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Faculty of Sport Research Center in Physical Activity, Health and Leisure
Faculty of Sport Research Center in Physical Activity, Health and Leisure
Faculty of Sport
Research Center in Physical Activity, Health and Leisure
CONTEXT OF OBESITY
The present dissertation was submitted in order to achieve the PhD degree
included in the doctoral course of Physical Activity and Health designed by the
Research Center in Physical Activity, Health and Leisure, Faculty of Sport,
University of Porto, according to the Law 74/2006 from March 24th.
BEIGING, ADIPOKINES
ii
Funding Sources
The candidate work was supported by a PhD Grant from Portuguese Foundation
for Science and Technology (FCT), SFRH/BD/89807/2012.
The experiments from the present work were performed by the support of the
projects PTDC/DES/113580/2009-FCOMP-01-0124-FEDER-014705,
PTDC/DTP/DES/1071/2012 and POCI-01-0145-FEDER-016690 PTDC/DTP-
DES/7087/2014.
The present work was conducted in the Research Centre in Physical Activity,
Health and Leisure (CIAFEL) (FCT, UID/DTP/00617/2013) and in the Metabolic
Research Laboratory (Clínica Universidad de Navarra) supported by Fondo de
Investigación Sanitaria-FEDER (FIS PI10/01677, PI12/00515 and PI13/01430)
from the Spanish Instituto de Salud Carlos III, the Department of Health of the
Gobierno de Navarra (61/2014).
iii
In memory of my dad, who I love and miss dearly.
v
Acknowledgements
First and foremost I want to thank my supervisors Professor José Magalhães and
Professor António Ascensão. I wish to express my sincere gratitude for your
support. I am grateful for all their contributions of time, ideas, and funding to make
my Ph.D. productive and creative. The enthusiasm they have for research was
very inspiring, even during tough moments in this doctoral process. I am grateful
for the opportunity you gave me 6 years ago when I began to take the first steps
in research field. I appreciate the exceptional example they have provided as a
successful researcher and professor.
To CIAFEL, Professor Jorge Mota and Professor José Oliveira, for all enthusiasm
and financial support during this Ph.D course.
vii
To D. Celeste for sharing all knowledge and experience in histology and her
concern about my work and to Ana Isabel for technical contribution in slot blot
analyses.
In the last scholar visit, I am grateful to Professor Gema Frühbeck, Amaia, Leire,
Javier, Beatriz, Sara, Victoria and Silvia from Metabolic Research Laboratory,
Clínica Universidad de Navarra for welcome and for making me feel part of the
group since I arrived. A special thanks to Amaia for your scientific contribution,
dedication, professionalism, simplicity that inspired and encouraged me during
this work.
A huge and sincere thank you to my friends who always have been with me,
Mónica for your sweetness and the moments that we lived in my first “American
adventure” and for many healthy discussions that I will always remember; Inês
for the good advices; Cristina for the words of incitement along our dinners;
Raquel for the smiles and laughers; Patrícia and Filipa for cheerfulness and
funny; Pilar for all kindness and confidence; Luana, Lu Souto, Carol, João,
Thuane, Mari, Sofia and Joana for the “crazy” and good times we had together;
Eduardo for the friendship; Bernardo for your generosity and technical help;
Diana, Andreia, Ana, Filipa, Ana Sousa for the friendship for so many years.
Thank you for always supporting me and believing in me.
viii
To Nuno, thank you for all support, friendship, dedication, affection and love you
gave me on this journey.
My biggest supporters have always been my family. I am very grateful for the
opportunities and encouragement provided by my parents, brothers over the
years. Thank you so much for your support, help, and advices through this
process. Thank you all for contributing to my success.
ix
Table of Contents
1. Introduction ............................................................................................ 3
2. Aims ..................................................................................................... 13
Study I ...................................................................................................... 97
xi
General Discussion ................................................................................ 227
References............................................................................................. 249
xii
List of Figures
Study I
Figure 2. Body weight, feed efficiency ratio, Lee index, visceral adiposity to
body weight, and relative weights of mWAT, rWAT and eWAT...…………......106
Figure 6. Plasma and eWAT adipQ expression. Plasma total adipQ, HMW
adipQ form, HWM/total adipQ ratio and eWAT adipQ protein expression…...110
Study II
Figure 1. Body weight, energy intake and relative fat depots weights…..133
xiii
Figure 3. Soleus, gastrocnemius muscle weights, total mass-body weight
ratio, and citrate synthase activity…………………………………………………135
Study III
Figure 3. The relative protein content of AMPK and ACC as well as their
phosphorylation at Thr 172 ans Ser 79, respectively……………………………..161
xiv
Study IV
Figure 2. Plasma cytokines. The protein content of IL-6, TNF-α, IL-10 and
IL-10/TNF-α ratio………………………………………………………………..…189
Figure 3. The pro- and anti-inflammatory cytokines in eWAT. The gene and
protein expression of IL-6, TNF-α, protein expression of IL-10, IL-10/TNF-α
ratio………………………………………………………………………………….191
Study V
Figure 1. Body weight over a period of 17 weeks, final body weight, energy
efficiency, visceral fat mass, and HOMA-IR…………………………………..….213
Figure 2. The frequency distribution of adipocyte size and gene and protein
expression of DLK1/PREF1…………………………………………………..……214
xv
List of Tables
Study II
Study III
Study IV
Table 1. Body weight, total energy intake, visceral adiposity, and adipocyte
size determinations…………………………………………………………………192
xvii
Resumo
O tecido adiposo visceral (TAV) é fisiologicamente reconhecido pela sua
capacidade em armazenar e libertar energia. No entanto, a sua acumulação
excessiva tem sido associada à manifestação patológica da obesidade e
doenças associadas. O exercício físico, por outro lado, é identificado como uma
estratégia importante para induzir adaptações positivas no TAV, que podem
decorrer da comunicação entre o eixo músculo esquelético e o tecido adiposo.
