Relationship between Innate Immune Response Toll-Like Receptor 4 (TLR-4) and the Pathophysiological Process of Obesity Cardiomyopathy

Abstract

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.

Full text

Arq Bras Cardiol. 2021; 117(1):91-99 91
Original Article
Relationship between Innate Immune Response Toll-Like
Receptor 4 (TLR-4) and the Pathophysiological Process of Obesity
Cardiomyopathy
Pedro Henrique Rizzi Alves,1 Artur Junio Togneri Ferron,1 Mariane Róvero Costa,1 Fabiana Kurokawa Hasimoto,1
Cristina Schmitt Gregolin,1,2 Jéssica Leite Garcia,1 Dijon Henrique Salomé de Campos,1 Antônio Carlos
Cicogna,1 Letícia de Mattei,1 Fernando Moreto,1 Silméia Garcia Zanati Bazan,1 Fabiane Valentini Francisqueti-
Ferron,1 Camila Renata Corrêa1
Universidade Estadual Paulista Júlio de Mesquita Filho Câmpus de Botucatu Faculdade de Medicina,1 Botucatu, SP - Brazil
Universidade Federal de Mato Grosso,2 Sinop, MT – Brazil
Mailing Address: Fabiane Valentini Francisqueti-Ferron •
Universidade Estadual Paulista, Faculdade de Medicina - Avenida Prof Mario
Rubens Guimarães Montenegro, s/n. Postal code 18618-687,
Botucatu, SP – Brazil
E-mail: fabiane_vf@yahoo.com.br
Manuscript received November 14, 2019, revised manuscript May 04, 2020,
accepted June 16, 2020
DOI: https://doi.org/10.36660/abc.20190788
Abstract
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.
Keywords: Cardiovascular Diseases/physiopathology; Obesity; Inflammation; Cytokines; Adipocytes; Fatty Acid
synthases; Nutrional Assessment.
Introduction
Obesity, defined as an excessive body fat accumulation that
may impair an individual’s health,1 is currently considered the
most important nutritional disorder in both developed and
underdeveloped countries.2 Estimates show that in 2025 at
least 18% of the adult population will be obese, a worrying
condition since it can lead to several comorbidities, including
cardiovascular diseases.3,4
One of the main causes responsible for the obesity epidemic
is the excessive consumption of hypercaloric diets, which are
rich in saturated fat and refined sugars, associated with a
sedentary health style.5,6 This modern lifestyle leads to adipose
tissue hypertrophy that triggers, initially, a local inflammatory
process, which may later affect and compromise the function
of other organs, such as the heart.7 This inflammation of
metabolic origin results in the synthesis of the proinflammatory
cytokines led by different pathways, among them: adipose
tissue lipolysis,8 adipocytes hypertrophy,9 fatty acids from
the diet,10 and intestinal lipopolysaccharides (LPS) due to
dysbiosis.11 Within this context, free fatty acids and LPS are
the main components involved in the inflammatory response,
acting as Damage-Associated Molecular Patterns (DAMPs) and
Pathogen-associated Molecular Patterns (PAMPs), respectively,
which are recognized by some receptors, especially the Toll-
Like Receptor 4 (TLR-4).12

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TLR-4 belongs to a family of receptors usually expressed
in cells of the innate immune system, such as macrophages,
neutrophils, and lymphocytes, and exerts an important role
to detect and recognize pathogens leading to the innate13
and acquired14 immune response activation. This receptor
is responsible for the activation of the nuclear factor kappa
B (NF-ĸB) with consequent synthesis of proinflammatory
cytokines involved in the pathophysiology of several diseases,
including heart diseases.15
However, the literature reports that the TLR-4 receptor is
not expressed only in immune system cells. Cardiomyocytes
also express this receptor and its activation, especially against
infectious cardiac pathogens, contributes to the myocardial
dysfunction process.16–18 Although the involvement of the
immune system in the development of cardiac pathologic
hypertrophy is well established,14,19,20 the TLR-4 pathway
participation in the development and progression of obesity-
related cardiac remodeling has not yet been clarified.21
Considering the lack of studies regarding this topic, the aim
of this study was to test the hypothesis that the TLR-4 receptor
activation participates in the obesity cardiomyopathy process,
due to production of cytokine through the NF-ĸB activation.
