 levels in EPI and adiponectin levels were higher in the CD + EGCG group than in the CD + placebo group. TNF-α levels were lower in the HFD + EGCG
group than in the HFD + placebo group. Furthermore, the CD + EGCG group exhibited a lower adipocyte area than the CD + placebo group. These indicate that the
effects of decaffeinated green tea extract on body mass may be related to the crosstalk between lipolytic and inflammatory pathways in normolipidic diet-fed mice but
not in HFD-fed mice.
© 2015 Elsevier Inc. All rights reserved.
Keywords: Epigalocatechin-3-gallate; obesity; lipolysis; lipid metabolism; adipose tissue
1. Introduction
Obesity is defined as excessive adipose mass and adipose tissue
expansion owing to adipocyte hypertrophy and hyperplasia [1]. In
general, chronic low-grade inflammation (particularly by elevated IL-6
and TNF-α levels) is found in adipose tissue in obese people, and this
adipose tissue inflammation is characterized by changes in immune cell
populations, giving rise to altered adipokine profiles, which in turn
induce skeletal muscle and hepatic insulin resistance [2].
In addition, IL-6 and TNF-α are known to play a pivotal role in lipid
metabolism by stimulating white adipocytes in white adipose tissue
(WAT), thereby increasing the release of nonesterified fatty acids
(NEFAs) through lipolytic processes and leading to elevated serum
⁎ Corresponding author at: Universidade Federal de São Paulo, Departamento
de Fisiologia, Rua Botucatu, 862 Vila Clementino, São Paulo/SP, Brasil, CEP:
04023062. Tel./fax: +55 11 5576 4765.
E-mail address: [email protected] (L. Oyama).
http://dx.doi.org/10.1016/j.jnutbio.2015.03.001
0955-2863/© 2015 Elsevier Inc. All rights reserved.
fatty acid levels [3]. Furthermore, NEFAs can stimulate adipokine
production, mainly in adipose tissue, via TLR-4 activation, leading to a
vicious cycle in obesity.
Adipocyte lipolysis is a complex process that is precisely controlled
through the integration of multiple diverse hormonal and biochemical
signals. Lipolysis typically occurs through catecholamine-mediated
stimulation of β-adrenergic receptors and adipocyte triglyceride
lipase (ATGL) and hormone-sensitive lipase (HSL), which catalyze
triglyceride hydrolase activities in adipose tissue [4]. In comparison
with lean individuals, the expression of both HSL and ATGL is reduced
in the adipose tissues of obese patients [5,6]. Therefore, the inhibition
of lipolysis promotes diet-induced obesity [7], whereas increases in
lipolysis prevent obesity [8].
Over the past two decades, chemicals derived from plants, referred
to as phytochemicals, have attracted the interest of the public and
scientific community because of their role in maintaining health and
preventing disease. Polyphenols derived from many components of
the human diet are among the leading phytochemicals, and some of
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A. Santana et al. / Journal of Nutritional Biochemistry 26 (2015) 893–902
Table 1
Composition of experimental diets, CD and HFD (AIN-93 modified) [11], growth (G),
and maintenance (M).
Ingredients
CD (G/M)
HFD (G/M)
Corn starch, %
Casein, %
Soybean oil, %
Lard, %
Cellulose, %
Mixture of vitamins, %
Mixture of mineral, %
L-cystine, %
Choline bitartrate, %
Hydroquinone, g/kg
Energy, Kcal/g
Treatment by oral gavage
EGCG
Water
62. 95/72.07
20. 0/14,0
7.0/4.0
–
5
1.0
3.5
0. 3/0.18
0.25
0.014/0.008
3.9 kcal/g
40. 92/40.87
13. 95/14.0
7.0/4.0
28. 08/31.2
5
1.0
3.5
0.18/0.18
0.25
0.014/0.008
5.36 kcal/g
(CD + E) 50 mg/kg/day
100 μl/day
(HFD + E) 50 mg/kg/day
100 μl/day
The EGCG was resuspended in water.
their potential preventive and therapeutic properties have been
extensively studied [9].
In recent years, the health benefit effects of green tea have been
reported to be primarily attributed to their high concentrations of
polyphenols, which are collectively referred to as catechins. The
antiobesity effects of green tea can be primarily attributed to
catechins, particularly epigallocatechin-3-gallate (EGCG). In previous
studies [10–12], we observed the beneficial effects of green tea extract
and EGCG on increasing lipolytic activity and reducing adipose tissue
weight, and this may explain the attenuation of low-grade inflammation in obese mice. In the present study, we investigated the effects of
decaffeinated EGCG on adipose tissue lipolysis and inflammatory
status in mice fed a high-fat diet (HFD).