Contudo, os efeitos do exercício físico, enquanto estratégia preventiva ou
terapêutica, na libertação de miocinas, nas adaptações metabólicas,
inflamatórias e autofágicas no TAV, bem como o seu potencial para induzir um
fenótipo brown adipocyte-like num contexto de obesidade está pouco estudado.
A presente dissertação é composta por uma revisão da literatura e cinco estudos
experimentais, desenvolvidos a partir de um modelo animal de obesidade, cujo
objetivo geral foi analisar o impacto de dois modelos distintos de exercício físico
contra as alterações adversas impostas por uma dieta rica em gordura na
adiposidade, desregulação das adipocinas (estudo I), fenótipo brown adipocyte-
like (estudo II), perfil dos ácidos gordos, reguladores de acumulação lipídica,
conteúdo, biogénese e fusão mitocondrial, e inflamação (estudos III e IV),
autofagia e apoptose (estudo V). Desta forma, recorremos a análises
histomorfométricas, espectrofotométricas e às técnicas de Western blot e PCR
quantitativo em tempo real para determinar a expressão de proteínas e genes,
respetivamente, envolvidos nos diferentes processos estudados. Os resultados
sugerem que o exercício físico, em particular o treino de endurance, preveniu e
reverteu características relacionadas com a obesidade, como a adiposopatia,
acumulação lipídica, produção e secreção de adipocinas, bem como a
inflamação em animais submetidos à dieta gorda. Além disso, a produção de
miocinas induzidas pelo programa de treino de endurance associou-se a um
fenótipo brown adipocyte-like e a um aumento do conteúdo, biogénese e fusão
mitocondrial. Estes resultados realçam a importância do tecido adiposo nas
adaptações induzidas pelo exercício e contribuem para um melhor conhecimento
dos mecanismos através dos quais o exercício atenua os efeitos adversos na
obesidade, reforçando a relevância desta estratégia no tratamento da obesidade
e doenças associadas.
xix
Abstract
The visceral adipose tissue (VAT) is well known both for its capacity to store and
release energy, particularly in conditions of excessive accumulation, due to its
detrimental role in obesity and related chronic disorders. On the other hand,
physical exercise is recognized as an important strategy to induce positive
adaptations in VAT, which possibly occur through the cross-talk between skeletal
muscle and adipose organ axis. However, the effects of physical exercise, as a
preventive or therapeutic strategy, on myokines release, metabolic, inflammatory,
autophagic and apoptotic adaptations in VAT, as well as its potential signaling
influence toward a brown adipocyte-like phenotype under obesity conditions are
scarcely studied. This dissertation comprising one review and five experimental
studies, and developed with an obese animal model, intended to analyze the
potential role of two distinct physical exercise regimens in counteracting the
adverse consequences imposed by a diet-induced obesity (DIO) in adiposity,
adipokines dysregulation (study I), brown adipocyte-like phenotype (study II),
fatty acids profile, lipid accumulation mediators, inflammation, mitochondrial
content, biogenesis and fusion-related proteins (study III and IV), autophagy and
apoptosis (study V). Therefore, we used histomorphometric and
spectrophotometric analyses, Western blot and real-time PCR to determine the
relative expression of genes and proteins, respectively, involved in the studied
processes. Our data suggest that physical exercise, particularly endurance
training (ET), prevented or reverted some obesity-related features, such as
adiposopathy, lipid accumulation, adipokines production and secretion and
inflammation in DIO animals. Moreover, ET-induced myokines production was
associated with a brown adipocyte-like phenotype and also improved
mitochondrial content, biogenesis and fusion-related proteins. These data
highlight the prominent role of adipose tissue in whole-body adaptations induced
by exercise and contribute to a better understanding of the mechanisms by which
exercise attenuate the adverse consequences of obesity, strengthening the
relevance of this strategy to treat obesity and related disorders.
xxi
List of Abbreviations
ACC Acetyl CoA
AdipQ Adiponectin
AQP7 Aquaglyceroporin 7
CS Citrate synthase
ET Endurance training
FA Fatty acid
xxiii
FNDC5 Fibronectin type III-domain containing 5
GH Growth hormone
IL-10 Interleukin 10
IL-6 Interleukin 6
IR Insulin resistance
MFN1 Mitofusin 1
MFN2 Mitofusin 2
xxiv
MUFA Monounsaturated fatty acids
PE Physical exercise
RT Resistance training
xxv
SIRT1 Sirtuin 1
SIRT3 Sirtuin 3
TG Triglycerides
xxvi
CHAPTER I. General Introduction
Chapter I. General Introduction
1. Introduction
Among the innumerous organs and tissues closely associated with the etiology
and/or the physiopathology of obesity, the white adipose tissue (WAT) assumes
nowadays a pivotal role in the disease, given the substantial alterations and the
plasticity occurring in/of this tissue when submitted to a variety of stimuli,
including energy turnover (un)balances. In fact, after the identification of leptin as
an adipose-derived hormone, WAT, rather than just an energy storage tissue,
has been considered an important endocrine organ that produces several
biologically active factors, collectively termed as adipokines, with local and/or
systemic actions and interacting with different organ systems (Rodriguez et al.,
2015). In mammals, adipocytes were formerly classified in two types, the white
adipocytes, which are highly adapted to store excess energy and the brown
adipocytes that use fatty acids to generate heat - thermogenesis - via
mitochondrial uncoupling protein 1 (UCP1) (Lin & Farmer, 2016). However, in the
last years, a third subtype of adipocytes, with an inducible brown-like phenotype
and thermogenic properties, was identified, the so-called “beige” adipocytes
(Bostrӧm et al., 2012; Knudsen et al., 2014; Stanford et al., 2015; Tiano et al.,
2015). This “browning” or “beiging” process has been receiving a lot of scientific
attention in the literature as it may represent a promising and attractive
3
Chapter I. General Introduction
Depending on its location in the body, the visceral adipose tissue (VAT), situated
around the internal organs, has specific and distinct inherent characteristics from
subcutaneous adipose tissue (SAT), such as cellular composition, tissue
dynamics, adipokine release, and hormonal responses (Guilherme et al., 2008).