Material and Methods
Experimental Protocol
Male Wistar rats (n=16), 21 days of age, were obtained
from Universidade Estadual Paulista (UNESP). Sample size
was calculated by using SigmaStat for Windows, version 3.5
(Systat Software Inc., San Jose, CA, USA), considering the
expected difference mean of 2.0, expected standard deviation
of 1.0, test power of 90%, and α of 0.05. This calculation
considered the adiposity index of previous studies published
by our group.22–24 The animals were randomly divided into
two experimental groups: the control group (C, n= 8 animals)
that received standard diet/water and the obese group (OB,
n= 8 animals) that received high sugar-fat (HSF) diet and
water plus 25% of sucrose for 30 weeks. The diet model is a
well-established model to induce obesity previously published
by our research group.25 The HSF diet contained soybean
meal, sorghum, soybean peel, dextrin, sucrose, fructose, lard,
vitamins, and minerals, plus 25% sucrose in drinking water.
The control diet contained soybean meal, sorghum, soybean
peel, dextrin, soy oil, vitamins, and minerals.
Food and water were offered ad libitum and the animals
were kept in individual cages, in an environment with
controlled temperature (24 ± 2°C), humidity (55 ± 5%),
and light-dark cycle (12-12h). The study protocol (CEUA
1265/2018) was approved by the Ethics Committee on Animal
Experimentation of the Botucatu School of Medicine, UNESP,
São Paulo, Brazil, and followed the recommendations from
the Guide for the Care and Use of Experimental Animals.26
At the end of 30 weeks, the animals were fasted for 8h, then
euthanized and the material was collected for analysis.
Nutritional Evaluation
Food and water consumption were evaluated daily. Caloric
intake was determined by multiplying the energy value of
each diet (g × Kcal) by daily food intake. For the OB group,
caloric intake also included water calories (0.25 × 4 × mL
consumed). The animals’ body weight was measured weekly.
After euthanasia, visceral fat (VAT), epididymal fat (EAT), and
retroperitoneal fat (RAT) were collected and used to calculate
the adiposity index (AI) by the formula: [(VAT + EAT + RAT)
/ ANIMAL WEIGHT] x100.
Obesity-related disorders analysis
Plasma glucose and triglycerides, (BioClin, Quibasa
Química Básica Ltda., Belo Horizonte, Minas Gerais, Brazil)
were measured by colorimetric-enzymatic method in an
automatic enzymatic analyzer system (Chemistry Analyzer
BS-200, MindrayMedical International Limited, Shenzhen,
China). Insulin (EMD Millipore Corporation, Billerica, MA,
USA) was analyzed in plasma by ELISA using a commercial kit.
The reading was performed by Spectra Max 190 microplate
spectrophotometer (Molecular Devices®, Sunnyvale, CA, USA).
The Homeostatic Model Assessment (HOMA-IR), which
allows evaluating insulin resistance, was also calculated
according to the following formula: fasting insulin (μUI / mL)
x fasting glucose (mmol / L) / 22.5.27,28
The Systolic blood pressure (SBP) evaluation was assessed
in conscious rats by the non-invasive tail-cuff method
with a NarcoBioSystems® Electro-Sphygmomanometer
(International Biomedical, Austin, TX, USA). The animals were
warmed in a wooden box (50 x 40 cm) between 38–40°C with
heat generated by two incandescent lamps for 4–5 minutes
to stimulate arterial vasodilation. After this procedure, a cuff
with a pneumatic pulse sensor was attached to the tail of each
animal. The cuff was inflated to a pressure of 200 mmHg and
subsequently deflated. Blood pressure values were recorded
on a Gould RS 3200 polygraph (Gould Instrumental Valley
View, Ohio, USA). The mean of three pressure readings was
recorded for each animal.
Since obesity is associated with an inflammatory condition
in adipose tissue, TNF-α and IL-6 levels were evaluated.
Epididymal adipose tissue (400 mg) was triturated with 2 ml
of phosphate buffer saline (PBS) (pH 7.4) and then centrifuged
at 3,000 rpm and 4°C for 10 min. Using the supernatants,
TNF-α and IL-6 were measured using commercial ELISA kits
(R&D Systems) according to the manufacturer’s instructions.
