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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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Metabolic syndrome and inflammation in adipose tissue occur at different
times in animals submitted to a high-sugar/fat diet
Fabiane Valentini Francisqueti
*, André Ferreira Nascimento
, Igor Otávio Minatel
, Marcos Correa Dias
Renata de Azevedo Melo Luvizotto
, Carolina Berchieri-Ronchi
, Ana Lúcia A. Ferreira
Camila Renata Corrêa
São Paulo State University (UNESP), Botucatu Medical School, Botucatu, São Paulo, Brazil
Institute of Health Sciences, Federal University of Mato Grosso (UFMT), Sinop, Mato Grosso, Brazil
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
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
The main factor that leads to this condition is overnutrition, espe-
cially when characterised by the excessive intake of carbohydrates
and fat
, 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
The primary cause of the MS appears to be increased adiposity
associated with insulin resistance
strong relationship between obesity and inammation
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
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
© The Author(s) 2017. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creative-, which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is
properly cited.
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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
, stimulating the production of
chemokines and proinammatory cytokines
, as well as
attracting immune cells from the circulation into the adipose
. 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
. 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
. 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
and it has
been published previously
, 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
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
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
). 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
: 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
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
. Samples (25 µg of protein) were heated
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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
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
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 %.
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)
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
38·0 469·0
40·3 499·3
42·3 562·7
54·0 568·4
66·7 708·0*
69·1<0·001 <0·001 0·006
Weight gain (g) 107·2
19·7 118·6
32·0 149·9
29·7 218·5*
57·7 215·6
58·7 358·0*
72·1<0·001 <0·001 0·002
Epididymal (g) 7·0
7·4<0·001 <0·001 0·7983
Retroperitoneal (g) 8·0
7·4<0·001 0·0010 0·6688
Visceral (g) 5·8
5·7<0·001 <0·001 0·9074
Total body fat (g) 20·9
25·6<0·001 <0·001 0·001
Adiposity index (%) 4·5
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
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.
Mean values within a row with unlike uppercase letters were significantly different among the C groups (6 v.12v. 24 weeks) (P<0·05).
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.
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
2014, Strissel et al.
associated obesity with chronic low-
grade inammation, called metainammation
, which differs
from the classic inammatory response against injury or
. Chronic consumption of an HSF is associated
with metabolic changes, since it triggers an increase in body
weight and obesity
. 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
. 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)
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
0·13 1·03*
0·32 0·43
0·04 0·93*
0·25 0·58
0·17 1·14*
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
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.
Mean values within a row with unlike uppercase letters were significantly different among the C groups (6 v.12v. 24 weeks) (P<0·05).
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.
(a) (b) 200
II-6 (pg/g protein)
TNF-a (pg/g protein)
HSF6 HSF12 HSF24C12 C24 C6 HSF6 HSF12 HSF24C12 C24
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
HOMA-IR has been used to assess overall insulin sensitivity
in human subjects and rats with different degrees of insulin
. 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
. 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
. 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
. 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
. 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
Group Mean SD Number of inflammatory cells/field Pdiet <0·001 Ptime <0·001 Pinteraction 0·004
C6 1·0
HSF6 1·4
C12 1·1
HSF12 1·4
C24 2·8
HSF24 5·0*
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.
Mean values within a column with unlike uppercase letters were significantly different among the C groups (6 v.12v. 24 weeks) (P<0·05).
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
. 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
. 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
. 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.
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
. 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
. 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
or by the change
in intestinal ora and lipopolysaccharide stimulus
. 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-
, 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
. 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
NF-kB p-65
NF-kB p-65
(a) (b)
Relative expression of protein
NF-kB phospho/total
Relative expression of protein
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
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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.
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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... C control diet group, HED highenergy diet group, HS highsugar diet group. *p < 0.05 vs C high-fat or combined high-sugar diets (Ainslie et al. 2000;Cha et al. 2000;Francisqueti et al. 2017;Moreno-Fernández et al. 2018). In addition, high-fat diets disrupted the daily pattern of leptin levels with significant changes in diurnal leptin peaks (Cha et al. 2000). ...
