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Metabolic syndrome and inflammation in adipose tissue occur at different times in animals submitted to a high-sugar/fat diet

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Obesity is associated with low-grade inflammation, triggered in adipose tissue, which may occur due to an excess of SFA from the diet that can be recognised by Toll-like receptor-4. This condition is involved in the development of components of the metabolic syndrome associated with obesity, especially insulin resistance. The aim of the study was to evaluate the manifestation of the metabolic syndrome and adipose tissue inflammation as a function of the period of time in which rats were submitted to a high-sugar/fat diet (HSF). Male Wistar rats were divided into six groups to receive the control diet (C) or the HSF for 6, 12 or 24 weeks. HSF increased the adiposity index in all HSF groups compared with the C group. HSF was associated with higher plasma TAG, glucose, insulin and leptin levels. Homeostasis model assessment increased in HSF compared with C rats at 24 weeks. Both TNF-α and IL-6 were elevated in the epididymal adipose tissue of HSF rats at 24 weeks compared with HSF at 6 weeks and C at 24 weeks. Only the HSF group at 24 weeks showed increased expression of both Toll-like receptor-4 and NF-κB. More inflammatory cells were found in the HSF group at 24 weeks. We can conclude that the metabolic syndrome occurs independently of the inflammatory response in adipose tissue and that inflammation is associated with hypertrophy of adipocytes, which varies according to duration of exposure to the HSF.
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RESEARCH ARTICLE
Metabolic syndrome and inflammation in adipose tissue occur at different
times in animals submitted to a high-sugar/fat diet
Fabiane Valentini Francisqueti
1
*, André Ferreira Nascimento
2
, Igor Otávio Minatel
3
, Marcos Correa Dias
2
,
Renata de Azevedo Melo Luvizotto
2
, Carolina Berchieri-Ronchi
1
, Ana Lúcia A. Ferreira
1
and
Camila Renata Corrêa
1
1
São Paulo State University (UNESP), Botucatu Medical School, Botucatu, São Paulo, Brazil
2
Institute of Health Sciences, Federal University of Mato Grosso (UFMT), Sinop, Mato Grosso, Brazil
3
São Paulo State University, Institute of Bioscience, Botucatu, São Paulo, Brazil
(Received 22 September 2016 Final revision received 23 May 2017 Accepted 29 June 2017)
Journal of Nutritional Science (2017), vol. 6, e41, page 1 of 8 doi:10.1017/jns.2017.42
Abstract
Obesity is associated with low-grade inammation, triggered in adipose tissue, which may occur due to an excess of SFA from the diet that can be recog-
nised by Toll-like receptor-4. This condition is involved in the development of components of the metabolic syndrome associated with obesity, especially
insulin resistance. The aim of the study was to evaluate the manifestation of the metabolic syndrome and adipose tissue inammation as a function of the
period of time in which rats were submitted to a high-sugar/fat diet (HSF). Male Wistar rats were divided into six groups to receive the control diet (C) or
the HSF for 6, 12 or 24 weeks. HSF increased the adiposity index in all HSF groups compared with the C group. HSF was associated with higher plasma
TAG, glucose, insulin and leptin levels. Homeostasis model assessment increased in HSF compared with C rats at 24 weeks. Both TNF-αand IL-6 were
elevated in the epididymal adipose tissue of HSF rats at 24 weeks compared with HSF at 6 weeks and C at 24 weeks. Only the HSF group at 24 weeks
showed increased expression of both Toll-like receptor-4 and NF-κB. More inammatory cells were found in the HSF group at 24 weeks. We can conclude
that the metabolic syndrome occurs independently of the inammatory response in adipose tissue and that inammation is associated with hypertrophy of
adipocytes, which varies according to duration of exposure to the HSF.
Key words: Obesity: Adipocytes: Inammation: Metabolic syndrome
The prevalence of obesity has increased strikingly during the
past three decades, particularly among minorities and socio-
economically disadvantaged populations around the world
(13)
.
The main factor that leads to this condition is overnutrition, espe-
cially when characterised by the excessive intake of carbohydrates
and fat
(49)
, which can trigger the metabolic syndrome (MS)
dened as a constellation of metabolic abnormalities for CVD
and diabetes. A consensus agreement by the International
Diabetes Federation and the American Heart Association/
National Heart, Lung and Blood Institute identies the criteria
of the MS as abdominal obesity, reduced HDL, elevated TAG,
glucose intolerance and hypertension; a diagnosis requires any
three of these ve criteria
(10)
.