Visceral adipose tissue, unlike SAT, is anatomically linked to liver, via the portal
vein, providing non-esterified fatty acids (NEFA) and adipokines/cytokines
directly into liver (Bjorntorp, 1990; Rodriguez et al., 2014). Therefore, an
excessive VAT (or visceral adiposity) has been linked to detrimental alterations
in hepatic metabolism, manifested by a set of obesity and related comorbidities,
such as dyslipidemia, insulin resistance, type 2 diabetes, and liver steatosis
(Guilherme et al., 2008; van der Poorten et al., 2008). Obesity is a worldwide
epidemic, with the prevalence of overweight and obese individuals dramatically
increasing in Western developed countries (Bray & Bellanger, 2006). Obesity
results from a positive caloric intake associated with low levels of physical activity,
interacting or not with genetic factors, and leads to adipocyte hypertrophy and
visceral adiposity accumulation (Lopes et al., 2016; Ye et al., 2007). In this
context, a pathological increase of visceral adiposity has been referred as
adiposopathy or “sick fat” (Bays et al., 2008), which usually results in adverse
metabolic consequences via biochemical processes involved in lipid uptake,
esterification, lipolysis and adipogenesis (Jacobs et al., 2016; Lopes et al., 2016).
Generally, “sick fat” secretes high levels of NEFA and glycerol into circulation as
a result of an increase or excessive basal adipocyte lipolysis (Frühbeck et al.,
2014), typically observed in obese individuals with adiposopathy (Jacobs et al.,
2016; Lopes et al., 2016). The NEFA transport across the membrane is facilitated
by several membrane proteins, including fatty acid binding proteins and fatty acid
translocase (FAT/CD36) (Frühbeck et al., 2014), which play an important role in
fatty acids uptake and intracellular lipid metabolism (Zhou et al., 2012). On the
other hand, the expression of aquaglyceroporin 7 (AQP7), the main glycerol efflux
channel (Rodríguez, Catalan, Gomez-Ambrosi, & Frühbeck, 2011), seems to be
elevated in the VAT of obese individuals reflecting an increase of lipolysis-derived
4
Chapter I. General Introduction
5
Chapter I. General Introduction
proteins also have an important role in the process, strongly reinforcing the link
between inflammation and metabolic adipocyte deregulation. Leptin, a product of
the ob gene, was discovered as an adipocyte-specific secreted protein that
regulates food intake and energy expenditure in an endocrine manner (Zhang et
al., 1994). Moreover, several other adipokines are secreted by WAT with
important functions involved in the regulation of nutrient metabolism, energy
expenditure, insulin sensitivity and inflammatory response (Choe et al., 2016;
Flores et al., 2006). However, the production and secretion of adipokines by VAT
are dysregulated in obesity (Choe et al., 2016; Gollisch et al., 2009; Lara-Castro
et al., 2006). For example, some studies reported that circulating adiponectin
(adipQ) levels were reduced in obese individuals (Lara-Castro et al., 2006) and
inversely correlated with the degree of adiposity and insulin resistance (Choe et
al., 2016), while leptin levels were elevated (Gollisch et al., 2009). On the other
hand, hypothalamic leptin resistance aggravates obesity status through appetite
control inhibition and lipid oxidation (Choe et al., 2016). Ghrelin was firstly
discovered in stomach as a ligand of the growth hormone secretagogue receptor
(GHS-R), which is expressed in WAT (Tsubone et al., 2005). Through its
receptor, ghrelin acts as a growth hormone (GH) releasing peptide with important
roles in appetite stimulation, energy and glucose homeostasis, autophagy and
immune function (Dixit et al., 2004; Mao et al., 2015; Tsubone et al., 2005).
Therefore, circulating levels of ghrelin may function as an adiposity signal that
contributes to weight regain in obese subjects as observed by its increased
secretion during weight loss (Aas et al., 2009). Under obese conditions,
adiposopathy has been associated with the development of inflammation by
increasing secretion of various pro-inflammatory chemokines and cytokines, such
as tumor necrosis factor (TNF)-α, interleukin-6 (IL-6) and monocyte chemotactic
protein (MCP-1) (Lopategi et al., 2016; Lumeng et al., 2007). Moreover, the
macrophage content of VAT is positively correlated with both adipocyte size and
body fat mass, and the expression of pro-inflammatory cytokines, such as TNF-
α, is mostly derived from macrophages rather than adipocytes (Weisberg et al.,
2003). Along with the increased number of macrophages in “sick” VAT, obesity
has been linked to a phenotypic switch from an anti-inflammatory “M2”
6
Chapter I. General Introduction
7
Chapter I. General Introduction
humans (Alkhouri et al., 2010) has been reported. In addition, the autophagic flux
seems to be increased in visceral fat of obese individuals (Kovsan et al., 2011)
as this process is required for adipocyte differentiation (Sarparanta et al., 2016).
Markers of autophagy are correlated with whole body adiposity, visceral fat
distribution, and adipocyte hypertrophy (Sarparanta et al., 2016).