... In addition, TLR4, COX-2, and E-selectin immunoexpression revealed a high positivity score in the liver of HED rats. TLR-4 is critical in glucose and lipid metabolism and its expression can result in systemic and local inflammation, which include an increased production of proinflammatory cytokines through the activation of the transcription factor NF-κB, leading to insulin sensitivity (Könner and Brüning 2012;Francisqueti et al. 2017;Porras et al. 2017). Expression of COX-2 is typically induced in response to cell stressors, such as NF-κB stimulation, being overexpressed in hepatocytes after liver injury and in liver pathology (Martín-Sanz et al. 2010;Francés et al. 2015;Motiño et al. 2019). ...
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Early-life consumption of high-fat and sugar-rich foods is recognized as a major contributor for the onset of metabolic dysfunction and its related disorders, including diabetes and nonalcoholic fatty liver disease. The lifelong impact of early unhealthy eating habits that start at younger ages remains unclear. Therefore, to better understand the effects of diet, it is essential to evaluate the structural and functional changes induced in metabolic organs and potential mechanisms underlying those changes. To investigate the long-term effects of eating habits, young male rats were exposed to high-sugar and high-energy diets. After 14 weeks, body composition was assessed, and histopathological changes were analyzed in the liver and adipose tissue. Serum biochemical parameters were also determined. Expression of inflammatory markers in the liver was evaluated by immunohistochemistry. Our results revealed that serum levels of glucose, creatinine, aspartate transaminase (AST), alanine transaminase (ALT), and lipid profile were increased in rats red high-sugar and high-energy diets. Histopathological alterations were observed, including abnormal hepatocyte organization and lipid droplet accumulation in the liver, and abnormal structure of adipocytes. In both unhealthy diet groups, hepatic expression of Toll-like receptor 4 (TLR4), cyclooxygenase 2 (COX-2), and E-selectin were increased, as well as a biomarker of oxidative stress. Together, our data demonstrated that unhealthy diets induced functional and structural changes in the metabolic organs, suggesting that proinflammatory and oxidative stress mechanisms trigger the hepatic alterations and metabolic dysfunction.
... Before and at the end of the study, body weight and blood pressure (BP) (Systola, Neurobotics, Russia) were measured in the animals. At the 12 th week of the experiment, a glucose tolerance test (GTT) was performed [15]. Fasted rats (12 h of food deprivation) were injected intragastrically with a glucose solution at a dose of 2 g / kg (D-glucose, Sigma-Aldrich, USA). ...
... A correlation was also established between the white blood cell count and the specific gravity of the adipose tissue. It is known that obesity, as one of the important components of MS, is accompanied by a chronic inflammatory process in the adipose tissue, infiltrated by leukocytes, with high production of proinflammatory cytokines [15,19]. ...
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Aim. To study the effects of a high-fat, high-carbohydrate diet on erythrocytes and platelets of rats. Materials and methods. Male Wistar rats (n = 23) were used for the study. The rats were divided into a control group and an experimental group. The rats from the control group were fed with standard rat chow. The rats from the experimental group had received a high-fat and high-carbohydrate diet for 12 weeks. In the rats, body weight and blood pressure (BP) were measured, an oral glucose tolerance test was carried out, and hematological and lipid metabolism parameters were analyzed. The conductance of erythrocyte KCa-channels was measured by the potentiometric method, and platelet aggregation was determined by the turbidimetric method. Results. Feeding the rats with a high-fat, high-carbohydrate diet for 12 weeks resulted in obesity, BP elevation, hyperglycemia, impaired glucose tolerance, and dyslipidemia with pronounced triglyceridemia. In the experimental group, a rise in the number of leukocytes, mainly due to granulocytes, and an increase in the number of platelets and their collagen-induced aggregation were observed. The red blood cell count in the rats of the experimental group did not significantly differ from that of the control group. In the experimental group, multidirectional changes in the membrane potential were observed in response to the stimulation of the KCa-channels in the erythrocyte membrane with the Ca2+ ionophore A23187 or artificial redox systems. Conclusion. The obtained data indicate that a high-fat, high-carbohydrate diet leads to metabolic and hemorheological disorders that are typical of metabolic syndrome.