The primary cause of the MS appears to be increased adiposity
associated with insulin resistance
(11,12)
.Furthermore,thereisa
strong relationship between obesity and inammation
(13)
,since
hyperadiposity produces adipokines, such as leptin, adiponectin
and resistin, as well as proinammatory cytokines such as IL-6,
TNF-αand plasminogen activator inhibitor type 1, which are
allinvolvedinproinammatory and prothrombotic responses
(14)
.
Abbreviations: C, control diet; C24, control diet for 24 weeks; HOMA-IR, homeostasis model assessment; HSF, high-sugar/fat diet; HSF6, high-sugar/fat diet for 6 weeks;
HSF12, high-sugar/fat diet for 12 weeks; HSF24, high-sugar/fat diet for 24 weeks; MS, metabolic syndrome; TLR-4, Toll-like receptor-4.
*Corresponding author: F. V. Francisqueti, fax +55 14 3881 6424, email fabiane_vf@yahoo.com.br
© The Author(s) 2017. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creative-
commons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is
properly cited.
JNS
JOURNAL OF NUTRITIONAL SCIENCE
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In obesity, inammation that is triggered in adipose tissue
may occur due to the excess of SFA in the diet. These fatty
acids can be recognised by Toll-like receptor-4 (TLR-4),
which is expressed by adipocytes and macrophages, leading
to activation of NF-κB
(1518)
, stimulating the production of
chemokines and proinammatory cytokines
(19)
, as well as
attracting immune cells from the circulation into the adipose
tissue
(16)
. TLR-4 is a cell surface receptor that generates innate
immune responses to pathogens by inducing signalling cas-
cades of kinase and transcription factor activation, leading to
the generation of proinammatory cytokines, chemokines,
eicosanoids and reactive oxygen species, all of which are effec-
tors of innate immunity.
Thus, we can propose that excessive sugar and fat intake are
factors that lead to inammation in adipose tissue. Several
studies have shown that inammation is involved in the devel-
opment of components of the MS associated with obesity,
especially insulin resistance
(2022)
. However, few experimental
studies that have emphasised the role of diet, obesity and
inammation evaluated the participation of the TLR-4 path-
way as a function of time
(2328)
. Therefore, additional studies
are needed to characterise the inammatory response in adi-
pose tissue and insulin resistance as a function of time.
Thus, the aim of the present study was to evaluate the mani-
festation of the MS and inammation adipose tissue as a func-
tion of time in rats submitted to a high-sugar/fat diet (HSF).
Materials and methods
Animals and experimental protocol
The experimental protocol was approved by the local Ethical
Committee for Animal Research of the University of Sao
Paulo State University (permit number PE-47/2011). Male
Wistar rats (10 weeks old, ±350 g) from the Animal Center
of Botucatu Medical School, Sao Paulo State University
(UNESP, Botucatu, SP, Brazil), were assigned to either a com-
mercial chow diet (control diet; C; 12 % energy from fat) or an
HSF (49·7 % energy from fat) with sucrose in the drinking
water (300 g/l) for a period of 6, 12 or 24 weeks (C6,
HSF6, C12, HSF12, C24, HSF24). The diet-induced obesity
model was adapted from our previous study
(29)
and it has
been published previously
(30)
, which was used to mimic obes-
ity from Western occidental dietary habits.
Rats were housed in individual cages in the animal facility at
the Internal Medicine Experimental Laboratory, Botucatu
Medical School, UNESP, under controlled ambient tempera-
ture (2226°C) and lighting (12 h light12 h dark) conditions.
Dietary and water consumption was measured daily, and body
weight was assessed weekly. Energy intake was calculated
according to the formula: energy intake (kJ/d) = food con-
sumption (g) × dietary energy (kJ/g). For the animals that
received sucrose in drinking water (30 %), the energy intake
was calculated according to the formula: volume consumed
(ml) × 0·3 (equivalent to 30 % sucrose) × 16·7 (kJ per g of
carbohydrate) + energy values offered by feeding (food con-
sumption (g) × dietary energy (kJ/g)).