2. Methods and materials
2.1. Animal treatment
The Experimental Research Committee of São Paulo Federal University approved
(no. 0097/12) all procedures and the care of the animals used in this study. Male Swiss
mice (n = 26) at 30 days of age were purchased from Central Lab. All mice were
maintained in collective cages in a room with controlled temperature (25 ± 2 C),
humidity (60 ± 5%), and lighting (12-h/12-h light–dark cycle) and given water and diet
ad libitum. After acclimation for 1 week, mice were randomly divided into four groups
and fed a control diet (CD, AIN-93) or HFD (AIN-93 adapted) [13] for 8 weeks (Table 1).
Placebo treatment consisted of a daily dose of 0.1 ml of water by gavage, and EGCG was
administered at 50 mg/kg/day resuspended in 0.1 ml of water by gavage (Table 1).
TEAVIGO was prepared as follows. Green tea leaves (Camellia sinensis) were extracted
with water followed by an extraction with ethyl acetate. The extract was concentrated and
spray dried to yield the green tea extract in powder form. TEAVIGO was produced via
absorption chromatography of the green tea extract. The eluate was concentrated,
crystallized, dried, filled into containers, and stored in its original unopened packaging.
The animals in the placebo group were weighed once a week, and the weight of
animals in the EGCG group was measured daily throughout the experimental period.
We consider the sum of three adipose tissues (mesenteric, epididymal, and
retroperitoneal) as an index of adiposity. To calculate the delta, we used the formula
(final weight − initial weight). At the end of the experimental period, mice were
euthanized after 12 h of fasting, blood was collected, and tissues (epididymal and
retroperitoneal adipose tissue and gastrocnemius muscle) were removed. The tissues
were weighed and stored at −80°C.
2.2. Measurement of serum biochemical parameters
The serum total cholesterol, High-density lipoprotein-cholesterol (HDL-C), glucose,
and triglyceride levels were determined using commercial kits (Labtest). NEFAs, free
glycerol (Zen-bio Inc.), insulin (Millipore Inc.), and adiponectin (R&D Systems) levels
were determined using specific commercial kits. Low-density lipoprotein-cholesterol
(LDL-c) levels were indirectly estimated using the Friedewald equation [LDL-C = total
cholesterol − (HDL-C) − (triglycerides/5)] [14]. The Homeostasis Model Assessment of
Basal Insulin Resistance (HOMA-IR) was calculated as the product of fasting glucose
level (mg/dl) multiplied by the insulin level (μIU/ml) divided by a constant (22.5), as
described previously [15]. The total lipid content of the gastrocnemius muscle was
determined according to the Folch method [16] (Table 2).
2.3. Histological analysis
Fragments of epididymal adipose tissue were fixed in 4% paraformaldehyde. After
24 h, the tissues were maintained in 70% alcohol for a day to be processed and put in
paraffin. Histological sections of 5 μM in thickness were stained with hematoxylin and
eosin. All slides were examined under an optical microscope by a pathologist who was
unaware of the origin of the material and of the objectives of the study. Using the
program Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA) for image analysis,
we were able to measure the area as described by Hidalgo et al. [17].
2.4. Western blot analysis
Epididymal and retroperitoneal adipose tissues were homogenized in lysis buffer
containing 100 mM Tris–HCl (pH 7.5), 1% Triton X-100, 10% sodium dodecyl sulfate,
10 mM EDTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM sodium
orthovanadate, 2.0 mM phenylmethylsulfonyl fluoride, and 0.1 mg/ml aprotinin. The
homogenate was centrifuged at 19,000×g for 45 min at 4°C, and the supernatant was
collected. The total protein concentration of each lysate was measured using Bradford
reagent. Proteins in the lysates were separated electrophoretically using precast 4–15% gels
(Mini-PROTEAN TGX Precast Gels, Bio-Rad). The membranes were blocked in 1% bovine
serum albumin overnight at room temperature and then incubated overnight with the
following primary antibodies: phospho-AMPK α1/2 (Thr-172), AMPK α1/2 (Thr-172), and
perilipin purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and
phosphor-acetyl-CoA carboxylase (p-ACC; Ser-79), phospho-HSL (Ser-563, Ser-660), and
tubulin obtained from Cell Signaling Technology (Beverly, MA, USA). The membranes were
then incubated with horseradish peroxidase-conjugated secondary antibodies overnight at
room temperature. The bands were visualized using an UVITec scanner (Cambridge) after
exposure to an enhanced chemiluminescence reagent (GE Healthcare Bio-Sciences AB, UK),
and the intensities of the bands were quantified using ImageJ software.
Table 2
Effects of EGCG on biochemical parameters of serum and tissues in the experimental groups.