... Although MetS is mainly a metabolic disorder, this condition can be regarded also as a state of oxidative stress. MetS is characterized not only by high blood glucose that contributes to the generation of free radicals, but also by elevated free fatty acids [5]. Oxidative stress and chronic inflammation in MetS cause a significant impairment of hemorheology, where erythrocytes play a special role. ...
... As a consequence, their key target enzymes regulating lipid synthesis such as Fatty Acid Synthase (FASN) and Acetyl-CoA Carboxylase (ACC) also increase as shown, for example, in a western diet where fructose is provided as a 30%-fructose containing beverage for eight weeks [22]. The results are consistent with the literature data on diet-induced MetS models and indicate that the chosen diet effectively reproduces the clinical signs of MetS [5], [13]. ...
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Metabolic syndrome (MetS) is a cluster of metabolic, hormonal and hemodynamic disorders that contribute to a change in the structural and functional status of erythrocytes and contribute to dysregulation of their cation transport function, where Ca2+ -dependent potassium channels (KCa channels) play an important role. A MetS model was performed using male Wistar rats, which were divided into control and experimental groups. Rats in the control group were fed standard rat chow. Rats in the experimental group were exposed to a high-fat and high-carbohydrate (HFHC) diet for 12 weeks. The data obtained indicate that the HFHC diet led to obesity, high blood pressure, hyperglycemia, impaired glucose tolerance, and dyslipidemia. The level of glutathione (GSH) decreased in the erythrocytes of rats suffering from MetS, but the level of malondialdehyde (MDA) increased. It was shown that the amplitude of the membrane potential of erythrocytes of rats with MetS changed depending on the acting agent: when stimulated with calcium ionophore A23187 it decreased, when the redox system ascorbat –phenazine methosulfate was used, it increased compared to the control group. The data obtained indicate that a HFHC diet leads to changes in the physical and chemical properties of the erythrocyte membrane.
... Epididymal adipose tissue was selected for analysis due its similar inflammatory patterns to visceral fat (Francisqueti et al., 2017b). Four hundred milligrams of tissue were homogenized in 2.0 mL of Phosphate-Buffered saline (PBS) pH 7.4 cold solution ULTRA-TURRAX T25 basic IKA Werke Staufen/Germany, and centrifuged at 800 g at 4 • C for 10 min. ...
... Carbonylated proteins were measured by an unspecific method that (Francisqueti et al., 2017b). The mean area of adipocytes was calculated using a method previously described by Osman in 2013 (Osman et al., 2013). ...
The literature has reported a higher prevalence of negative clinical outcomes due to Coronavirus disease 19 (COVID-19) in obese individuals. This can be explained by the cytokine storm, result from the cytokine production from both obesity and viral infection. Gamma-oryzanol (γOz) is a compound with anti-inflammatory and antioxidant activities. However, little is known about the γOz action as a possible agonist of peroxisome proliferator-activated receptor gamma (PPAR-γ). The aim of this study was to test the hypothesis that γOz attenuates the cytokine storm by stimulating PPAR-γ in the adipose tissue. Methods: Male Wistar rats were randomly divided into three experimental groups and fed ad libitum for 30 weeks with control diet (C, n = 6), high sugar-fat diet (HSF, n = 6) or high sugar-fat diet + γOz (HSF + γOz, n = 6). HSF groups also received water + sucrose (25%). The γOz dose was 0.5% in the chow. Evaluation in animals included caloric intake, body weight, adiposity index, plasma triglycerides, and HOMA-IR. In adipose tissue was evaluated: PPAR-γ gene and protein expression, inflammatory and oxidative stress parameters, and histological analysis. Results: Adipose tissue dysfunction was observed in HSF group, which presented remarkable PPAR-γ underexpression and increased levels of cytokines, other inflammatory markers and oxidative stress. The γOz treatment prevented adipose tissue dysfunction and promoted PPAR-γ overexpression. Conclusion: Natural compounds as γOz can be considered a coadjutant therapy to prevent the cytokine storm in COVID-19 patients with obesity conditions.