The animals were killed by decapitation after anaesthesia with
sodium pentobarbital Q4 (50 mg/kg, intraperitoneal injection)
and all efforts were made to minimise suffering. Blood from
fasted animals was collected in tubes containing EDTA and
centrifuged at 3500 rpm and the plasma was collected for ana-
lysis. Epididymal adipose tissue was selected for analysis
because of its similar inammatory patterns to visceral fat
(31)
.
Adiposity index
The adiposity index was used as an indicator of obesity
because it enables the precise evaluation of body fat percent-
age. Epididymal, retroperitoneal and visceral fat deposits
were dissected from the rats. The sum of the fat deposits,
normalised by body weight, was calculated to obtain the adi-
posity index: ((epididymal + retroperitoneal + visceral)/body
weight) × 100
(5,32)
.
Plasma analysis
Biochemical. After 12 h of overnight fasting, plasma analysis
were carried out. An enzymic colorimetric kit was used to
measure glucose (Bioclin
®
; Belo Horizonte), TAG (Bioclin
®
;
Belo Horizonte) and NEFA (WAKO
®
;HRSeriesNEFA-
HR
(2)
). Spectrophotometry was performed with the Chemistry
Analyser BS 200 automatic spectrophotometer (Mindray
Medical International Ltd).
Insulin resistance. Insulin resistance was determined using
the index of homeostasis model assessment (HOMA-IR)
using the following formula
(33)
: HOMA-IR = fasting insulin
(μU/ml) × fasting glucose (mmol/l)/22·5.
Hormones and inflammatory cytokines. Plasma levels of
insulin, leptin, adiponectin, TNF-αand IL-6 were
measured by ELISA. Insulin, leptin and adiponectin
ELISA kits were purchased from Millipore Corporation
and TNF-αand IL-6 ELISA kits were purchased from
R&D Systems. A microplate spectrophotometer reader
(SpectraMax 190; Molecular Devices) was used according
to the manufacturers instructions.
Analysis of epididymal adipose tissue
Adipokine levels. Epididymal adipose tissue (400 mg) was
triturated with 2 ml of PBS (pH 7·4) and then centrifuged at
3000 rpm and 4°C for 10 min. Using the supernatants,
TNF-αand IL-6 were measured using commercial ELISA
kits (R&D Systems) according to the manufacturers
instructions. The results were normalised to protein amounts
of each sample, quantied by the Bradford method
(34)
.
Western blotting. The protein concentration of the whole
epididymal adipose tissue extract (including the cell
membrane, cytoplasm and nucleus) was determined by the
Bradford method
(34)
. Samples (25 µg of protein) were heated
2
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in Laemmli buffer at 100°C for 5 min, then loaded onto a 10
% SDSpolyacrylamide gel. Transfer to a nitrocellulose
membrane was carried out at 4°C in the presence of
methanol. Incubation with the primary antibodies (purchased
from Santa Cruz Biotechnology) was performed overnight at
4°C in Tris-buffered saline solution containing Tween
20 (TBS-T) and 3 % non-fat dried milk. Antibody dilutions
were: 1:200 for mouse anti-TLR-4 sc293072, 1:200 for
mouse anti-β-actin sc47778, 1:100 for rabbit anti-
phosphorylated NF-κB (ser536) sc33020, and 1:200 for
mouse anti-total NF-κB sc8008. After incubation overnight
at 4°C in TBS-T containing 1 % non-fat dried milk with the
Abcam secondary antibodies (dilution 1:10 000) anti-rabbit
ab97069 and anti-mouse ab98808, protein was revealed
using the chemiluminescence method according to the
manufacturers instructions (ECL SuperSignal
®
West Pico
Chemiluminescent Substrate; Thermo Scientic). Band
intensities were evaluated using Scion Image Software (Scion
Corporation).
Histological analysis
Adipose tissue was xed in 4 % formaldehyde and embedded
in parafn. Two consecutive sections from each sample were
cut (4 µm) and stained with haematoxylin/eosin. The entire
slide was scanned using a 3DHISTECH Panoramic MIDI
System attached to a Hitachi HV-F22 colour camera and
ten elds/slide were analysed under 40× magnication in a
blinded manner. The inammatory reactions are reported
as the number of inammatory cells per high-power eld.
