Oxidative stress and
inflammation are key features in a number of chronic diseases, most notably in
those with metabolic alterations [1-3]. Epidemiological studies have identified
fruits and vegetables as
key components of dietary patterns that reduce the risk for the development of
chronic diseases, including cardiovascular disease, insulin resistance and type
II diabetes and the incidence of many tumors [4-6].
Euterpe oleracea Martius is a large palm tree found in South
especially in the Amazon. Its fruit,
commonly known as açai, is a round, black-purple berry  and its pulp is
traditionally consumed in Brazil. Açai has gained international attention as a
functional food owing to its high content of polyphenols and potential health
Açai beneficial effects are
related mainly to secondary metabolites such as flavonoids, including
anthocyanins and proanthocyanidins, which provide antioxidant activity [9-11].
Several studies showed that açai consumption slows the progression of oxidative
stress [12-16] and presents
Although the açai compounds
show positive health effects, most of the commercial beverages containing this
berry also present high sugar content. The consumption of sugar-sweetened
beverages has been associated with obesity and weight gain , impaired
glucose and lipid metabolism and promotion of inflammation . Therefore, the
high glucose concentration in the açai juices could inhibit the described
health effects of this fruit.
Based on this data, we
investigated the effects of short-term consumption (6 weeks) of two different
commercial açai beverages available in Brazil. First, we examined the effects
of short-term consumption of a glucose-sweetened
açai beverage on plasma lipid profile of rats. After detecting deleterious
metabolic effects, we also studied the effects of short-term consumption of a
commercial honey-sweetened açaí beverage in lipid profile, as well as oxidative status and cytokine expression in the visceral white
adipose tissue, liver and muscle.
Materials and Methods
Male adult Wistar rats
obtained from the Institute of Biomedical Sciences, University of São Paulo,
were maintained in metabolic cages, in a 12 h light:12 h dark cycle, under controlled temperature conditions (22 ± 2oC).
Animals were acclimated to their environment for 1 week before the beginning of
the experiment. The Ethical Committee for Animal Research from the University
of São Paulo approved all the adopted procedures, which were carried out in
accordance with the ethical principles stated by the Brazilian College of
Animal Experimentation - Protocol n. 041/2005.
Two studies were carried
out in different moments. In both studies animals were randomly divided into 2
groups, a control group and an açai group. Control group received water and
food (NuvilabCR1-Nuvital, Curitiba, Paraná, Brazil) ad libitum. Açai group received commercial açai beverage and food
ad libitum (these animals had no acess to water).
Study 1: In the first study, animals received a
commercial sugar-sweetened açai beverage (Açai-S), containing 40 % acai pulp,
15 % glucose, citric acid and water.
Study 2: Animals received a commercial
honey-sweetened açai beverage (Açai-H), containing 70 % açai pulp, 18 % honey,
10 % acerola (Malpighia emarginata),
2 % (Citrus limon), 0.1 % powdered
guarana seeds (Paullini)
Açai beverages were always
offered in dark bottles to maintain the sensory characteristics and to prevent
oxidative processes. Animals were weighed 3 times per week, and their food and
liquid intake was recorded daily. After six weeks in each treatment, animals
were sacrificed by decapitation after 12 h fasting. Then, the weight of
visceral white adipose tissue depots (epidydimal, retroperitoneal and
mesenteric), liver and gastrocnemius muscles were measured. Blood and tissues
were collected and immediately stored at - 80oC until the
experiments were carried out.
Plasma Measurements and
Liver Lipid Content Assessment
Blood plasma was isolated
by centrifugation at 3,000 x g for 15 min and stored at - 80oC.
Total cholesterol, HDL-cholesterol, triacylglycerol and glucose were quantified
using commercial colorimetric kits (Labtest®, Brazil). Adiponectin
and leptin plasma levels were determined by ELISA (Invitrogen, USA) and
radioimmunoassay (LincoReasearch Inc., USA), respectively. Liver
triacylglycerol content was assessed with the method described by Folch et al.