Given the social, health and economic impact of obesity, clinicians and
researchers have focused their attention on a variety of preventive and
therapeutic countermeasures to antagonize such phenomenon. These include
the development of many pharmacological agents and chemicals and, on the
other hand, the study of the powerfulness that life style changes have on the
prevention, attenuation and reversion of obesity-associated features. Similarly to
its impact against many other pathological disorders, including
neurodegenerative, cardiovascular and metabolic diseases (Bertram et al., 2016;
Safdar et al., 2016), physical exercise is one of the most powerful lifestyle
interventions used to prevent and/or mitigate overweight and visceral adiposity
accumulation associated with obesity (Bajer et al., 2015; Way et al., 2016). Even
when body weight or visceral adiposity is not reduced, physical exercise has
significant impact on metabolic health. Accumulating evidence reveal that
physical exercise induced favorable metabolic and inflammatory adaptations in
VAT, strengthening the metabolic relevance of adipose tissue on whole-body
adaptations to physical exercise and being a promising direct target in the
treatment of obesity and associated disorders (De Matteis et al., 2013; Giles et
al., 2016; Hirshman et al., 1989; Holland et al., 2016; Stanford et al., 2015;
Tanaka et al., 2015). Moreover, it has been reported that physical exercise-
induced myokines release mediates some physiological adaptations in VAT,
including the modulation of a brown adipocyte-like phenotype (Rodríguez et al.,
2016). In the present dissertation, we focused in the cross-talk between skeletal
muscle and WAT, and in the potential mediators of this process in an attempt to
have a more global view of the benefits of physical exercise on the obesity-related
underlying pathways.
Although structural and functional differences between white and brown adipose
tissues, they have a remarkable plasticity and can acquire features of one another
8
Chapter I. General Introduction
under specific physiological conditions and stimuli (Wu et al., 2014), which
include physical exercise. Therefore, white adipose cells can initiate adaptive
responses to physical exercise and acquire a brown adipocyte-like phenotype
(Bostrӧm et al., 2012; Nakhuda et al., 2016; Wu et al., 2014). The browning
process relates to an increase in mitochondrial density and function (Laye et al.,
2009; Sutherland et al., 2009; Xu et al., 2011), and to an uncoupling of the
oxidative phosphorylation (OXPHOS) caused by the increase of uncoupling
protein-1/thermogenin (UCP1) expression. Morphologically, brown adipocyte-like
phenotype cells are characterized by the presence of small lipid droplets typical
of brown adipocytes (Cao et al., 2011). Animals-based studies showed that the
expression of browning genes (e.g. Ucp1 and Prdm16) increases in VAT after a
chronic ET (Tiano et al., 2015; Wu et al., 2014) or voluntary wheel running (Cao
et al., 2011; Tiano et al., 2015), which suggests that physical exercise enhances
brown adipocyte progenitor cells in adipose tissue. One potential mechanism
underlying the development of brown adipocyte-like phenotype includes the
myokines secreted by exercised skeletal muscle (Bostrӧm et al., 2012). In fact,
some skeletal muscle-derived myokines, e.g. irisin and IL-6, exert endocrine
effects on adipocytes as positive regulators of brown adipocyte-like phenotype
(Bostrӧm et al., 2012; Cao et al., 2011; Knudsen et al., 2014), and thus, emerged
as new potential candidates to treat obesity and related disorders. However, to
our best knowledge, the underlying mechanisms of physical exercise-induced
myokines release and its potential signaling influence on WAT metabolism is still
a matter of debate in the context of obesity. An increasing number of studies
demonstrate that physical exercise exerts important effects by reducing visceral
adiposity and large-sized adipocytes, which consequently improves hypoxia-
responsive markers, e.g. HIF-1α and VEGF, in obesity (Baynard et al., 2012; Yan
et al., 2012). Small-sized adipocytes have been associated with a positive impact
on the production and secretion of adipose-derived hormones, including leptin
and adipQ, which are involved in the regulation of several physiological functions,
such as energy balance, appetite, inflammation, and metabolism (Choe et al.,
2016). Short-term ET-induced decreases in leptin levels were associated with fat
mass loss (Miyatake et al., 2004) and long-term of ET (more than 12 weeks) had
9
Chapter I. General Introduction
10
Chapter I. General Introduction
Existing evidence showed that physical exercise increased the expression of VAT
mitochondrial content and biogenesis markers (Laye et al., 2009; Sutherland et
al., 2009; Xu et al., 2011), such as peroxisome proliferator-activated receptor
gamma coactivator 1-alpha (PGC-1α) and mitochondrial transcript factor A
(TFAM) (Sutherland et al., 2009; Xu et al., 2011). In addition, the protein and gene
expression of OXPHOS subunits increased after voluntary free-wheel and ET in
DIO mice (Xu et al., 2011), as well as in hyperphagic obese rats (Laye et al.,
2009). These physical exercise-induced benefits in the metabolism of VAT
mitochondria seem to be explained by increased 5' AMP-activated protein kinase
(AMPK) activation (Canto et al., 2009). The AMPK activation has been associated
with increases of PGC-1α expression, which stimulates mitochondrial biogenesis
(Chen et al., 2015), and also potentially shifts adipocyte metabolism toward fat
utilization instead of storage (Chen et al., 2015). These metabolic effects of
physical exercise are regularly accompanied by a reduction of the inflammatory
status (Goto-Inoue et al., 2013; Oliveira et al., 2011). The anti-inflammatory
impact of regular physical exercise is well documented (Gollisch et al., 2009;
Jenkins et al., 2012; Kawanishi et al., 2015), and rely on several modulatory