The values were normalized by the protein amounts of
each sample quantified by the Bradford method29 and the
results are expressed in picogram/g protein (pg/g protein).
Epididymal adipose tissue was selected for presenting a similar
inflammation pattern to that found in visceral fat.30
Echocardiographic Analysis
The analysis was performed on live animals by transthoracic
echocardiography, with a Vivid S6 system equipped with a
multifrequency ultrasonic transducer of 5.0 to 11.5 MHz
(General Electric Medical Systems, Tirat Carmel, Israel). The
animals were lightly anesthetized by intraperitoneal injection
with a mixture of ketamine (50 mg/kg) and xylazine (1 mg/
kg) and placed in a left decubitus position. The structural
measurements of cardiac images were obtained in the
one-dimensional mode (M-mode) guided by the images

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Alves et al.
TLR-4 and obesity cardiomyopathy
in the two-dimensional mode with the transducer in the
parasternal position, minor axis. Left ventricular (LV) evaluation
was performed with the M-mode cursor just below the
mitral valve plane at the level of the papillary muscles. All
examinations were performed by the same examiner and
obtained according to the main method recommended by
the American Society of Echocardiography. The aorta and
left atrium images were obtained by positioning the M-mode
cursor to plan the aortic valve level. The following cardiac
structures were evaluated: left ventricular diastolic diameter
(LVDD); left ventricular posterior wall thickness at end-systole
(LVPWS); Left ventricular systolic interventricular septum
thickness (LVSIS); aorta diameter (AD); left atrium (LA); and
the relative wall thickness (RWT) of the LV. The LV systolic
function was evaluated by cardiac output and also by heart
rate (HR), since it is a cardiac systolic function modulator. The
LV diastolic function was assessed by the transmitral flow early
peak velocity (E); deceleration time of E wave (Dec. time). The
study was supplemented by evaluation by early (E’) and late
(A’) diastolic tissue Doppler of the mitral annulus (arithmetic
mean travel speeds of lateral and septal walls), and the wave
ratio (E/E’ and E’/A’).
Cardiac tissue analysis
Inflammation
One hundred milligrams (100mg) were homogenized
in PBS and the supernatant was used. TNF-α and IL-6
quantification were performed by ELISA using a commercial
kit (Linco Research Inc., R & D Systems, Millipore and B-Brigde
International Inc.). The reading was performed by Spectra Max
190 microplate spectrophotometer (Molecular Devices®,
Sunnyvale, CA, USA). The results were corrected by the total
protein amount according to the Bradford method.
Real time PCR
The frozen left ventricular fragment was homogenized in
TRIzol® for extraction of ribonucleic acid (RNA). Afterward,
the RNA was subjected to reverse transcription, conversion
of RNA to complementary deoxyribonucleic acid (cDNA),
by the action of the enzyme reverse transcriptase, using the
SuperScript II First-Strand Synthesis System for RT-PCR®
Invitrogen, São Paulo, Brazil. The obtained cDNA was used in
the polymerase chain reaction (PCR) using ready-made assays
(Applied Biosystems, CA, USA) containing TaqMan MGB (FAM)
primer and primer specific for TLR-4 (Rn00569848_m1).
Western Blot
The heart fragments were homogenized in lysis buffer and
centrifuged. The supernatant was collected and the protein
concentration was analyzed by the Bradford method.29 After
quantification, the cardiac protein extracts were diluted in
a buffer solution containing 50 mM Tris-HCl (pH 6.8), 200
mM 2-Mercaptoethanol, 2% Sodium Dodecyl Sulfate (SDS),
0.1% bromophenol blue, and 10% glycerol. The dilutions
(50ug) were heated and subjected to sodium dodecyl
sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) in
10% polyacrylamide gels. After electrophoresis, the proteins
were electrotransferred to nitrocellulose membranes (Bio-
Rad Biosciences; NJ, USA). Non-specific binding sites of
the primary antibody to the membrane were blocked by
incubation with 0.5% skimmed-milk powder solution,
dissolved in TBS-T buffer pH 7.4. The membrane was then
washed three times in basal solution and incubated overnight
with TLR-4 specific primary antibody (sc293072), total NF-ĸB
(sc8008), phosphorylated NF-ĸB (ser536) (sc33020). β- actin
was used as an internal control (sc47778). After incubation, the
membranes were washed and incubated with the respective
secondary antibodies. Finally, immunodetection was
performed by the chemiluminescence method, according to
the manufacturer’s instructions (ECL SuperSignal® West Pico
Chemiluminescent Substrate – Thermo Scientific, Rockford, IL,
USA, 34080), and analyzed by means of a densitometer (GS-
710 calibrated imaging densitometer, Bio-Rad lab, CA, USA).