Using the same slides, the mean area of adipocytes was cal-
culated using a method previously described by Osman
et al. in 2013
(35)
.
Statistical analysis
Results are expressed as means and standard deviations.
Comparisons among groups were performed using two-way
ANOVA for independent groups and were completed using
Tukeyspost hoc test. SigmaPlot 11.0 software (Systat
Software Inc.) was used for statistical analyses. Differences
were considered signicant at P<0·05. The statistical power
for the main outcome variables was above 80 %.
Results
Body weight and body fat
There was no difference in energy intake between the C and
HSF groups at any time (Table 1). The HSF caused changes
in the body composition of the animals. At the end of 24
weeks, the HSF group had a greater average weight than the
rats in the C24, HSF6 and HSF12 groups. The adiposity
index was higher in all HSF groups compared with their
respective controls, and in HSF12 and HSF24 compared
with HSF6 (Table 1).
Plasma biochemical and hormonal measurements
An increase in TAG, glucose, insulin and leptin levels and a
decreased level of adiponectin was detected in all animals in
the HSF group compared with the C rats. However, when
comparing animals subjected to the same diet for different
periods of time, only leptin was increased in HSF12 and
HSF24 rats compared with the HSF6 group. Animals that
received HSF showed insulin resistance only at 24 weeks com-
pared with control animals characterised by increased
HOMA-IR (Table 2). There was no difference in NEFA levels
among the groups.
Table 1. Nutritional profile of the control diet (C) group and high-sugar/fat diet (HSF) group
(Mean values and standard deviations, n8)
Group
C6 HSF6 C12 HSF12 C24 HSF24
Variables Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Pdiet Ptime Pinteraction
IBW (g) 348·840·5 350·433·0 349·528·8 344·223·1 352·833·3 350·022·50·81 0·91 0·25
FBW (g) 456·0
A
38·0 469·0
a
40·3 499·3
A
42·3 562·7
b
54·0 568·4
B
66·7 708·0*
c
69·1<0·001 <0·001 0·006
Weight gain (g) 107·2
A
19·7 118·6
a
32·0 149·9
A
29·7 218·5*
b
57·7 215·6
B
58·7 358·0*
c
72·1<0·001 <0·001 0·002
Epididymal (g) 7·0
A
1·810·8*
a
3·28·9
A
2·218·4*
b
5·311·7
B
4·426·7*
c
7·4<0·001 <0·001 0·7983
Retroperitoneal (g) 8·0
A
2·615·6*
a
5·310·7
B
4·624·7*
b
7·312·2
B
4·726·7*
c
7·4<0·001 0·0010 0·6688
Visceral (g) 5·8
A
1·89·9*
a
3·48·1
B
2·516·8*
b
6·49·8
B
3·724·0*
c
5·7<0·001 <0·001 0·9074
Total body fat (g) 20·9
A
5·936·4*
a
11·627·7
A
8·559·9*
b
17·436·6
A
12·794·8*
c
25·6<0·001 <0·001 0·001
Adiposity index (%) 4·5
A
1·07·6*
a
1·85·5
A
1·510·5*
b
2·35·8
B
1·710·9*
c
2·3<0·001 0·0029 0·9288
Food intake (g/d) 28·32·912·3* 2·027·72·212·7* 2·129·94·115·7* 1·4<0·001 0·0060 0·6283
Water intake (ml/d) 35·85·027·9
A
7·536·64·629·4
A
8·845·35·343·2
B
8·80·0064 <0·001 0·3913
Energy intake
kcal/d 106·610·894·611·4 104·38·299·910·6 107·212·4 102·59·50·2032 0·4762 0·5623
kJ/d 446·045·2 395·847·7 436·434·3 418·044·4 448·551·9 428·939·70·2032 0·4762 0·5623
C6, control diet for 6 weeks; HSF6, high-sugar/fat diet for 6 weeks; C12, control diet for 12 weeks; HSF12, high-sugar/fat diet for 12 weeks; C24, control diet for 24 weeks; HSF24,
high-sugar/fat diet for 24 weeks; IBW, initial body weight; FBW, final body weight.