Parameters
Blood triacylglycerol (mg/dl)
Blood cholesterol (mg/dl)
HDL-C (mg/dl)
LDL-C (mg/dl)
Glucose (mg/dl)
Insulin (μUi/ml)
HOMA-IR
TG gastrocnemius muscle (mg/100 g of tissue)
NEFA (mmol/l)
Glycerol (mmol/l)
Relative mass
Gastrocnemius muscle (%)
Liver (%)
CD
202.41 ± 17.98
126.76 ± 8.13
92.35 ± 6.75
74.55 ± 5.74
94.37 ± 1.81
6.57 ± 0.60
1.60 ± 0.12
206.04 ± 11.55
5.28 ± 0.29
0.08 ± 0.00
0.47 ± 0.01
2.93 ± 0.27
CD + E
HFD
HFD + E
9.24 ± 1.11
2.91 ± 0.26
229.75 ± 32.71
4.49 ± 0.45
0.09 ± 0.00
187.07 ± 7.05
146.87 ± 3.63 †
108.54 ± 5.93
98.36 ± 6.64 †
117.57 ± 4.59 †
15.75 ± 0.13 †
4.31 ± 0.13 ††
214.3 ± 26.51
5.41 ± 0.25
0.10 ± 0.01 †
170.28 ± 4.92
162.84 ± 6.55
105.28 ± 12.10
106.79 ± 8.02
155.85 ± 7.47 #
24.17 ± 3.42 #
8.67 ± 0.94 #
200.53 ± 22.86
5.29 ± 0.29
0.09 ± 0.01
0.44 ± 0.00
4.08 ± 0.26
0.40 ± 0.02
3.08 ± 0.12
0.36 ± 0.03
3.42 ± 0.10
170.2 ± 10.60 ⁎
136.02 ± 7.99
86.96 ± 10.48
76.49 ± 6.52
123.01 ± 8.27 ⁎⁎
Serum and muscle were collected from the mice after fasting for 12 h at the end of study. Values are expressed as mean ± EPM.
⁎ P b .05 significantly different from the CD + E compared to CD.
⁎⁎ P b .01 significantly different from the CD + E compared to CD.
†
††
#
P b .05 significantly different from the HFD versus CD.
P b .01 significantly different from the HFD versus CD.
P b .01 HF + E compared to HFD.
A. Santana et al. / Journal of Nutritional Biochemistry 26 (2015) 893–902
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2.5. Cytokine concentrations in adipose tissue
Epididymal and retroperitoneal adipose tissues were collected after euthanasia and
stored at −80°C. The adipose tissues were homogenized in protein extraction buffer
and centrifuged at 19,000 ×g for 45 min at 4°C. The supernatant was collected to
measure the concentrations of TNF-α and IL-10 following the recommendations of the
manufacturer (ELISA duoset, R&D Systems).
3. Statistical analyses
The normality of data were verified using the Shapiro–Wilk test.
Analysis of variance (ANOVA) was performed using Levene’s test.
Descriptive analysis was performed using the mean ± S.E.M. To verify
the interactions between groups, we used two-way ANOVA followed
by Bonferroni’s post hoc test. The level of significance was P ≤ .05.
Statistical analyses were performed using SPSS version 19.
4. Results
4.1. Effect of EGCG on body weight
In the fourth and sixth weeks of the experimental period, the body
weights of the mice fed HFD + EGCG were significantly reduced
compared with those fed HFD alone (P b .01). The body weights of the
mice that were fed CD + EGCG were affected in the sixth and seventh
weeks. A reduction in body weight was observed of the CD + EGCG
group compared with that in the CD group (P b .01; Fig. 1A). Similar
results were found for the delta (HFD + EGCG vs. HFD and CD + EGCG
vs. CD, P b .01; Fig. 1B).
4.2. Effect of EGCG on tissues
The relative weights of the liver, gastrocnemius muscle (Table 2),
and mesenteric adipose tissue (Fig. 1C) of the mice were not altered.
The relative mass of retroperitoneal adipose tissue was lower in the
HFD + EGCG group than in the HFD group (P ≤ .01). No difference was
observed when the animals received EGCG. However, the relative
mass of epididymal adipose tissue was significantly reduced in the
CD + EGCG group compared with that in the CD group (P ≤ .01;
Fig. 1C). The adiposity index was similar for the HFD-fed groups;
however, it was reduced in the CD + EGCG-fed mice compared with
that in CD-fed mice (P b .01; Fig. 1D).
4.3. Effect of EGCG on biochemical parameters in serum
The effects of EGCG on biochemical markers are listed in Table 2.
The total serum cholesterol (P b .05), glucose (P b .05), insulin (P b .01),
LDL-C (P b .01), and glycerol (P b .05) levels, and HOMA-IR (P b .05)
were higher in the HFD group than in the CD group. NEFA and HDL-C
levels were not significantly different between the treatment groups.