effects, such as decreased expression of pro-inflammatory cytokines (TNF-α and
IL-6) and macrophage recruitment and infiltration (Gollisch et al., 2009; Kawanishi
et al., 2010), independently of body weight reduction (Vieira, Valentine, Wilund,
Antao, et al., 2009). Furthermore, physical exercise increases “M2” macrophages
activation, which was proposed as a potential mechanism by which exercise
reduces inflammation in adipose tissue (Kawanishi et al., 2010). The “M2”
macrophages release anti-inflammatory cytokines and are associated with
increased FFA oxidation and decreased availability of potentially toxic lipid
species. Interestingly, Kawanishi et al (Kawanishi et al., 2010) demonstrated that
mice feed with DIO and submitted to ET exhibited reduced pro-inflammatory
cytokines in epididymal WAT even without reductions on fat mass. Those
changes were associated both with a suppression of macrophages infiltration and
with macrophages phenotype switch from M1 to M2 (Kawanishi et al., 2010). The
specific changes on fatty acid profile in adipose tissue triglycerides may also
be an important adaptation induced by physical exercise. In fact, this may
11
Chapter I. General Introduction
contribute to the attenuation of the inflammatory state as some fatty acids have
been described to be involved in the inflammatory process (Oliveira et al., 2015;
Vaughan et al., 2015). In fact, ET programs seem to induce fatty acids profile-
specific changes in WAT triglycerides by increasing the percentage of long chain
and PUFA (Petridou et al., 2005; Thorling & Overvad, 1994; Wirth et al., 1980),
possibly due to stimulated chain elongation, polydesaturation and/or depressed
monodesaturation. Therefore, physical exercise-mediated fatty acids profile
changes may represent a relevant mechanism to unrevealing the anti-
inflammatory effects of physical exercise. The improved inflammatory conditions
induced by physical exercise have also been associated with positive remodeling
in VAT mass. This putative association is reinforced by studies showing that
physical exercise reduced autophagy activity (Tanaka et al., 2015) and adipose
progenitors (Sertie et al., 2013), as well as increased the expression of anti-
apoptotic markers in VAT (Sertie et al., 2013). Both autophagy and apoptotic-
related cell death tend to dampen inflammation (Sarparanta et al., 2016), which
may represent an important mechanism by which physical exercise improve
inflammation in obesity. So far, only few studies addressed this issue and the
evidence for the effectiveness of physical exercise on these mechanisms is
scarce. Moreover, the role of mitochondria in the inflammation, autophagy and
apoptosis has been recently discussed (Vieira-Potter et al., 2015) and the
physical exercise-mediated mitochondrial functional improvements have been
reported. Therefore, the potential effects of physical exercise on the interaction
of these processes might provide new information regarding the mechanisms
underlying whole adipose tissue metabolism and mitochondrial function and
dynamics.
12
Chapter I. General Introduction
2. Aims
This general purpose encompasses specific objectives designed for each original
study, which are included in the third chapter of this thesis, as follows:
Study 1
i) adiposopathy-related features;
ii) hypoxia-related markers (HIF-1α and VEGF) in eWAT;
iii) circulating and eWAT adipokines content (total adipQ, high molecular
weight (HMW) adipQ and leptin);
iv) circulating ghrelin content and protein expression of growth hormone
secretagogue receptor (GHS-R) in eWAT.
13
Chapter I. General Introduction
Study 2
Study 3
Study 4
To analyse the impact of physical exercise on VAT fatty acids profile and to
ascertain whether these exercise-induced changes in specific FA have significant
repercussions on the inflammatory response in VAT of HFD feeding rats. In
particular, the effects of VPA and ET on:
14
Chapter I. General Introduction
Study 5
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CHAPTER II. Theoretical Background
Chapter II. Theoretical Background
Review Article
Submitted
31
Chapter II. Theoretical Background
Abstract
The white adipose tissue (WAT) is well known for its capacity to store and release
energy, and therefore has a detrimental role in obesity and related pathological
conditions. Physical exercise (PE) adaptations affects not only skeletal muscle
but also other non-contractile organs, such as WAT improving systemic
metabolism and whole-body fat mass. This review summarizes current
knowledge of the cellular and molecular mechanisms involved in PE-induced
myokines signaling to “browning” adipose tissue as well as the counteractive
effects against obesity-induced adiposopathy. A complete understanding of the
effects of PE on obesity is important to improve preventive and therapeutic
strategies to combat the increasing incidence of obesity and its complications.
32
Chapter II. Theoretical Background
Introduction
33
Chapter II. Theoretical Background
response, energy balance, as well as lipid and glucose metabolism (Harms &
Seale, 2013; Rodriguez et al., 2015). The WAT is primarily composed of white
adipocytes and is distributed throughout the body with two representative types,
the subcutaneous (SAT) and the visceral adipose tissue (VAT). The SAT is
located under the skin and provides insulation from heat or cold. On the other
hand, VAT can be found around internal organs (e.g. stomach, liver, intestines
and kidneys) (Guilherme et al., 2008). Visceral adipose tissue, unlike SAT, is
anatomical linked to liver via the portal vein providing non-esterified fatty acids
(NEFA) and adipokines/cytokines directly into liver (Bjorntorp, 1990; Rodriguez
et al., 2014). Therefore, an excessive VAT (or visceral adiposity) has been linked
to a detrimental alterations in hepatic metabolism, manifested by a set of obesity
and related comorbidities, such as dyslipidemia, insulin resistance, type 2
diabetes, and liver steatosis (Guilherme et al., 2008; van der Poorten et al., 2008).