Cytokines Protein Content Assessment
After euthanasia, the
tissues (liver, gastrocnemius and visceral white adipose tissue depots) were
rapidly removed and frozen. These tissues (0.1 - 0.3 g) were homogenized in a
RIPA buffer (0.625% Nonidet P-40, 0.625% sodium deoxycholate, 6.25 mM sodium phosphate,
and 1 mM ethylene-diamine tetra acetic acid at pH 7.4) containing 10 μg/ml of a
protease inhibitor cocktail (Sigma-Aldrich, USA). Homogenates were centrifuged
at 12,000 × g for 10 min at 4°C, the supernatant was saved, and the
protein concentration was determined using a BCA protein assay reagent (Thermo
Scientific, USA). Quantitative assessment of TNF-alpha (CRC3013), IL-6
(CRC0063) and IL-10 (CRC0103) proteins content was carried out by ELISA
Thiobarbituric Acid Reactive Species (TBARS)
As an index of lipid
peroxidation in the tissues, we measured the formation of TBARS during an
acid-heating reaction . Briefly, tissue samples were mixed with 8.1%
tricholoacetic acid (2.5 M, pH 3.4; Sigma, USA) and 0.8% thiobarbituric acid
(Sigma, USA). The tubes were covered with aluminium foil and kept in a dry bath
for 30 min, followed by centrifugation at 3,000 rpm for 10 min at 4oC.
The absorbance of the supernatant was read at 532 nm with Malondialdehyde (MDA)
as an external standard. Data are reported as mmol of MDA/mg of protein.
Real time PCR
Total RNA was obtained from
aliquots of 100 mg of liver by Trizol® reagent extraction according
to the manufacturer’s instructions. The primers used were: GLUT-2 [NM_012879.2]
(sense TTAGCAACTGGGTCTGCAAT, antisense GGTGTAGTCCTACACTCATG); glycogen synthase
2 [NM_013089.1] (sense GTTTCCTGGGAAGTGACCAA, antisense CCATGTTTGTTCATGGCATC),
SREBP-2 [NM_005106.4] (sense GGCCTGACAGGTGAAATCAG, antisense
ATAGGGGGCATCAAATAGGC); MTP [NM_000253] (sense AATGACCGGCTGTACAAGCTCAC,
antisense CCTTTGAAGATGCTCTTCTCTC); Glyceraldehyde-3-Phosphate Dehydrogenase
(GAPDH) [NM_017008.3] (sense AGACAGCCGCATCTTCTTGT, antisense
CTTGCCGTGGGTAGAGTCAT). Quantitative real-time PCR was carried out with an ABI
7300 Real Time PCR Systems (Applied Biosystems) and the mRNA levels were
determined by a comparative Ct method.
Data are expressed as means
± s.e.m. Statistical analysis was performed using the Graph Pad Prism
statistics software package version 5.0 for Windows. Results were analyzed by
Student’s t test, followed by Tukey’s post-test. The 0.05 probability level was
considered to indicate statistical significance.
Food and beverage intake
Food intake during the
experimental period was decreased in Açai-S compared to Control (15.76 ± 0.04 g
/day vs. 22.23 ± 0.88 g / day, respectively; p < 0.05). However,
supplemented animals consumed 58.88 ± 0.52 mL / day of acai beverage. The
beverage intake resulted in a higher total caloric intake by the supplemented
rats (103.75 ± 1.77 kcal/day vs. 75.61 ± 2.64 kcal/day; p< 0.05).
Body and tissues relative
The increased caloric
intake resulted in higher body weight gain in Açai-S compared to Control (Table
1). The weight of all visceral white adipose tissue depots and liver was
increased in Açai-S (Table 1).
content and plasma measurements
The triacylglycerol liver
content was augmented in supplemented rats (Açai-S = 74.34 ± 16.53 vs. Control
= 39.66 ± 3.82 mg triacylglycerol /mg of tissue, P<0.05). Similarly,
triacylglycerol plasma levels were also higher in Açai-S than (Table 2). Despite these negative
effects, the chronic consumption of a sugar-sweetened commercial açai beverage
promoted an increase in HDL-cholesterol plasma levels and there was no
total cholesterol and glucose levels (Table 2).
The leptin plasma w augmented in the supplemented animals (Table 2), positively correlated with body weight gain (r=0.6; p<0.05).
Since plasma leptin levels are reported to be related with increases on adipose
tissue mass this result was already expected. However, adiponectin plasma
levels were surprisingly higher in supplemented rats (by 60%, p<0.05) (Table
2). Moreover, adiponectin plasma concentration was also positively correlated
with body weight gain (r=0.7; p<0.05).