Triglyceride levels were significantly lower in the CD + E group than in
the CD group (P b .05). Increases in glucose and insulin levels were
found in the CD and HFD groups (P b .05 and P b .01) after EGCG
treatment compared with the findings in the corresponding placebo
groups. Because of the concomitant increases in glucose and insulin
content, HOMA-IR was significantly higher in the HFD + E group than
in the HFD (P b .01).
4.4. Effect of EGCG on the content of cytokines involved in
inflammatory pathways
TNF-α (P b .01 and P b .05) and IL-10 (P b .01 and P b .05) levels in
retroperitoneal and epididymal adipose tissues, respectively, and serum
adiponectin levels (P b .05) were increased in the CD-fed mice after
EGCG supplementation. The TNF-α level was lower in the HFD + E
group than in the HFD group (P b .05; Fig. 2A–C).
Fig. 1. Effect of EGCG supplementation on (A) body weight, (B) body weight gain, (C) relative
mass of adipose tissue, and (D) adiposity index. Values are expressed as means, with their
S.E. represented by vertical bars.*P b .05; **P b .01; ***P b .00 between CD + E and CD group.
$
P b .01 between HFD and CD group; #P b .01 between HFD + E and HFD group.
4.5. Effect of EGCG on the expression of proteins involved in lipolysis and
lipogenesis in adipose tissue
No statistical differences were found in the expression of the
analyzed proteins among the HFD groups. In addition, in the CD
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A. Santana et al. / Journal of Nutritional Biochemistry 26 (2015) 893–902
Fig. 2. Measurements of epididymal and retroperitoneal adipose tissues and serum
adiponectin. Concentrations of (A) TNF-α, (B) IL-10, and (C) adiponectin. The bars
represent the mean, and the vertical lines represent the S.E.M.. *P ≤ .05; **P b .01
comparing CD + E versus CD group; #P ≤ .05 between HFD + E and HFD group.
group, EGCG supplementation increased the phosphorylation of only
AMPK (P b .05) and total AMPK expression (P b .05) in epididymal
adipose tissue. In retroperitoneal adipose tissue, EGCG treatment
increased HSL phosphorylation (Ser-660) in the CD group. Perilipin
and pHSL (Ser-563) expression was similar in both adipose tissue
types. EGCG treatment increased ACC (Ser-79) phosphorylation in
both adipose tissue types in the CD-fed mice (P b .01; Fig. 3A–F). The
intensity of each protein band and the respective housekeeping
protein is shown in Fig. 4.
4.6. Effect of EGCG on the adipocyte area in epididymal adipose tissue
No differences were found in the diameters of adipocytes from
epididymal adipose tissue in the HFD-fed mice. However, we found
significant differences in the control group supplemented with EGCG.
Fig. 3. Quantification of protein expression (AMPK) and activity (HSL and ACC) of
epididymal and retroperitoneal adipose tissues. Protein expression of (A) pAMPK,
(B) AMPK, (C) pHSL(660), (D) pHSL(563), (E) perilipin, and (F) pACC. The bars
represent the mean, and the vertical lines represent the S.E.M.. *P b .05; **P b .01
comparing CD + E versus CD group.
This group exhibited a smaller adipocyte area than the placebo group
(Fig. 5A). Histological analyses are shown in Fig. 5A, and the adipocyte
area data are presented in Fig. 6.
A. Santana et al. / Journal of Nutritional Biochemistry 26 (2015) 893–902
}
Fig. 3. (continued)
5. Discussion
In the present study, we investigated the effect of EGCG treatment
on metabolic and inflammatory pathways in Swiss mice that were fed
HFD. Higher levels of total cholesterol, glucose, insulin, HOMA-IR, LDL-C,
and glycerol were found in the mice fed HFD, with lard as the primary
source, compared with the findings in the CD group.
This finding indicates that HFD treatment for 8 weeks was effective
in promoting variations in serum biochemical parameters. However,
HFD feeding did not modify the levels of HDL-C, NEFAs, and TAG
deposits in gastrocnemius muscle and the area of adipocytes within
this treatment time.