... Body mass index is calculated by dividing body weight (g) by the square of tailless body length (m 2 ), while the adiposity index is the total body fat (the sum of epididymal, retroperitoneal, and visceral fat depots) divided by the final body weight and multiplied by 100. The adiposity index is used as a marker of obesity because the degree of fat tends to increase with obesity, allowing accurate assessment of body fat percentage [158,159]. The whole-body composition can also be measured using imaging methods such as the magnetic resonance imaging system [157] and the dual energy X-ray absorptiometry [156]. ...
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Obesity, classified as an epidemic by the WHO, is a disease that continues to grow worldwide. Obesity results from abnormal or excessive accumulation of fat and usually leads to the development of other associated diseases, such as type 2 diabetes, hypertension, cancer, cardiovascular diseases, among others. In vitro and in vivo models have been crucial for studying the underlying mechanisms of obesity, discovering new therapeutic targets, and developing and validating new pharmacological therapies against obesity. Preclinical animal models of obesity comprise a variety of species: invertebrates, fishes, and mammals. However, small rodents are the most widely used due to their cost-effectiveness, physiology, and easy genetic manipulation. The induction of obesity in rats or mice can be achieved by the occurrence of spontaneous single-gene mutations or polygenic mutations, by genetic modifications, by surgical or chemical induction, and by ingestion of hypercaloric diets. In this review, we describe some of the most commonly used murine models in obesity research.
... 39 In obesity, the adipose tissue lipolysis represents an important source of free fatty acids, capable of activating the inflammatory pathway. 40,41 This catabolic process can occur due to insulin ...
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Background: Obesity is a chronic low-grade inflammation condition related to cardiac disorders. However, the mechanism responsible for obesity-related cardiac inflammation is unclear. The toll-like receptor 4 (TLR-4) belongs to a receptor of the transmembrane family responsible for the immune response whose activation stimulates the production of proinflammatory cytokines. Objective: To test whether the activation of the TLR-4 receptor participates in the obesity cardiomyopathy process, due to cytokine production through NF-ĸB activation. Methods: Male Wistar rats were randomized into two groups: the control group (C, n= 8 animals) that received standard diet/water and the obese group (OB, n= 8 animals) that were fed a high sugar-fat diet and water plus 25% of sucrose for 30 weeks. Nutritional analysis: body weight, adiposity index, food, water, and caloric intake. Obesity-related disorders analysis: plasma glucose, uric acid and triglycerides, HOMA-IR, systolic blood pressure, TNF-α in adipose tissue. Cardiac analysis included: TLR-4 and NF-ĸB protein expression, TNF-α and IL-6 levels. Comparison by unpaired Student's t-test or Mann- Whitney test with a p-value < 0.05 as statistically significant. Results: The OB group showed obesity, high glucose, triglycerides, uric acid, HOMA, systolic blood pressure, and TNF-α in adipose tissue. OB group presented cardiac remodeling and diastolic dysfunction. TLR-4 and NF-ĸB expression and cytokine levels were higher in OB. Conclusion: Our findings conclude that, in an obesogenic condition, the inflammation derived from cardiac TLR-4 activation can be a mechanism able to lead to remodeling and cardiac dysfunction.
... A limitation of our study is the difference in visceral adipose tissue depot: in vivo mouse studies utilized gonadal adipose tissue, while our human adipose tissue was pericardial. Although literature comparing the pericardial and gonadal/epididymal fat depots, specifically, were not found, both fat depots exhibit inflammatory and/or metabolic dysfunction parameters (78)(79)(80). Future work is needed to verify a similar inflammatory profile between the two adipose tissue depots. Several studies report that approximately one-third to one-half of MHO individuals develop MUO, showing that MHO may not be a stable condition for a significant portion of obese patients. ...