The liver plays a central
role in glucose homeostasis and lipid metabolism; thereby we assessed the
effects of short-term sugar-sweetened a
beverage consumption important key genes: GLUT-2,
glycogen synthase-2, MTP and SREBP-2. Liver GLUT-2 and glycogen synthase-2 mRNA
expression was reduced in Açai-S. Reduced GLUT-2 mRNA content in the liver has been
insulin resistance in this tissue  and a decrease in glycogen synthase-2
gene expression suggests modulation of glucose homeostasis and possible
impairment of hepatic glycogen synthesis. There were no alterations regarding the
other studied genes (Figure 1).
Food and beverage intake
Food intake during the
experimental period was decreased in Açai-H, when compared with control (14.27
± 0.37g/day vs. 20.75 ± 1.14g/day, respectively; p<0.05). Moreover,
supplemented rats consumed 84.24 ± 3.72 mL of açai beverage per day. When total
drink and food consumption was analysed, Açai-Htotal caloric intake than rats (107.29 ± 3.20 K
vs. 70.66 ± 275 Kcal/day, respectively; p<0.05).
Body and tissue
relative weight gain
Despite the Açai-H
augmented caloric intake, both groups showed similar body weight gain (Table
1). Similarly, no differences were found regarding the relative weight of
liver, retroperitonial white adipose tissue and mesenteric white adipose
tissue, after the experimental period (Table 1). Açai beverage consumption
presented solely an effect on the relative weight of the epididymal white
adipose tissue and gastrocnemius. It is important
to note that some supplemented rats showed moderate diarrhea episodes in the first
week of supplementation, which was completely abolished along the following
Table 2 shows the plasma
lipid profile, as well as glucose, leptin and adiponectin plasma concentration.
There was no difference between the groups regarding all these measurements.
Cytokine protein expression
To assess the possible
anti-inflammatory role of the açai beverage we measured cytokine expression in
the visceral white adipose tissue (retroperitoneal, epididymal and mesenteric
pads), liver and gastrocnemius. The protein content of IL-6 and TNF-alpha, two
important inflammatory cytokines, as well as of IL-10, the major
anti-inflammatory cytokine, is described in Table 3. Cytokine expression showed
the white adipose tissue in Açai-H. In the retroperitoneal depot, a
reduced TNF-alpha expression was observed, resulting in a modified
IL10/TNF-alpha ratio. Moreover, IL-10 were increased by 56 % in the mesenteric white adipose tissue of Açai-H.
reactive species (TBARS)
studies have shown that the açai fruit presents in vitro [9,19] and in vivo [20,21] antioxidant capacity.
Therefore, lipid peroxidation in the visceral white adipose tissue pads, liver
and gastrocnemius was
(Figure 2). Açai-H had a
significant reduction in TBARS formation in the retroperitoneal white adipose
tissue and epididymal white adipose tissue 4 9.75
% and 44.90 % respectively (p<0.05), in relation to the Control.
Açai is one of
Amazon´s most popular functional food
and widely in the world . Commercial açai beverages claim to have health
benefits due to the antioxidant and anti-inflammatory properties of this fruit.
However, our results show that the benefits of consuming these beverages
depends on the sweetener used.
We demonstrated that
short-term consumption of a honey-sweetened açai beverage is able to modulate
cytokine levels in the visceral white adipose tissue, in a depot-specific
manner. Results also revealed that this supplementation was able to reduce
oxidative stress markers in two visceral white adipose tissue pads.
White adipose tissue is an
important endocrine organ being involved in the regulation of many pathological
processes . The white adipose tissue is able to secrete a plethora of
factors, including cytokines (e.g. TNF-alpha, IL-10, IL-6) and hormones (e.g.
leptin and adiponectin), acting locally and distally, with autocrine, paracrine
Severaldifferences have been reported among intra-abdominal visceral white adipose
tissue and peripheral subcutaneous white adipose tissue . Visceral adipose secretes cytokines, such as
TNF- alpha and IL-6, which are associated with many disease conditions (e.g.
obesity, metabolic syndrome, diabetes, etc.).