A,B
Mean values within a row with unlike uppercase letters were significantly different among the C groups (6 v.12v. 24 weeks) (P<0·05).
a,b,c
Mean values within a row with unlike lowercase letters were significantly different among the HSF groups (6 v.12v. 24 weeks) (P<0·05).
* Mean value was significantly different from that for the C group at the same time point (P<0·05).
Comparisons among groups were performed using two-way ANOVA for independent groups and were completed using Tukeyspost hoc test.
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Adipose tissue and serum adipokine measurements
No differences were found in the plasma levels of TNF-αand
IL-6 when comparing different diets or different durations
(data not shown). In adipose tissue, animals in the HSF24
group exhibited elevated levels of these cytokines compared
with the C24 group, as well as the HSF6 and HSF12 groups
(Fig. 1). However, HSF12 had lower levels of TNF-αand
IL-6 compared with C12.
Adipose tissue area
Table 3 shows the mean area of adipocytes in control animals
and those fed the HSF in the three periods of the experiment.
Note that there was an increase in the area of the adipocytes in
animals fed the HSF only at 24 weeks compared with the C24,
HSF6 and HSF12 groups. Fig. 2 shows images taken for the
assessment of inammatory cell inltration. A greater number
of cells was observed in the HSF24 group.
Western blotting
Fig. 3 shows the protein expression of TLR-4 and NF-κBin
epididymal adipose tissue. At the end of 24 weeks, the animals
in the HSF24 group showed higher TLR-4 expression than
those in the C24 and HSF6 groups. Similar to TLR-4, the
expression of NF-κB increased in epididymal adipose tissue
after 24 weeks in the HSF group when compared with animals
in the C24 group.
Discussion
In the present study using Wistar male rats fed an HSF, we
evaluated the temporal relationship between the manifestation
of metabolic parameters and the effects of inammation in
adipose tissue via TLR-4. According to the WHO, obesity is
dened as an excessive accumulation of body fat
(36)
.In
2014, Strissel et al.
(37)
associated obesity with chronic low-
grade inammation, called metainammation
(38)
, which differs
from the classic inammatory response against injury or
pathogens
(39)
. Chronic consumption of an HSF is associated
with metabolic changes, since it triggers an increase in body
weight and obesity
(9)
. These factors are associated with high
blood pressure, as well as biochemical and hormonal changes,
such as increased blood glucose, TAG, NEFA, leptin and
insulin, as well as reduced adiponectin
(40,41)
. However, it is
still unclear how the duration of this diet is related to metabolic
Table 2. Plasma biochemical and hormonal measurements
(Mean values and standard deviations, n8)
Groups
C6 HSF6 C12 HSF12 C24 HSF24
Variables Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Pdiet Ptime Pinteraction
TAG (mmol/l) 0·47
A,B
0·13 1·03*
a,b
0·32 0·43
A
0·04 0·93*
a
0·25 0·58
B
0·17 1·14*
b
0·25 <0·001 0·0150 0·2970
NEFA (mmol/l) 0·30·10·30·10·40·10·40·10·30·10·40·10·0192 0·0515 0·4190
Glucose (mmol/l) 4·77 0·58 5·68* 0·40 6·53 1·17 7·77* 1·39 5·38 0·04 6·51* 0·71 <0·001 0·0069 0·7055
Insulin (ng/ml) 1·90·83·9* 1·62·72·14·2* 1·32·40·85·4* 1·6<0·001 0·1487 0·3074
HOMA-IR 0·90·53·82·52·54·44·22·41·41·17·0* 4·8<0·001 0·232 0·194
Leptin (ng/ml) 2·5
A
0·86·2*
a
0·82·8
A,B
0·711·4*
b
5·83·7
B
2·413·9*
b
3·2<0·001 0·0053 0·5502
Adiponectin (ng/ml) 19·47·211·4* 1·620·14·411·7* 2·619·04·013·1* 1·6<0·001 0·724 0·370
C6, control diet for 6 weeks; HSF6, high-sugar/fat diet for 6 weeks; C12, control diet for 12 weeks; HSF12, high-sugar/fat diet for 12 weeks; C24, control diet for 24 weeks; HSF24,
high-sugar/fat diet for 24 weeks; HOMA-IR, homeostasis model assessment.