34
Chapter II. Theoretical Background
The dynamic of WAT in response to distinct stimulus, such as HFD (or nutrient
availably) or PE (Applegate & Stern, 1987; Askew & Hecker, 1976; Bailey et al.,
1993; Gollisch et al., 2009; Guerra et al., 2007; Hatano et al., 2011; Miyazaki et
al., 2010; Peres et al., 2005; Sakurai et al., 2010; Sakurai et al., 2007; Sertie et
al., 2013; Speretta et al., 2012; Stallknecht et al., 1993; Vinten & Galbo, 1983;
Zachwieja et al., 1997) rely on hypertrophy (increase of existing adipocytes size)
or hyperplasia (increase of adipocytes number, i.e. adipogenesis) (Jo et al., 2009)
related-mechanisms. In adults-onset obesity, an increased visceral adiposity is
primarily associated with hypertrophy with minimal contribution of fat cell number,
due to a relative lack of progenitor cell activity. This explains VAT accumulation,
particularly of hypertrophic dysfunctional adipocytes and why is associated with
a high risk of onset and progression of obesity-associated diseases (Joe et al.,
2009). On the other hand, data from animal studies showed that distinct models
of PE (voluntary physical activity - VPA and ET) reduced adipocyte size, which
resulted in decreased visceral adiposity both in lean (Hatano et al., 2011; Sakurai
et al., 2010) and in HFD-fed animals (Gollisch et al., 2009; Guerra et al., 2007;
Speretta et al., 2012). From a molecular point of view, data showed that a long-
term ET program decreased the expression of adipocyte differentiation-related
genes (such as Glycerol-3-phosphate dehydrogenase), regulators of G protein
signaling-2, β-catenin and Wtn10b genes in epididymal stromal vascular fraction
(SVF) cells (Sakurai et al., 2010), and increased delta-like 1/pre-adipocyte factor
1 (Dlk1/Pref1) gene expression (Sakurai et al., 2010; Sertie et al., 2013), a
gatekeeper of adipogenesis. Experiments conducted in mouse models that mimic
skeletal muscle adaptation to PE demonstrated that skeletal muscle, particularly
35
Chapter II. Theoretical Background
36
Chapter II. Theoretical Background
37
Chapter II. Theoretical Background
38
Chapter II. Theoretical Background
3.2 Leptin
Leptin functions rely primarily in the suppression of appetite and in the increase
of energy expenditure through metabolism-mediated effects on the endocrine
and autonomic nervous systems (Flores et al., 2006). An increase of circulating
leptin levels is commonly observed in obesity (Gollisch et al., 2009; Linden et al.,
39
Chapter II. Theoretical Background
2014), being these levels positively correlated with body fat and adipocyte size
(Choe et al., 2016). Unfortunately, this chronic condition promotes a
hypothalamic leptin resistance that aggravates the obesity status through the
inhibition of the appetite control and lipid oxidation (Munzberg et al., 2004).
40
Chapter II. Theoretical Background
(Klimcakova et al., 2006; Phillips et al., 2012), which suggest a direct impact of
exercise on leptin secretion and/or protein turnover independent of changes in
body weight.
3.3 Ghrelin
Regarding the impact of exercise on ghrelin levels, conflicting data has been
published. In fact, previous studies observed reduction (Broom et al., 2009;
Broom et al., 2007; Ghanbari-Niaki et al., 2011; King et al., 2011), increment
(Christ et al., 2006; Larson-Meyer et al., 2012; Russell & Misra, 2010) or no
alterations (King et al., 2010) in plasma ghrelin concentrations following an acute
bout of exercise. In addition, ET-based studies reported increases in ghrelin
levels in women who lost weight (Leidy et al., 2004), and absence of changes in
obese individuals (Ebrahimi et al., 2013; Ghanbari-Niaki et al., 2011; Morpurgo
et al., 2003). These inconsistent findings may be due, at least in part, to the
exercise intensity, degree of obesity, gender differences, as well as to time points
of plasma and tissue collection as human’s plasma ghrelin concentration
increases before meal and decreases in the postprandial state (Fathi et al., 2010).
41
Chapter II. Theoretical Background
2014), which may be sufficient to influence metabolic activity and overall energy
balance with positive metabolic consequences (Fatouros et al., 2005). On the
other hand, high-intensity ET results in significant increases in plasma glucose
and in a peak of insulin concentrations immediately post-exercise, which may
have different effects on adipokines and ghrelin secretion (Erdmann et al., 2007).
In addition, lactate production from skeletal muscle during high-intensity exercise,
and its consequent accumulation in plasma, also seems to have an important
impact on the secretion of appetite regulating hormones (Erdmann et al., 2007;
Hsu et al., 2011). Nevertheless, being the mechanisms responsible for
altering adipokines and ghrelin secretion in response to PE still very unclear,
additional studies are clearly needed.