In our study, triglyceride levels were greater in the CD-fed animals
than in the HFD-fed animals. We did not expect this result, but we
believe that this finding could have arisen because carbohydrates and
lipids are inextricably connected (despite belonging to two different
biochemical classes). Metabolically, the elevation of blood lipid
concentrations in response to the high consumption of carbohydrates,
especially sugars, has been recognized since the 1960s [18–21]. As
macronutrients, dietary sugars appear to modulate the manner in which
the human body handles dietary fat. This metabolic interaction forms
the basis of carbohydrate-induced hypertriacylglycerolemia (HPTG), a
condition that occurs because of lowered dietary fat and increased
dietary carbohydrate intake. Low-fat high-carbohydrate diets have been
demonstrated to elevate plasma TAG concentrations and depress HDL-C
concentrations in the short term, as well as induce long-lasting effects
[22]. This response appears to be similar to HPTG, which is often
observed in diabetes, and the development of heart disease, which
may be a common consequence of consuming diets high in fats [23]. It
is unknown whether carbohydrate-induced HPTG confers a level of
atherogenic risk similar to that associated with other forms of HPTG [24].
Although HFD feeding did not cause hypertriglyceridemia in the present
study, we know that the prolonged consumption of unbalanced diets
rich in saturated fatty acids such as HFDs can result in increases in total
cholesterol and LDL-C levels, thus elevating cardiovascular risk.
897
No differences were found in the body weights of HFD-fed mice
between the groups. However, we found differences in the relative
mass of retroperitoneal adipose tissue when the animals received
HFD. These animals exhibited a high relative mass compared with the
CD-fed mice, thus proving the role of HFD in promoting greater fat
accumulation in this tissue.
We believe that energy density (hypercaloric or isocaloric) and the
quantity of lipids can be influenced by dietary intake. One limitation of
our study is that we did not evaluate the level of dietary intake of these
animals. This could have clarified whether dietary consumption was
different among the animals that received the CD or HFD. Chen et al.
[25] found a reduction on food consumption in animals fed HFD
compared with CD-fed animals. In addition, Akagiri et al. [26] did not
detect significant differences in weight gain between animals fed HFD
and those fed a CD in a 14-week study, although HFD-fed animals
tended to gain more weight. Buettner et al. [27] observed that
prolonged lipid-rich diet feeding induces weight gain in susceptible
rats on the order of 10–20% compared with control animals. According
to these authors, the induction of obesity is more effective when HFD
feeding is started at a young age and continued for several weeks.
However, some authors suggested that rats fed HFD maintain
metabolic homeostasis for approximately 6 weeks, following which
they start to develop obesity and insulin resistance [28].
A possible explanation for the influence of the duration of HFD feeding
on weight gain is that HFD feeding induces a positive fat balance on a
short-term basis because of the loss of adjustment between fat oxidation
and consumption [29]. In the long term, this positive accumulation can
lead to weight gain; therefore, body weight gain and fat accumulation
increase with an increasing duration of HFD feeding. However, there is
controversy about the optimal timing of diet-induced obesity in rodents.
There is substantial controversy about the weight gain of rats fed
HFD, and a possible explanation for the wide variation in results could
be the absence of standardization of the HFDs used in the various
studies [30]. Many of these studies did not describe the exact quantity
of lipids contained in the HFDs, their energy density, the duration of
HFD feeding, or the types of fat used.
HFDs have been used for decades to develop models of obesity in
rodents. These diets can lead to major metabolic complications
associated with the pathogenesis of obesity. Fat-enriched diets mainly
consisting of saturated fatty acids have been demonstrated to increase
the levels of total cholesterol, triglycerides [31], and LDL-C [32], as well
as to elevate body weight and insulin resistance [33,34] in experimental animal and human models of obesity.
Obesity is clearly a chronic low-grade inflammatory state as
initially proposed by Hotamisligil [35]. Furthermore, the effect of HFD
feeding on inflammatory markers depends on different factors such as
gender [36], biological age [37,38], the intervention time [39], and the
type of fat used [40]. Our data demonstrated that HFD feeding for
8 weeks did not alter the production of anti- and proinflammatory
cytokines in adipose tissue and serum, which is typically observed in
obesity-associated metabolic disorders.
We believe that the inflammatory response to HFD feeding
develops in distinct stages, namely, an acute phase followed by an
inflammatory response in the liver and WAT (chronic). Both the acute
phase and the inflammatory response are mainly transient in the liver,
whereas the inflammatory responses in WAT increase over time,
indicating that WAT is an important source of inflammation in the long
term. This chronic inflammatory response of WAT involves the
increased expression of genes encoding inflammatory cytokines. The
acute phase is defined as a systemic change occurring in response to
injury or inflammation, which can be split into two major effects:
alterations in the concentrations of a number of plasma proteins and
certain physiological and behavioral modifications. The acute phase
response plays a fundamental role in avoiding the harmful effects of
external challenges and pathogenic events in mammals with the
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A. Santana et al. / Journal of Nutritional Biochemistry 26 (2015) 893–902
Fig. 4. The intensity of each band of the proteins analyzed and respective housekeeping protein (tubulin) in both adipose tissues.
function of reestablishing homeostasis. Therefore, our study illustrated that short-term HFD feeding may trigger different responses in the
pathogenesis of inflammation. The treatment period of 8 weeks was
not sufficient to impair plasma proteins involved in inflammatory
signaling; however, this treatment period was sufficient to induce
changes in some metabolic parameters, indicating that the effects of
the HFD are dependent on the treatment time, type of tissue, and
inflammatory stimulus [41–44].