A,B
Mean values within a row with unlike uppercase letters were significantly different among the C groups (6 v.12v. 24 weeks) (P<0·05).
a,b
Mean values within a row with unlike lowercase letters were significantly different among the HSF groups (6 v.12v. 24 weeks) (P<0·05).
* Mean value was significantly different from that for the C group at the same time point (P<0·05).
Comparisons among groups were performed using two-way ANOVA for independent groups and were completed using Tukeyspost hoc test.
8000
(a) (b) 200
150
100
50
0
6000
4000
2000
C6
II-6 (pg/g protein)
TNF-a (pg/g protein)
HSF6 HSF12 HSF24C12 C24 C6 HSF6 HSF12 HSF24C12 C24
0
Fig. 1. Cytokine levels (pg/g protein) in epididymal adipose tissue in control diet (C) and high-sugar/fat diet (HSF) groups over 6, 12 and 24 weeks (n8 animals/
group). (a) IL-6 level; (b) TNF-αlevel. Values are means, with standard deviations represented by vertical bars. * Mean values were significantly different (P<0·05).
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changes and whether these changes occur before or after
inammation.
HOMA-IR has been used to assess overall insulin sensitivity
in human subjects and rats with different degrees of insulin
sensitivity
(42)
. Therefore, HOMA-IR is a good predictor of
whole-body insulin sensitivity. In our study, the modication
of parameters including glucose, TAG, leptin and insulin, as
well as low adiponectin, started as early as 6 weeks on the
diet. There was also a greater adiposity index observed in
these animals. All these conditions persisted up to 24 weeks,
even without higher energy intake. This underscores the
notion that diet components are an important factor in the
manifestation of the MS. The HSF is a combination of
palatable foods with high energy density, reecting the
Western dietary pattern. These data corroborate the HSF as
a trigger of metabolic and hormonal changes, abdominal cir-
cumference, and expansion of fat mass
(43,44)
. More specically,
a higher proportion of SFA (myristic (C14), palmitic (C16) and
stearic (C18)) in relation to unsaturated fats (mono- and poly-
unsaturated) is associated with high adiposity and central fat
deposit
(45)
. High levels of carbohydrates are also able to mobil-
ise fat from the periphery to central deposits and reduce the
activity of adiponectin in peripheral tissues
(46)
. When there is
an expansion of fat mass, there is also adipocyte hypertrophy,
which is responsible for the production of adipokines, includ-
ing TNF-αand IL-6
(47)
. In our work, at 6 and 12 weeks,
Table 3. Mean area of adipocytes in epididymal adipose tissue of control diet (C) and high-sugar/fat diet (HSF) groups
(Mean values and standard deviations, n8)
Area of
adipocytes (mm
2
)
Group Mean SD Number of inflammatory cells/field Pdiet <0·001 Ptime <0·001 Pinteraction 0·004
C6 1·0
A
0·215
HSF6 1·4
a
0·215
C12 1·1
A
0·415
HSF12 1·4
a
0·12040
C24 2·8
B
1·2520
HSF24 5·0*
b
0·9 >40
C6, control diet for 6 weeks; HSF6, high-sugar/fat diet for 6 weeks; C12, control diet for 12 weeks; HSF12, high-sugar/fat diet for 12 weeks; C24, control diet for 24 weeks; HSF24,
high-sugar/fat diet for 24 weeks.
A,B
Mean values within a column with unlike uppercase letters were significantly different among the C groups (6 v.12v. 24 weeks) (P<0·05).
a,b
Mean values within a column with unlike lowercase letters were significantly different among the HSF groups (6 v.12v. 24 weeks) (P<0·05).
* Mean value was significantly different from that for the C group at the same time point (P<0·05).
Comparisons among groups were performed using two-way ANOVA for independent groups and were completed using Tukeyspost hoc test.
Fig. 2. Inflammatory cells in adipose tissue. (a) Control group; (b) group fed high-sugar/fat diet for 6 weeks (HSF6); (c) group fed high-sugar/fat diet for 12 weeks
(HSF12); (d) group fed high-sugar/fat diet for 24 weeks (HSF24) (n8 animals/group). 40× Magnification.