Statistical analysis
The data were submitted to Kolmogorov-Smirnov normality
test. Parametric variables were compared by unpaired
Student’s t-test and the results are expressed as mean ±
standard deviation. Non-parametric variables were compared
by Mann- Whitney test and the results are expressed as median
(interquartile range 25-75). Statistical analyses were performed
using Sigma Stat for Windows, version 3.5 (Systat Software
Inc., San Jose, CA, USA). A p-value < 0.05 was considered
statistically significant.
Results
Table 1 shows the nutritional parameters. OB animals
consumed less food, but more water compared to the control
group, reflecting a similar caloric intake. The OB group also
presented obesity characterized by increased body weight
and adiposity index.
Regarding the obesity-related disorders, the OB group
presented higher glucose, triglycerides, and uric acid, insulin
resistance, increased systolic blood pressure, and adipose
tissue inflammation, with elevated TNF-α and IL-6 levels
compared to the control group (Table 2).
The echocardiographic analysis is presented in Table
3. At the end of 30 weeks, the OB group showed cardiac
remodeling, characterized by reduced LVDD and increased
LVPWS, LVSIS, LA, and AD. Moreover, OB animals presented
diastolic dysfunction, represented by changes in the E wave,
E wave deceleration time, and E’/A’.
Regarding the TLR-4 gene and protein expression in cardiac
tissue, it is possible to verify that both were increased in the
OB group (Figure 1).
NF-ĸB phosphorylation in cardiac tissue was also higher in
the OB group (Figure 2), resulting in increased cytokines, since
this group showed higher TNF-α and IL-6 levels compared to
the C group (Figure 3).
Discussion
This study hypothesized that the TLR-4 receptor activation
participates in obesity-related cardiac disease by triggering
cytokine production via NF-ĸB. In order to induce obesity and

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disorders related to excessive body fat, the animals in the OB
group were submitted to a HSF diet for 30 weeks. At the end
of the experimental period, the results show that these animals
presented higher adiposity index and several disorders, such
as hyperglycemia, increased uric acid, insulin resistance,
hypertriglyceridemia, elevated SBP, increased TNF-α and
IL-6 levels in both heart and adipose tissue, confirming the
efficacy of the diet model used. 22–25
The coexistence of obesity-related disorders — such as
insulin resistance, diabetes, and dyslipidemia — associated
with adipose tissue dysfunction, characterized by adipokine
imbalance, promote maladaptive responses in the heart, such
as myocyte hypertrophy, contractile dysfunction, and cardiac
remodeling, which contribute to both the development and
progression of chronic heart failure.31–33 This condition was
confirmed in this study, since the echocardiogram evaluation
showed cardiac remodeling and diastolic dysfunction in the
OB group. It is important to emphasize that the concentric
characteristics of cardiac remodeling observed in the OB
group are the result of an increased functional overload and
the maintenance of this condition, leading to cardiac diastolic
dysfunction. Similar results have been related to the diet-
induced obesity model in the literature.34
The literature reports that cardiac remodeling can also
be led by high concentrations of proinflammatory cytokines,
such as TNF-α and IL-6.35,36 TNF-α has been implicated in the
development of left ventricular dysfunction, left ventricular
remodeling, increased cardiac myocyte apoptosis and its direct
action is exerted by TNF-α receptors, which are expressed by
almost all nucleated cells. Increased IL-6 levels can also induce
myocyte hypertrophy and myocardial dysfunction.37 It is
important to emphasize that these cytokines can be produced
in response to TLR-4 pathway activation, and also L-6 seems
to be released in direct response to TNF-α,35 exacerbating the
cardiac changes due to inflammatory condition. Therefore, our
findings confirm the evidence regarding obesity cardiopathy
and inflammation, since the cardiac levels of TNF-α and IL-6
were higher in the OB group.18,37
The involvement between proinflammatory cytokines and
obesity-related cardiac remodeling induced by diet can be
attributed to several causes.18,38 Thus, the aim of this study
was to evaluate cardiac TLR-4 activation as responsible for
triggering the inflammatory process. It was observed that the
OB group presented higher TLR-4 gene and protein expression
together with increased NF-ĸB phosphorylation, confirming
the activation of this pathway as a mediator of inflammation.