Fig. 5. Histology of groups: (A) CD; (B) CD + EGCG; (C) HFD; (D) HFD + EGCG. Magnification = ×40.
A. Santana et al. / Journal of Nutritional Biochemistry 26 (2015) 893–902
Fig. 6. Adipocyte area in the epididymal adipose tissue.
Reductions in adipose tissue anabolism, for example, lipogenesis or
adipogenesis, and enhancements of lipid catabolism, for example,
lipolysis, serve as strategies to reduce excess fat and prevent fat
accumulation [45]. In the present study, no variations were found in
the expression of AMPK, perilipin, HSL, and ACC in the HFD-fed mice.
Although some studies have reported reduced HSL expression in the
adipose tissue of obese humans [46,47], others have reported no
alteration in the levels of HSL and ATGL in visceral adipose tissue in
morbidly obese men [48]; a few studies have reported an increase in
obesity [49].
More pronounced physiological effects were found when EGCG
supplementation was used in the control group. High-quality diets,
including bioactive compounds, are becoming particularly important
for the prevention of noncommunicable diseases, particularly when
accompanied by physical activity. These diets represent the primary
promising factor in the primary and secondary prevention of obesity.
Differences in the dosage, type of extract (decaffeinated tea or
caffeinated tea extract), supplier, treatment time, and bioavailability
result in different health effects. In the present study, we did not test
EGCG levels in plasma, but we used published data as a reference.
In a report by Lee et al. [50], EGCG content was analyzed after the
administration of a single oral dose of green tea or decaffeinated green
tea (20 mg tea solids/kg) to volunteers. The dose of green tea used
in the study (20 mg tea solids/kg) corresponds to 195 mg of EGCG.
The maximum plasma concentration of EGCG was 77.9 ± 22.2 ng/ml.
The time needed to reach the peak concentrations was in the range of
1.3–1.6 h. The elimination half-life was 3.4 ± 0.3 h.
In a study by Oritani et al. [51], in which EGCG was orally
supplemented (100 mg/kg) to rats, the catechin was detected in
7.6 min in plasma at a concentration of 23 μg/l, and according to the
findings of Yang et al. [52] in volunteers humans, after injecting 1.5,
3.0, and 4.5 g of decaffeinated green tea solids (dissolved in 50 ml of
water), the maximum plasma concentration of EGCG was 326 ng/ml
at 1.4–2.4 h after ingestion.
Considerable differences in the bioavailability of EGCG in experiments
with humans and animal models were observed in the literature, and we
believe that this may be caused by the instability of EGCG in different
conditions; specifically, factors as cool and dry storage, fasting conditions,
albumin, soft water, vitamin C, fish oil, and piperine appear to enhance the
plasma levels of EGCG, and oxidation via air contact, gastrointestinal
inactivation, sulfation, glucuronidation, and metals (calcium and
magnesium) appear to diminish the bioavailability of EGCG. Most of
these factors are easily modifiable, although the bioavailability and
metabolic fate of tea polyphenols are not clearly understood [53].
In the present study, the body weight of the CD + E group was
reduced in the sixth and eighth weeks. EGCG supplementation
significantly suppressed adiposity in this group, possibly because of
the low visceral WAT weight, especially concerning epididymal adipose
899
tissue, the relative mass of which was reduced. In addition, animals
supplemented with EGCG in the CD group displayed a lower adipocyte
area in epididymal adipose tissue. A few studies have reported the effect
of decaffeinated green tea extract on body weight. Brown et al. reported
that 530 mg of decaffeinated green tea taken twice daily for 6 weeks
(800 mg total catechins) does not affect the apparent body weight [54].
Similarly, Hsu et al. found no differences between the decaffeinated
green tea extract and placebo groups regarding waist circumference,
body weight, and body mass index (BMI) after 16 weeks of administration. However, numerous studies have reported the different effects
of caffeinated green tea extract on body weight [55]. Basu et al. observed
that green tea consumption (four cups per day) or extract supplementation (two capsules per day) with similar EGCG dosing for 8 weeks
significantly reduced body weight and BMI [56]. Similarly, Nagao et al.
found that the daily ingestion of green tea containing 583 mg of
catechins decreases the body weight, BMI, body fat ratio, body fat mass,
waist circumference, hip circumference, visceral fat area, and subcutaneous fat area [57]. Janle et al. reported a lower weight gain in diabetic
rats administered green tea extract containing 50–125 mg/kg EGCG
starting at 7 weeks of age [58].