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although the animals presented an increase in the adiposity
index, hypertrophy was not present and was only observed
at 24 weeks, accompanied by inammation and increased
TNF-αand IL-6 levels. Therefore, the inammatory state
was observed only together with adipocyte hypertrophy, sug-
gesting that adipose tissue is made up of mature and immature
adipocytes (low fat content). Under excessive energy supply
conditions, immature adipocytes would be responsible for
the accumulation of fat, giving the adipose tissue a uniform
appearance that still does not characterise hypertrophy.
Two distinct macrophage populations can be identied in
adipose tissue: M1 and M2
(48)
. The M1 prole produces proin-
ammatory cytokines that affect cell proliferation and promote
insulin resistance, while the M2 population is associated with
an anti-inammatory phenotype that protects against meta-
bolic disorders
(49,50)
. Lean individuals express a balance in
the M1/M2 prole in adipose tissue, while obese individuals
initially show an increase in the M2 prole, a defence mechan-
ism to combat possible inammation
(50)
. Our data show that,
at 12 weeks, the animals showed a reduction in TNF-αand
IL-6, providing evidence for this compensatory response in
adipose tissue. This mechanism can occur to reduce inamma-
tion and metabolic deterioration of the tissue. However, at 24
weeks, due to the hypertrophy of adipocytes and increased
body fat, a shift may have occurred in the prole of these
macrophages towards M1, causing an increase in proinam-
matory cytokines. Corroborating this hypothesis, a murine
study by Shaul et al.
(51)
also showed an enhanced M2 pheno-
type in adipose tissue in obese mice after 12 weeks on a high-
fat diet compared with mice fed the same diet for 8 weeks.
Increased adiposity is associated with the activation and
migration of inammatory cells into the adipose tissue, as
well as proinammatory cytokine secretion and development
of low-grade chronic inammation
(52)
. Besides the hyper-
trophy of adipose tissue and macrophage prole in tissue,
another factor that can enhance this inammatory condition
is the activation of TLR-4 receptors
(19)
. Our results show an
increase in expression of adipocytes at 24 weeks. The literature
shows that this activation can occur by an increased release of
fatty acids by adipose tissue, by SFA intake
(51)
or by the change
in intestinal ora and lipopolysaccharide stimulus
(53)
. In the
present study, we can attribute the increase in TLR-4 to the
dietary fat stimulus, since elevated NEFA in circulation were
not found. TLR-4 activates the transcription factor NF-κB,
leading to increased production of proinammatory cyto-
kines
(54)
, reinforcing the importance of this pathway.
In obesity, the degree of inammation correlates with the
extent of insulin resistance, a mechanism involving TNF-α,
which interferes with the phosphorylation of the insulin recep-
tor, impairing its glucose uptake function
(55)
. Our work shows
that insulin resistance is present, together with the inamma-
tory response, in adipose tissue after 24 weeks, along with adi-
pocyte hypertrophy. Thus, these data allow us to hypothesise
that insulin resistance is inuenced by inammation of the adi-
pose tissue, which in turn is associated with adipocyte hyper-
trophy and TLR-4 activation. We can conclude that the MS
occurs independently of inammation in adipose tissue and
that inammation is associated with adipocyte hypertrophy,
which varies according to the duration of exposure to an HSF.
Final considerations
Although the present study was carried out in rats, the
mechanisms related to the development of the MS and inam-
mation may be similar to those involved in clinical obesity,
since after the onset of inammation in adipose tissue,
dependent on adipocyte hypertrophy, the organism may
develop new co-morbidities over time, and potentiate the pre-
existing ones. The results of the present study are clinically
important because they provide information that may promote
C6 HSF6 HSF12 HSF24C12 C24 C6 HSF6 HSF12 HSF24C12 C24
C6 HSF6 HSF12 HSF24C12 C24
C6 HSF6
NF-kB p-65
phospho
NF-kB p-65
total
b-Actin
TLR-4
6
(a) (b)
4
2
0
5
Relative expression of protein
NF-kB phospho/total
Relative expression of protein
TLR-4/b-actin
4
3
2
1
0
HSF12 HSF24C12 C24
Fig. 3. Relative expression of protein in epididymal adipose tissue in control diet (C) and high-sugar/fat diet (HSF) groups over 6, 12 and 24 weeks (n8 animals/
group). (a) Toll-like receptor-4 (TLR-4); (b) NF-κB. Values are means, with standard deviations represented by vertical bars. * Mean values were significantly different
(P<0·05).