The sympathetic nervous system (SNS) is highly activated during exercise and
affects many of the mechanisms involved in lipid homeostasis (de Glisezinski et
al., 2009). Generally, acute endurance training (ET) increases the circulating
levels of several hormones with the ability to enhance lipolysis and lipid oxidation,
while chronic exercise blunts these hormonal responses, although increasing the
sensitivity to theses hormones, therefore also facilitating lipolysis (McMurray &
Hackney, 2005). As an example, an acute bout of ET induced an increased
expression of the glycerogenic enzymes pyruvate dehydrogenase lipoamide
kinase isoenzyme 4 (PDK4) and phosphenolpyruvate carboxykinase (PECK) in
epididymal WAT (eWAT) (Wan et al., 2012). Moreover, several studies
demonstrated that epinephrine-mediated adrenergic signaling stimulate lipolysis
in WAT, which results in increased fatty acids release during ET and recovery
(Horowitz, 2003). During ET performed at moderate-intensity (40-65% VO2 max),
the levels of catecholamines increase, which induce the phosphorylation of
proteins functioning in β-adrenergic signaling, including the β3-adrenergic
receptor and neuron-derived orphan receptor (NOR1) (Stephenson et al., 2013),
and the downstream lipolysis-regulators perilipin, adipose triglyceride lipase
(ATGL) and hormone-sensitive lipase (HSL) (Hashimoto et al., 2013). In addition,
42
Chapter II. Theoretical Background
some studies reported that high-intensity ET (≥70% VO2 max) increases lipid
metabolism at a higher percentage of maximal capacity, which seems to be
related to i) improved cellular capacity to metabolize lipids, ii) improved oxygen
availability, and iii) attenuated SNS/catecholamine response to submaximal
exercise (Hurley et al., 1986; Rahkila et al., 1980). In the context of obesity, some
studies suggest that obese individuals show a distinct hormonal response
compared to normal weight individuals, including abnormalities of
cathecolamines-induced SAT lipolysis (Marion-Latard et al., 2003; Schiffelers et
al., 2003; Schrauwen et al., 1998). Studies reported that ET have an inhibitory
effect on adipocyte lipolytic activity in overweight and obese individuals (Mardare
et al., 2016) and rats (Pistor et al., 2015). Winder and co-workers (Winder et al.,
1979) showed that the catecholamine levels at rest and in response to exercise
diminished in obese individuals engaged in a chronic ET regimen; however, the
sensitivity of the adipocytes to catecholamines increased via adrenoceptor signal
transduction changes (De Glisezinski et al., 2001; Stich et al., 1999). Similarly,
insulin levels appear to decline with ET (Lange, 2004), but insulin sensitivity rises
(Straczkowski et al., 2001), at least in part, mediated by increases in glucose
transporter 4 (GLUT4) (Caponi et al., 2013; Haczeyni et al., 2015; Marcinko et
al., 2015; Stallknecht et al., 1993). In accordance, it has been also reported that
small white adipocytes from endurance-trained rats express higher levels of
insulin receptors (Craig et al., 1981). Moderate-intensity ET performed during 10
wks improved insulin sensitivity, increased adiponectin and decreased retinol
binding protein (RBP)-4 concentrations, in healthy middle-aged women (Lim et
al., 2008). Moreover, resistance training (RT) programs improved insulin
sensitivity in different populations (Ishii et al., 1998; Miller et al., 1994; Ryan et
al., 2001) and also reduced RBP4 levels in obese subjects (Klimcakova et al.,
2006).
43
Chapter II. Theoretical Background
5.1 Meta-inflammation
It is now clearly recognized that the expression of several critical factors involved
in energy metabolism, such as PPARs, toll-like receptors, and fatty acid-binding
proteins also act as crosslinking agents between metabolic regulation and
inflammatory signaling pathways (Johnson et al., 2012). Moreover, increasing
evidence associate over-nutrition and obesity conditions with a chronic low-grade
inflammatory response, also referred as meta-inflammation (Hotamisligil, 2006).
Accordingly, several studies report that increased plasma NEFA are involved in
the inflammatory response associated to obesity (Frühbeck et al., 2014; Johnson
et al., 2012). The NEFA-mediated activation of IκB kinase β (IKKβ) induces the
translocation of NF-κB to the nucleus and the posterior transcription of its target
genes, which results in an increase of pro-inflammatory cytokines and other
factors, such as TNF-α and MCP-1 (Rodriguez et al., 2014). In fact, it is well
established that the IKKβ/NF-κB pathway is activated in humans (Tantiwong et
al., 2010) and animals (da Luz et al., 2011; Oliveira et al., 2011) in the context of
obesity. The activation of this pro-inflammatory signaling pathway seems to be a
key pathogenic mechanism in obesity and associated disorders (Guilherme et al.,
2008; van der Poorten et al., 2008).
44
Chapter II. Theoretical Background
45
Chapter II. Theoretical Background
5.2 IL-6
The IL-6 belongs to a family of cytokines that collectively have an important role
in immune reactions and metabolism in both adipose tissue and skeletal muscle,
being one of the major cross-talk mediators between these two tissues axis
(Pedersen & Febbraio, 2012).
In adipose tissue, acute physiological elevation of IL-6 was observed immediately
after a single bout of acute exhaustive exercise and after at least 6 hours of rest
(Rosa Neto et al., 2009), suggesting a stimulation of fat metabolism that
contributes to the energy supply for muscle and other tissues (Carey et al., 2006).
In the context of obesity, an excessive production of the adipokine IL-6 has an
adverse effect on glucose metabolism and insulin sensitivity (Golbidi & Laher,
2014). Moreover, an increased IL-6 in obesity has been reported to be a
secondary response to the elevated amount of TNF-α, which has been described
to stimulate IL-6 release (Golbidi & Laher, 2014). Regarding exercise, the Il6 gene
is upregulated in exercised muscle and the transcriptional rate of the Il6 gene is
also markedly enhanced in the exercised skeletal muscle (Keller et al., 2001).
Nevertheless, conflicting data exist in literature. While some studies reported that
circulating IL-6 levels decreased in response to an acute high intensity interval
training (HIIT) session (Leggate et al., 2012) and long-term ET (Baturcam et al.,
2014; Bruun et al., 2006; Donges et al., 2013; Kohut et al., 2006; Oberbach et al.,
2008), others showed that remained unchanged (Klimcakova et al., 2006; Nassis
et al., 2005; Nicklas et al., 2004; Polak et al., 2006) in overweight or obese
individuals engaged in distinct physical exercise routines.