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Metabolically healthy obesity (MHO) accounts for roughly 35% of all obese patients. There is no clear consensus that has been reached on whether MHO is a stable condition or merely a transitory period between metabolically healthy lean and metabolically unhealthy obesity (MUO). Additionally, the mechanisms underlying MHO and any transition to MUO are not clear. Macrophages are the most common immune cells in adipose tissues and have a significant presence in atherosclerosis. Fas (or CD95), which is highly expressed on macrophages, is classically recognized as a pro-apoptotic cell surface receptor. However, Fas also plays a significant role as a pro-inflammatory molecule. Previously, we established a mouse model (ApoE -/- /miR155 -/- ; DKO mouse) of MHO, based on the criteria of not having metabolic syndrome (MetS) and insulin resistance (IR). In our current study, we hypothesized that MHO is a transition phase toward MUO, and that inflammation driven by our newly classified CD95 ⁺ CD86 ⁻ macrophages is a novel mechanism for this transition. We found that, with extended (24 weeks) high-fat diet feeding (HFD), MHO mice became MUO, shown by increased atherosclerosis. Mechanistically, we found the following: 1) at the MHO stage, DKO mice exhibited increased pro-inflammatory markers in adipose tissue, including CD95, and serum; 2) total adipose tissue macrophages (ATMs) increased; 3) CD95 ⁺ CD86 ⁻ subset of ATMs also increased; and 4) human aortic endothelial cells (HAECs) were activated (as determined by upregulated ICAM1 expression) when incubated with conditioned media from CD95 ⁺ -containing DKO ATMs and human peripheral blood mononuclear cells-derived macrophages in comparison to respective controls. These results suggest that extended HFD in MHO mice promotes vascular inflammation and atherosclerosis via increasing CD95 ⁺ pro-inflammatory ATMs. In conclusion, we have identified a novel molecular mechanism underlying MHO transition to MUO with HFD. We have also found a previously unappreciated role of CD95 ⁺ macrophages as a potentially novel subset that may be utilized to assess pro-inflammatory characteristics of macrophages, specifically in adipose tissue in the absence of pro-inflammatory miR-155. These findings have provided novel insights on MHO transition to MUO and new therapeutic targets for the future treatment of MUO, MetS, other obese diseases, and type II diabetes.
... (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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This study investigated the effects of tocotrienol on inflammation, oxidative stress and non-alcoholic fatty liver disease (NAFLD). Male rats were fed with high-carbohydrate high-fat (HCHF) diet and treated with either 60 or 100 mg/kg annatto tocotrienol or palm tocotrienol. Activated Toll-like receptor (TLR) and its downstream targets, increased lipid peroxidation, decreased antioxidant levels in liver and higher serum C-reactive protein (CRP) level were detected in the HCHF rats. Higher steatosis, lobular inflammation, hepatocyte ballooning, NAFLD activity score and lipid deposition was noted. Tocotrienol inhibited TLR activation, increased interleukin-10, reduced lipid peroxidation and raised antioxidant activities in the liver. Lower serum CRP level was observed after tocotrienol administration. Palm tocotrienol (100 mg/kg) reduced steatosis grade in the HCHF rats. In conclusion, tocotrienol potentially mitigates inflammatory response, oxidative stress and steatosis in this animal model of NAFLD. The alleviation of inflammation may be in part mediated through the suppression of TLR activation.
Background Obesity is increasing rapidly affecting half billion adult's population. Pathophysiology of obesity involves low grade inflammation sustained by Toll like receptor 2 (TLR-2) the innate immune adapters. This study was conducted for detection and association of TLR-2 gene mutations with obesity. Methods In this case-control study 228 individuals with obesity and 228 controls were enrolled based on Body Mass Index (BMI) ≥25 and 18-24 kg/m² respectively. The variations in TLR-2 gene were detected by Sanger sequencing. These identified TLR-2 variants were further analyzed in silico for change in miRNA binding and mRNA strucutre. Results Four novel single base substitutions (153688371 T >C, 153702295 T >C, 153703504 T >C and 153705074 C >A) were identified in exon 3 and 4 of TLR-2 gene affecting splice site and poly-A tail. The genotypic and allelic frequencies of the variants were strongly associated with increasing obesity susceptibility. Only variant 153703504 T >C was significantly associated with preobesity. Despite variations in gene sequence, no change in miRNA binding except for variant 153688371 T >C of Exon 3 where a novel binding site for hsa-miR-4523 was created. Furthermore, mRNA stability and secondary structure were also compromised in identified variants. Conclusion All detected variants of TLR-2 gene were significantly associated with and posed risk for development of obesity. Furthermore, in silico analysis revealed generation of new miRNA (hsa-miR-4523) binding site and change in mRNA structure/stability which needs to be further investigated for possible role in altering TLR-2 gene regulation/expression in obesity.