6
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interventions to prevent adipocyte hypertrophy and inamma-
tion, being a preventive measure in the development of
co-morbidities arising from this process.
Acknowledgements
We thank Mario B. Bruno, José Carlos Georgette and Renata
Capela for their technical support.
We thank Fundação de Amparo a Pesquisa do Estado de
São Paulo FAPESP (2011/14132-0, 2011/14593-8, 2015/
10626-0) for providing nancial support.
The author contributions were as follows: A. F. N. and
C. R. C. designed the research; F. V. F., C. B.-R., I. O. M.,
M. C. D., R. A. M. L. and A. L. A. F. conducted the research;
A. F. N. and C. R. C. analysed the data; F. V. F., A. F. N. and
C. R. C. wrote the paper.
The authors declare no conicts of interest.
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... (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) and/or cholesterol resulted in higher expression of TLR2, MyD88, phosphorylated NF-κB and inflammatory cytokines [including tumour necrosis factor-alpha (TNF-α), IL-6, interleukin-1 (IL-1) and monocyte chemoattractant protein-1 (MCP-1)] (Bhaskar & Helen, 2016;Francisqueti et al., 2017;Han et al., 2016;Kim, Choi, Choi, & Park, 2012). We postulated the increase in inflammatory response in MetSassociated NAFLD was mainly attributed to the activation of TLR2 and TLR4. ...
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Adipose tissue is the major storage sites of energy deposition which can be recruited in times of need to provide fuel for other organs (reviewed in Gunawardana 2014). When normalized to volume, adipose tissue is mainly composed of so-called mature adipocytes which are cells that have the capacity to store energy in the form of triacylglycerols (TAGs) in lipid droplets. When normalized to cell number, only 20–30% of the adipose tissue is made up from mature adipocytes; the other 70–80% are composed of the so-called stromal vascular fraction (SVF), which consists of fibroblasts, adipocyte precursors, endothelial cells, and immune cells (Rosenwald et al. 2013; Wang et al. 2013). This cell heterogeneity clearly demonstrates that adipose tissue is a complex organ with various different functions in the regulation of whole body metabolism. In line with this, over the past several years, our understanding of adipose tissue has changed. Only 20 years ago adipose tissue was considered to be an inert energy storage organ, while nowadays it is accepted that besides its role in energy storage and dissipation, adipose tissue serves as a key organ for the regulation of whole body energy metabolism by cross talk with other organs through the secretion of adipokines, such as tumor necrosis factor α, (TNF-α), interleukin-6 (IL-6), adiponectin, leptin, and resistin, just to mention a few (Bluher and Mantzoros 2015).
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Low-grade inflammation in the obese AT (AT) and the liver is a critical player in the development of obesity-related metabolic dysregulation, including insulin resistance, type 2 diabetes and non-alcoholic steatohepatitis (NASH). Myeloid as well as lymphoid cells infiltrate the AT and the liver and expand within these metabolic organs as a result of excessive nutrient intake, thereby exacerbating tissue inflammation. Macrophages are the paramount cell population in the field of metabolism-related inflammation; as obesity progresses, a switch takes place within the AT environment from an M2-alternatively activated macrophage state to an M1-inflammatory macrophage-dominated milieu. M1-polarized macrophages secrete inflammatory cytokines like TNF in the obese AT; such cytokines contribute to insulin resistance in adipocytes. Besides macrophages, also CD8(+) T cells promote inflammation in the AT and the liver and thereby the deterioration of the metabolic balance in adipocytes and hepatocytes. Other cells of the innate immunity, such as neutrophils or mast cells, interfere with metabolic homeostasis as well. On the other hand, eosinophils or T-regulatory cells, the number of which in the AT decreases in the course of obesity, function to maintain metabolic balance by ameliorating inflammatory processes. In addition, eosinophils and M2-polarized macrophages may contribute to "beige" adipogenesis under lean conditions; beige adipocytes are located predominantly in the subcutaneous AT and have thermogenic and optimal energy-dispensing properties like brown adipocytes. This chapter will summarize the different aspects of the regulation of homeostasis of metabolic tissues by immune cells.