An important mechanism related to the body weight-modifying
effects of EGCG is the compound's tendency to modulate lipogenic
enzymes and stimulate lipolysis. Lee et al. reported that EGCG depletes fat
accumulation via the stimulation of lipolysis by increasing HSL gene
expression in 3 T3-L1 adipocytes [59]. Cunha et al. reported an increase in
HSL and ATGL protein levels in the adipose tissues of mice fed a chow diet
supplemented with caffeinated green tea (400 mg/kg body weight/day)
for 8 weeks [10]. In the current study, EGCG did not significantly affect the
expression of HSL (Ser-563, Ser-660) and perilipin in epididymal adipose
tissue. However, EGCG affects retroperitoneal adipose tissue by enhancing the phosphorylation of HSL (Ser-660). Numerous agents control
lipolysis in adipocytes by modulating the activity of HSL and ATGL [60].
Furthermore, the lipolytic pathway can be activated when TNF-α, a
pleiotropic cytokine involved in different signaling pathways, regulates
TAG hydrolysis in adipocytes. This cytokine has been implicated in some
of the direct effects of lipolysis; however, whether it is involved in the
regulation of lipolysis must be established. Moreover, EGCG supplementation significantly increased TNF-α concentrations in both adipose
tissues in the control group. We hypothesized that the effects of EGCG on
body weight may be related to this cytokine because its action is
independent of the activation of lipases. The lipolytic effect of TNF-α is
most likely dependent on transcriptional regulation [61], and the
cytokine is also considered to be an important modulator of energy
metabolism, particularly in adipocytes [62]. Gasic et al. proposed that
some of the effects of TNF-α can be mediated by the downregulation of
membrane Gi, thus resulting in abrogated antilipolytic signals [63]. Other
researchers reported that TNF-α down-regulates the expression of
perilipins and facilitates the access of lipases to lipid droplets [64,65].
The influence of members of the MAPK family on the expression of Gi or
perilipins must be established. Moreover, TNF-α-mediated lipolysis is
most likely dependent on numerous other downstream effectors, and
further studies are needed to define them [61].
Many regulatory effects of adipocyte-derived hormones on
different biological systems have been identified, and maintaining
the systemic energy homeostasis remains the essential function of
most adipocyte-derived hormones [66].
Adiponectin is a hormone secreted by adipocytes that regulates
energy homeostasis as well as glucose and lipid metabolism [66]. It
affects the activation of AMPK in different tissues, such as adipose tissue
[67]. In humans, adiponectin levels can be regarded as a marker of the
total triglyceride lipolytic rate per adipose tissue mass [68]. Low adipose
tissue adiponectin expression, as observed in obese people, may
contribute to the progression of obesity and its comorbidities by
modulating HSL activity and fatty acid oxidation [69]. In our study,
EGCG supplementation in the CD-fed mice was found to enhance the
900
A. Santana et al. / Journal of Nutritional Biochemistry 26 (2015) 893–902
levels of adiponectin in serum compared with those in the CD group. The
increase of adiponectin levels corroborates our other findings and
confirms the effects of EGCG on different signaling pathways related
to lipid metabolism. As expected, a concomitant increase was found in
IL-10 expression in the same group. IL-10 is secreted by activated
monocytes/macrophages and lymphocytes and is known to possess
multifaceted anti-inflammatory properties [70]. Furthermore, we
hypothesized that the increase in IL-10 levels in adipose tissue is
associated with weight loss, as found in other studies [71–74]. Although
the increased levels of TNF-α are linked in some inflammatory disorders,
this cytokine also participates in numerous cellular functions not
associated with inflammatory processes. In our study, the increase of
TNF-α levels was accompanied by increased levels of anti-inflammatory
cytokines such as adiponectin and IL-10, demonstrating that its effects
were not related to inflammatory processes; however, we believe that
its effect is possibly associated with the regulation of metabolism.
In addition, the positive effects of green tea extract on plasma lipids
in human subjects have been reported in some studies. Maron et al.
found that 375-mg green tea extract capsules taken daily decreased
total cholesterol and LDL-C levels after 4 weeks. This reduction was
more significant after 12 weeks of treatment [75]. Batista et al. observed
reductions of 3.9 and 4.5% in total cholesterol and LDL-C concentrations,
respectively, when 250-mg capsules of dry green tea extract were
ingested for 8 weeks. However, no significant variations were found in
HDL-C and triglyceride levels [76]. In the present study, EGCG
supplementation conferred beneficial effects by reducing triglyceride
in the CD group, but this was not observed in the HFD group.