Short-term consumption of a
commercial honey-sweetened açai beverage induced an increase in IL-10 protein
content in the mesenteric white adipose tissue, as well as a reduction in
TNF-alpha protein levels in the retroperitoneal pad. Moreover, the reduced
TNF-alpha expression in the retroperitoneal depot resulted in a modified
IL-10/TNF-alpha ratio. Consistent with our observation, Xie et al.  showed
a reduction on serum levels, gene expression and protein levels of TNF-alpha
and IL-6 in resident macrophages from mice fed with açai. Other authors also
described that oral administration of an açai stone extract reduced the
increase in TNF-alpha expression in the lung of animals exposed to cigarette
smoke . Furthermore, a recent research described that açai frozen pulp
ingestion prevented increase in IL-1beta, IL-18 and TNF-alpha, reducing the
carbon tetrachloride-induced damage in rat brain tissue .
TNF-alpha is the
most-studied cytokine in white adipose tissue. This cytokine is involved in
metabolic, physiological and immunological regulation in this tissue, acting as
a mediator of inflammation. On the other hand, IL-10 secreted by adipocytes,
white adipose tissue stromal vascular fraction and tissue matrix, inhibits the
production of several cytokines, such as TNF-alpha, IL-1beta and IL-6 .
IL-10/TNF-alpha ratio has been adopted as an indicator of the inflammatory
status and disease-associated morbidity, with lower values associated with
poorer prognoses .
Açai has been reported to
contain many bioactive compounds. Major polyphenolic components in açai pulp
include anthocyanins, proanthocyanidins, other flavonoids and lignans [30,31].
Among them, the flavonoids were found to be the major polyphenols. Flavonoids
from açai pulp present anti-inflammatory effects at least in part through
inhibition of NF-kB activation  and by modulating the Toll-Like Receptor-4
(TLR-4) and NF-kB protein expression . NF-kB is a key mediator of
inflammation in adipocyte cells and studies have shown a close relationship
between TLR-4 and the activation of the NF-κB pathway, which leads to the
elevation of pro-inflammatory adipokine genes and protein expression in adipose
Flavonoids have been shown,
as a group, to exhibit strong antioxidant capacities. The mechanism responsible
for the antioxidant activity of flavonoids involves the direct scavenging or
quenching of oxygen free radicals or excited oxygen species, as well as the
inhibition of oxidative enzymes that generate these reactive oxygen species
. We found lower MDA levels in two visceral white adipose tissue pads
(retroperitoneal and epididymal) in Açai-H. Studies have shown a similar
reduction in MDA content in different tissues of animals treated with
anthocyanins or proanthocyanidins- rich fruit extracts [37-39]. Moreover, the
açai antioxidant effect was previously shown in the serum and in the liver of
ApoE deficient mice , in the serum of healthy adults  and liver of mice
fed with a high-fat diet .
The improvement of
anti-inflammatory/antioxidant profile of all visceral white adipose tissue
depots indicates a protective effect of the short-term honey-sweetened
commercial açai beverage consumption against chronic diseases. It is
interesting to note that even with the higher epididymal white adipose tissue
absolute and relative weight, the Açai-H cytokine profile was not altered. In
addition, the MDA content was increased in this tissue.
Adipokines play a role in a
wide variety of physiological and pathological process, including immunity and
inflammation, in addition to having significant effects on metabolism. Among
them, leptin and adiponectin are the most widely investigated. Leptin has a
pivotal role in the control of food intake and plasma leptin levels are related
with increases on adipose tissue mass. Therefore, an increase in leptin plasma
levels only in Açai-S was already expected.
Adiponectin is an anti-inflammatory and insulin-sensitising adipokine,
playing a central role in glucose and lipid metabolism . Considering that
generally adiponectin plasma levels are inversely correlated with body weight,
the increased adiponectin plasma level in Açai-S are very intriguing. Studies
have reported that chronic consumption of procyanidins or plant sterols, both
compounds present in açai, is able to modulate inflammation and oxidative
stress by reducing inflammatory markers and increasing adiponectin plasma
levels [41-43]. We hypothesize that this increase in adiponectin plasma levels
could be a physiological response to counteract the inflammatory profile
induced by the consumption of a glucose-rich beverage. In this respect, a
recent study described a similar increase in adiponectin serum levels of rats
fed with a high glucose diet . The authors suggest that this effect is due
to the pro-inflammatory microenvironment of the adipose tissue of these rats.