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Background Thousands of publications in recent years have addressed the induction of metabolic syndrome (MetS) in rodents, however, the criteria and the reference values for diagnosing this disease have not been defined. Objective Our main objetive was to carry out a systematic review to gather evidence about the criteria for biochemical and anthropometric parameters in which scientific studies have relied to report that rats developed MetS from a previous dietary manipulation. Methods We compiled characteristics and findings of diet induced MetS with high-fat, high-carbohydrate, high-fat/high-carbohydrates and cafeteria diet from PubMed and Science Direct databases published in the last 5 years. Results The results on the principal determinants for the syndrome, published in the reviewed articles, were chosen to propose reference values in the rat models of food induction. Conclusion The values obtained will serve as reference cut-of points in the development of the disease; in addition, the compilation of data will be useful in planning and executing research protocols in animal models.
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Obesity is an excessive accumulation of body fat that may be harmful to health. Today, obesity is a major public health problem, affecting in greater or lesser proportion all demographic groups. Obesity is estimated by body mass index (BMI) in a clinical setting, but BMI reports neither body composition nor the location of excess body fat. Deaths from cardiovascular diseases, cancer and diabetes accounted for approximately 65% of all deaths, and adiposity and mainly abdominal adiposity are associated with all these disorders. Adipose tissue could expand to inflexibility levels. Then, adiposity is associated with a state of low-grade chronic inflammation, with increased tumor necrosis factor-α and interleukin-6 release, which interfere with adipose cell differentiation, and the action pattern of adiponectin and leptin until the adipose tissue begins to be dysfunctional. In this state the subject presents insulin resistance and hyperinsulinemia, probably the first step of a dysfunctional metabolic system. Subsequent to central obesity, insulin resistance, hyperglycemia, hypertriglyceridemia, hypoalphalipoproteinemia, hypertension and fatty liver are grouped in the so-called metabolic syndrome (MetS). In subjects with MetS an energy balance is critical to maintain a healthy body weight, mainly limiting the intake of high energy density foods (fat). However, high-carbohydrate rich (CHO) diets increase postprandial peaks of insulin and glucose. Triglyceride-rich lipoproteins are also increased, which interferes with reverse cholesterol transport lowering high-density lipoprotein cholesterol. In addition, CHO-rich diets could move fat from peripheral to central deposits and reduce adiponectin activity in peripheral adipose tissue. All these are improved with monounsaturated fatty acid-rich diets. Lastly, increased portions of ω-3 and ω-6 fatty acids also decrease triglyceride levels, and complement the healthy diet that is recommended in patients with MetS.
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Obesity has been shown to impair myocardial performance. Some factors have been suggested as responsible for possible cardiac abnormalities in models of obesity, among them beta-adrenergic (βA) system, an important mechanism of regulation of myocardial contraction and relaxation. The objective of present study was to evaluate the involvement of βA system components in myocardial dysfunction induced by obesity. Thirty-day-old male Wistar rats were distributed in control (C, n = 25) and obese (Ob, n = 25) groups. The C group was fed a standard diet and Ob group was fed four unsaturated high-fat diets for 15 weeks. Cardiac function was evaluated by isolated papillary muscle preparation and βA system evaluated by using cumulative concentrations of isoproterenol and Western blot. After 15 weeks, the Ob rats developed higher adiposity index than C rats and several comorbidities; however, were not associated with changes in systolic blood pressure. Obesity caused structural changes and the myocardial responsiveness to post-rest contraction stimulus and increased extracellular calcium (Ca2+) was compromised. There were no changes in cardiac function between groups after βA stimulation. The obesity was not accompanied by changes in protein expression of G protein subunit alpha (Gsα) and βA receptors (β1AR and β2AR). In conclusion, the myocardial dysfunction caused by unsaturated high-fat diet-induced obesity, after 15 weeks, is not related to βAR system impairment at the receptor-signalling pathway.