The literature reports that this receptor can be activated by LPS
of gram-negative bacteria, but also by fatty acids.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
Table 1 – Nutritional parameters
Characteristics Groups
Control (n=8) OB (n=8) p value
Final body weight (g) 493 ± 50.8 583 ± 75.9 0.001*
Adiposity index (%) 4.79 ± 0.73 8.68 ± 1.76 0.0004*
Food consumption (g/day) 23.92 ± 2.37 13.0 ± 2.20 0.0001*
Water consumption (ml/day) 34.6 ± 5.08 41.6 ± 3.47 0.001*
Caloric intake (Kcal/day) 85.9 ± 8.52 98.3 ± 8.35 0.07
OB: obese group. Data expressed as mean ± standard deviation. Comparison by unpaired Student’s t-test. *indicates p < 0.05.
Table 2 – Obesity-related disorders
Characteristics GROUPS
Control (n=8) OB (n=8) p-value
Glucose (mg/dL) 75.7 ± 2.02 105 ± 17.9 0.02*
Triglycerides (mg/dL) 63.5 ± 18.6 104 ± 25.4 0.003*
Uric acid (mg/dL) 0.44 ± 0.09 0.62 ± 0.20 0.04*
HOMA-IR 5.90 ± 2.32 30.9 ± 17.0 0.004*
Systolic Blood Pressure (mmHg) 121 ± 5.77 128 ± 6.48 0.03*
TNF-α Adipose tissue (pg/g protein) 52.7 (46.6 – 62.5) 152 (117 – 219) 0.001*
IL-6 Adipose tissue (pg/g protein) 13.0 ± 9.2 94.5 ± 33.3 < 0.001*
OB: obese group; HOMA-IR: Homeostatic Model Assessment. Data expressed as mean ± standard deviation or median (interquartile range).
Comparison by unpaired Student’s t-test or Mann-Whitney. * indicates p < 0.05.

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Table 3 – Echocardiographic analysis
Characteristics GROUPS
Control OB p-value
LVDD (mm) 7.20 ± 0.20 6.70 ± 0.56 0.031*
LVDD/BW 14.7 ± 1.6 11.9 ± 1.3 0.002*
LVPWS (mm) 3.03 ± 0.31 3.35 ± 0.21 0.033*
LVSIS (mm) 3.35 ± 0.21 3.57 ± 0.19 0.045*
RWT 0.45 ± 0.05 0.51 ± 0.09 0.098
AD (mm) 3.73 ± 0.12 3.99 ± 0.13 0.001*
LA (mm) 4.75 ± 0.12 5.17 ± 0.38 0.011*
HR (bpm) 234 ± 39 295 ± 26 0.002*
Cardiac Output 0.91 ± 0.01 0.88 ± 0.06 0.142
Deceleration time (ms) 47.2 ± 3.0 50.6 ± 2.7 0.035*
E wave 68.3 ± 5.2 73.1 ± 3.3 0.046*
A wave 40.7 ± 3.7 45.7 ± 6.1 0.062
E/A ratio 1.68 ± 0.08 1.61 ± 0.016 0.327
E'/A' medium ratio 1.53 ± 0.14 1.26 ± 0.25 0.018*
E/ E’ medium ratio 12 ± 1.3 13.5 ± 1.86 0.094
OB: obese group; LVDD: left ventricular diastolic dysfunction; BW: body weight; LVPWS: left ventricular posterior wall thickness at end-systole; LVSIS:
left ventricular systolic interventricular septum thickness; RWT: relative wall thickness; AD: aorta diameter; LA: left atrium; HR: heart rate. Transmitral
flow early peak velocity (E wave); deceleration time: deceleration time of the E wave. Doppler early (E’) and late (A’) diastolic of the mitral annulus
(arithmetic average travel speeds of lateral and septal walls), and the ratio (E/E’ and E’/A’). Data expressed as mean ± standard deviation. Comparison
by unpaired Student’s t-test. * indicates p < 0.05.