Diets rich in sucrose have
been strongly associated with an increased prevalence of obesity, type 2
diabetes and cardiovascular risk factors. High sucrose feeding is able to
induce steatosis, hepatic insulin resistance and hypertriglyceridemia [44,45].
In this study, the short-term consumption of a glucose-sweetened açai beverage
resulted in high liver and plasma triacylglycerol content. Despite these
negative effects, Açai-S animals showed increase in HDL-cholesterol plasma
levels. Similarly, an increase in HDL-cholesterol in apolipoprotein deficient
mice fed with açai was previously described . On the other hand, the consumption of the
honey-sweetened açai beverage failed to show any improvement on plasma lipid profile.
Some studies reported açaihypocholesterolemic effect in pathological conditions
[7,14,20]. Thus, it could be possible that the beneficial effect of açai is
more pronounced when some alteration in plasma lipid profile is present.
The liver is the primary
organ responsible for glycogen and lipid metabolism. Biosynthesis of glycogen
and lipids is the primary means by which the body stores excess nutrients.
Under normal conditions, glycogen is the primary storage form of excess energy.
Glycogen production is regulated primarily via enzymes such as glycogen
synthase . The reduction in the glycogen synthase and GLUT-2 gene
expression observed in Açai-S indicates impaired liver glucose metabolism in
these animals. Enhanced lipogenesis and decreased glycogen synthesis are
hallmarks of hepatic insulin-resistance, which might subsequently lead to the
development of type 2 diabetes mellitus . Adipose tissue physiology is an
important contributor to the regulation of insulin resistance and fatty liver
disease . Many of the interactions among adipose tissue, insulin
resistance, and hepatic steatosis are orchestrated by adipokines. Adiponectin
expression was associated with the up-regulation of insulin-sensitizing genes
in the liver (i.e., GLUT-2 and PPARγ) in an obesity model of insulin resistance
. Therefore, we can hypothesize that the increase in adiponectin plasma
levels could be a physiological response to counteract not only the
inflammatory response, but, as well the disruption in liver glucose metabolism
induced by the consumption of a glucose-rich beverage.
It is important to note
that commercial beverages used in our study contained other bioactive compounds
besides açai. Antioxidant effects have been described for acerola (Malpighia emarginata) [49,50], lime (Citrus limon) , guarana (Paulliniacupana)  and honey .
Interestingly, a recent study showed that the intake of acerola juice decreased
the level of inflammatory proteins in the adipose tissue of obese rats .
The antioxidant effect of acerola is attributed to the high vitamin C level, as
well as to the polyphenols content in this fruit . In addition, guarana
also exhibits an important stimulant property because of its high caffeine
content . Açai-H animals had a higher caloric intake, but the weight gain
during the experimental period was similar to control animals. We can speculate
that the presence of guarana in the beverage could be responsible for such
intriguing effect. Guarana has a high caffeine content, which varies from 3% to
6% in the dried seeds [56-60]. Numerous studies described the beneficial effect
of caffeine on energy expenditure [61-65], therefore, it can potentially be
considered as a body-weight regulator.
In summary, the short-term
consumption of a honey-sweetened açai beverage was able to modulate cytokine
levels and reduce oxidative stress markers in the visceral white adipose
tissue, in a depot-specific manner. These data suggest a protective effect of
the short-term consumption of a honey-sweetened açai beverage against
conditions characterized by oxidative stress and inflammation. Moreover, the
short-term consumption of an açai beverage containing a high glucose
concentration, as that present in most commercially available beverages, leads
to alteration of body composition, lipid and carbohydrate metabolism.
Nevertheless, the consumption of this beverage interestingly induced an
enhancement in adiponectin plasma levels, which could represent a compensatory
response aimed at controlling the metabolic disruption induced by
R.X.N., F.O.R., M.J.A. and
R.G.C. carried out all animal studies; R.S., A.F.A.M. and E.C. designed the
study; R.S. and M.S. have written the manuscript; M.S. has supervised the
The study was financially
supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo)
grant number 2012/50079-0 and CAPES (Coordenação de Aperfeiçoamento de Nível
“The authors have declared
no conflicts of interest”.