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Obesity and type 2 diabetes are now recognized as chronic pro-inflammatory diseases. In the last decade, the role of the macrophage in particular has become increasingly implicated in their pathogenesis. Abundant literature now establishes that monocytes get recruited to peripheral tissues (i.e., pancreas, liver, and adipose tissue) to become resident macrophages and contribute to local inflammation, development of insulin resistance, or even pancreatic dysfunction. Furthermore, an accumulation of evidence has established an important role for macrophage polarization in the development of metabolic diseases. The general view in obesity is that there is an imbalance in the ratio of M1/M2 macrophages, with M1 "pro-inflammatory" macrophages being enhanced compared with M2 "anti-inflammatory" macrophages being down-regulated, leading to chronic inflammation and the propagation of metabolic dysfunction. However, there is emerging evidence revealing a more complex scenario with the spectrum of macrophage states exceeding well beyond the M1/M2 binary classification and confused further by human and animal models exhibiting different macrophage profiles. In this review, we will discuss the recent findings regarding macrophage polarization in obesity and type 2 diabetes.
Objectives: Obesity is a significant quality of life-impairing health problem affecting industrialized nations. However, despite carrying a large fat mass, some very obese individuals exhibit normal metabolic profiles (metabolically healthy obesity). The physiological factors underlying their protective and favorable metabolic profiles remain poorly defined. Methods: A search of the National Library of Medicine PubMed database was performed using the following keywords: Metabolically healthy obese, metabolically normal obese, insulin resistance, metabolically unhealthy normal weight, and uncomplicated obesity. Results: This article reviewed factors associated with severe obesity that lacks complications, and suggests putative activities by which these obese individuals avoid developing the clinical features of metabolic syndrome, or the metabolic complications associated with severe obesity. Conclusions: Despite the knowledge that visceral fat deposition is the seminal factor that ultimately causes insulin resistance (IR) and the detrimental inflammatory and hormonal profile that contributes to increase risk for cardiovascular disease, it remains unknown whether metabolically healthy obesity (MHO) has genetic predisposing factors, and whether MHO ultimately succumbs to IR and the metabolic syndrome, indicating a need for prophylatic bariatric surgery.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Metabolic syndrome is a cluster of conditions that synergistically increase the risk of cardiovascular disease, type 2 diabetes, and premature mortality. The components are abdominal obesity, impaired glucose metabolism, dyslipidemia, and hypertension. Prediabetes, which is a combination of excess body fat and insulin resistance, is considered an underlying etiology of metabolic syndrome. Prediabetes manifests as impaired fasting glucose and/or impaired glucose tolerance. Impaired fasting glucose is defined as a fasting blood glucose level of 100 to 125 mg/dL; impaired glucose tolerance requires a blood glucose level of 140 to 199 mg/dL 2 hours after a 75-g oral intake of glucose. In patients with prediabetes, the rate of progression to diabetes within 3 years can be decreased by approximately 58% with lifestyle modifications. These include weight loss through exercise (30 minutes or more of moderate physical activity on most, preferably all, days of the week) and dietary modifications. Recommended diets are high in fruits, vegetables, whole grains, and fish. Consumption of sweetened beverages, including diet soda, should be avoided. For patients who do not achieve goals with lifestyle modifications, metformin can be considered. Weight loss drugs and bariatric surgery are appropriate for select patients. Hypertension and dyslipidemia should be managed according to current guidelines. Written permission from the American Academy of Family Physicians is required for reproduction of this material in whole or in part in any form or medium.
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).
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.