We believe that the decrease in triglyceride levels was promoted by
signaling pathways involving both adiponectin and AMPK because these
two proteins together modulate lipid oxidation and decrease lipid
synthesis in the liver and possibly in adipose tissue. The current study
found that the expression of both adiponectin and phosphorylated AMPK
was decreased in retroperitoneal, epididymal, and mesenteric adipose
tissues in the HFD-fed animals treated with placebo or EGCG, whereas
that in the CD-fed animals, it was elevated. Taken together, these results
may indicate that adiponectin and AMPK signaling pathways together
are necessary to control metabolism in adipose tissue and decrease
triglyceride levels. Adiponectin reduces triglyceride content in adipose
tissue by binding to its receptors, which increases AMPK phosphorylation, activating the AMPK signaling pathway. Phosphorylated AMPK
activates PPAR, which up-regulates the expression of its target genes,
thereby increasing lipid oxidation. Furthermore, phosphorylated AMPK
inhibits the expression and transcriptional activity of SREBP-1c and
ChREBP, reducing the expression of their target genes and thereby
inhibiting lipid synthesis in hepatocytes [77]. Therefore, this response
appears to mirror lipid metabolism in adipose tissue. In the present
study, the activity of ACC (Ser-79), which inhibits lipid synthesis, was
decreased in retroperitoneal and epididymal adipose tissues in both HFD
groups and were elevated in CD-fed animals. Thus, according to
published data, obesity is a major risk factor for disorders involving the
regulation of lipid metabolism [78–81]. Our study clearly demonstrated
that EGCG has beneficial effects on lipid metabolism, but its effects were
possible only in animals fed a healthy diet.
However, no difference was observed in total cholesterol, LDL-C,
HDL-C, NEFA, and glycerol levels. Similar results were reported by
Serisier et al., who recorded no significant differences in NEFA, total
cholesterol, LDL-C, and HDL-C levels of dogs administered green tea
extract (80 mg/kg/day) orally for 12 weeks [82].
Numerous studies reported the influence of green tea and tea
catechins on the regulation of glucose homeostasis; however, the results
remain controversial. Igarashi et al. illustrated that the consumption of
dietary catechins lowered blood glucose levels at 6 and 9 weeks [83].
In contrast, Fukino et al. reported that an intervention group that
consumed a packet of green tea extract powder containing 544 mg of
polyphenols (456 mg of catechins) daily for the first 2 months did
not display variations in fasting serum glucose and insulin levels and
HOMA-IR [84]. Josic et al. did not observed a reduction in glucose or
insulin levels in participants who consumed 300 ml of green tea [85].
Another study reported that the ingestion of green tea is associated with
reduced levels of fasting blood glucose only among nonobese elderly
people [86]. A meta-analysis found that the consumption of green tea
extract with or without caffeine promoted significant reductions in
fasting blood glucose concentrations but did not affect fasting blood
insulin levels or HOMA-IR when the follow-up time exceeded a median
of 12 weeks [87]. Our results revealed that EGCG supplementation
increases fasting serum glucose levels, HOMA-IR, and serum insulin
levels compared with the corresponding placebo groups. These
biochemical changes may lead to the development of diseases such
as diabetes.
Dietary supplementation with EGCG in HFD-fed animals affected
body weight only in the fourth and sixth weeks. Similarly, Bajerska et al.
reported that rats fed HFD enriched with 2.0% green tea aqueous extract
for 8 weeks displayed reduced body weight gain and the absence of
visceral fat accumulation [88]. We also found reduced cytokine TNF-α
concentrations in retroperitoneal adipose tissue. We demonstrated that
normocaloric diet feeding was associated with a similar percentage
increase in body weight, as observed in the HFD group. These data can
be explained, at least in part, by the increase in skeletal muscle mass, in
contrast with the increase in body adiposity in the HFD group.
In summary, these results illustrated that HFD feeding, as opposed to
CD feeding, for 8 weeks leads to increases in biochemical parameters,
and EGCG supplementation prevented greater weight gains but failed to
improve the parameters modified by HFD. These results indicate that a
nutritional intervention consisting of 50 mg/kg/day decaffeinated EGCG
for 8 weeks cannot modulate the negative effects of an unbalanced diet.
The most significant results were found with CD feeding and EGCG
supplementation, which effectively reduced body weight, probably
owing to the crosstalk between lipolytic and inflammatory pathways in
adipose tissue.
Potential conflicts of interest
All authors declare no conflicts of interest.
Acknowledgments
This work was supported by Conselho Nacional de Desenvolvimento
Científico, Tecnológico (CNPq), Fundação de Amparo a Pesquisa do
Estado de São Paulo (FAPESP 2012/03713-5), and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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