Figure 1 – TLR-4 expression in cardiac tissue. (A) Gene expression by real-time PCR; (B) Protein expression by Western Blot. Results expressed as mean
± standard deviation. Comparison by unpaired Student’s t-test. * indicates p < 0.05; n = 8 animals/group.

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Figure 2 – Phospho and total NF-ĸB protein expression in cardiac tissue. Results expressed as mean ± standard deviation. Comparison by unpaired
Student’s t-test. *indicates p < 0.05; n = 8 animals/group.
Figure 3 – Cardiac cytokines in cardiac tissue. (A) IL-6 (pg protein/g); (B) TNF-α. Results expressed as median and interquartile range. Comparison by
Mann-Whitney test. * indicates p < 0.05; n = 8 animals/group.

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resistance, once the adipose tissue becomes resistant to the
hormone antilipolytic effect. Together with insulin resistance,
increased TNF-α and IL-6 levels are also able to induce lipolysis
in adipose tissue.42,43 These two lipolytic conditions were
presented by the OB group. Moreover, associated with these
mechanisms described above, some researchers report that the
Western diet pattern is associated with changes in the intestinal
microbiota, making this organ more permeable, allowing
the translocation of pathogenic bacteria to the circulation.42
Bacterial LPS can be recognized by TLR-4, triggering the NF-
ĸB activation with consequent cytokine synthesis. Although
this mechanism was not evaluated in this experiment, this
relationship is already well established.43–45 In addition, the
diet composition used in this study is also directly related to
the TLR-4 receptor activation, since the saturated fatty acids
offered in the diet have similar structures to bacterial LPS,
being also able to be recognized, leading to an inflammatory
process via TLR-4 pathway.46,47
Conclusion
In summary, all these mechanisms may have been
activated in a synergistic way, enhancing the production of
cytokines that play a fundamental role in the development
of cardiomyopathies. Thus, this study shows that the innate
immune response through TLR-4 receptor activation is one
of the mechanisms that can contribute to the onset of the
myocardial inflammatory process in obesity. Therefore,
since no studies were found in the literature showing an
interaction between this inflammatory pathway, heart disease
and obesity, our findings conclude that in an obesogenic
condition, the inflammation derivative from cardiac TLR-4
activation is a new mechanism which can lead to remodeling
and cardiac dysfunction.
Acknowledgments
The authors thank Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP) (2016/13592-1; 2015/10626-
0) and Unidade de Pesquisa Experimental (UNIPEX) from the
Botucatu School of Medicine (UNESP), Botucatu.
Study limitations
This study has few limitations. One of them is related to
the absence of plasma fat-free acid evaluation.
Author Contributions
Conception and design of the research: Ferron AJT, Campos
DHS, Cicogna AC, Francisqueti F, Corrêa C; Acquisition of
data: Alves PHR, Costa MR, Hasimoto FK, Gregolin C, Garcia
JL, Campos DHS, Mattei L, Moreto F, Bazan SGZ; Analysis and
interpretation of the data: Alves PHR, Ferron AJT, Campos
DHS, Bazan SGZ, Francisqueti F, Corrêa C; Statistical analysis:
Ferron AJT; Obtaining financing: Corrêa C; Writing of the
manuscript: Alves PHR, Ferron AJT, Francisqueti F, Corrêa C;
Critical revision of the manuscript for intellectual contente:
Ferron AJT, Cicogna AC, Francisqueti F, Corrêa C.
Potential Conflict of Interest
No potential conflict of interest relevant to this article was
reported.
Sources of Funding
This study was partially funded by Fundação de Amparo
à Pesquisa do Estado de São Paulo (FAPESP) (2016/13592-1;
2015/10626-0)
Study Association
This study is not associated with any thesis or dissertation work.
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Arq Bras Cardiol. 2021; 117(1):91-99 99
Original Article
Alves et al.
TLR-4 and obesity cardiomyopathy
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