5.3 TNF-α
46
Chapter II. Theoretical Background
47
Chapter II. Theoretical Background
2001), while others reported no changes in the levels of TNF-α (Leggate et al.,
2012; Nicklas et al., 2004; O'Leary et al., 2006; Polak et al., 2006). Accordingly,
although one study showed that TNF-α expression in SAT of severely obese men
and women decreased after 15 wks of aerobic exercise (Bruun et al., 2006); a 12
wks program of ET did not change TNF-α mRNA expression in SAT of obese
individuals, despite a reduction of body weight and body fat (Christiansen et al.,
2010). Furthermore, a 12-wks RT program did not induce alterations on plasma
and SAT TNF-α levels in obese individuals (Klimcakova et al., 2006).
Surprisingly, the combination of 12 wks ET and RT programs resulted in
decreased circulating levels of TNF-α in obese women (Donges et al., 2013), but
no changes were observed after 18 months of intervention (Nicklas et al., 2004).
Taken together, data suggest that the molecular mechanisms and consequences
underpinning the role of the different exercise approaches on TNF-α expression,
in the context of obesity, are clearly elusive and further studies are needed to
clarify the misunderstandings and conflicting results.
48
Chapter II. Theoretical Background
5.4 Hypoxia
49
Chapter II. Theoretical Background
The unsaturation index (UI) of FA was generally either slightly higher in adipose
tissue of trained animals and humans (Bailey et al., 1993; Danner et al., 1984;
Rocquelin & Juaneda, 1981; Thorling & Overvad, 1994; Wirth et al., 1980), or
50
Chapter II. Theoretical Background
unchanged between trained and untrained humans (Danner et al., 1984; Petridou
et al., 2005; Thorling & Overvad, 1994). In contrast, Simko et al (Simko et al.,
1970) reported lower UI in adipose tissue of trained rats. Regarding stearoyl-CoA
desaturase or (∆9-desaturase) activity, which catalyzes the conversion of SFA to
MUFA, most studies found a reduced activity in adipose tissue of trained animals
and humans (Danner et al., 1984; Simko et al., 1970; Wirth et al., 1980).
51
Chapter II. Theoretical Background
6.2 Myokines
52
Chapter II. Theoretical Background
6.3 Irisin
53
Chapter II. Theoretical Background
Nevertheless, data from studies performed with humans are not so consistent.
Some studies showed that chronic ET, RT or the combination of both exercise
models did not affect skeletal muscle FNDC5 (Nygaard et al., 2015; Pekkala et
al., 2013) or circulating irisin (Hecksteden et al., 2013; Pekkala et al., 2013;
Timmons et al., 2012) levels in sedentary healthy individuals or in obese children
(Palacios-Gonzalez et al., 2015). In contrast, other studies revealed an increased
FNDC5 and PGC-1α expression in response to an acute bout of high-intensity
ET and RT (Nygaard et al., 2015) and to 12-wks of combined ET and RT
(Norheim et al., 2014). In fact, although increased skeletal muscle FNDC5 has
been detected without any changes in circulating irisin after chronic exercise
(Pekkala et al., 2013; Vosselman et al., 2015), acute increases in irisin levels
have been reported during exercise in sedentary individuals (Bostrӧm et al.,
2012; Daskalopoulou et al., 2014; Huh et al., 2014; Jedrychowski et al., 2015;
Kraemer et al., 2014; Miyamoto-Mikami et al., 2015; Norheim et al., 2014;
Tsuchiya et al., 2014). Kraemer et al (Kraemer et al., 2014) found that plasma
irisin levels increased during the course of a 90 min (60% of VO 2 max) treadmill
running, reaching the peak values at 54 min; however, returned to baseline levels
immediately post-exercise. Moreover, Norheim et al (Norheim et al., 2014)
reported a peak concentration of irisin after 45 min of ergometer cycling without
a concomitant increase in Fndc5 gene expression, which suggest that increases
in irisin levels during acute exercise may be associated with protein post-
translational modifications.
54
Chapter II. Theoretical Background
Actually, the type, duration, and particularly the intensity of the exercise sessions
also seems to influence the expression of circulating irisin. High-intensity exercise
(80% VO2max for 20 min) promoted greater irisin response compared with low-
intensity exercise (40% VO2max for 40 min) under similar energy consumption
(Tsuchiya et al., 2014). In accordance, it has been demonstrated that circulating
irisin levels increased when the muscle adenosine triphosphate (ATP) levels
acutely dropped, but remain unchanged when muscle ATP content is restored,
suggesting that irisin may contribute to ATP homeostasis (Egan & Zierath, 2013).
Based on this hypothesis, the lack of irisin changes in some studies may be
explained by the exercise intensity as short-term low-to-moderate intensity
exercise induces low ATP depletion (Egan & Zierath, 2013). Of note, plasma irisin
levels seem to be progressively elevated in response to increasing exercise
workloads. In fact, physically active individuals with higher VO 2max showed
greater concentrations of irisin during maximal workload exercise (Daskalopoulou
et al., 2014; Huh et al., 2014; Tsuchiya et al., 2014). Furthermore, irisin levels
were positively correlated with skeletal muscle volume, fat-free mass, glucose,
ghrelin and insulin-like growth factor I (IGF-I) levels (Anastasilakis et al., 2014;
Ellefsen et al., 2014; Huh et al., 2012; Kurdiova et al., 2014).
55
Chapter II. Theoretical Background
On the other hand, in the context of obesity, the related excessive energy uptake
typically leads to abnormal VAT mitochondrial function (Boudina & Graham,
2014). Studies reported that VAT mitochondria exhibited altered morphology
(Cummins et al., 2014) and reduced mitochondrial content and activity in obese
animals (Laye et al., 2009; Rong et al., 2007; Sutherland et al., 2008). In humans,
downregulated OXPHOS content and activity has been correlated with the level
of obesity (Heinonen et al., 2015), while lower mitochondrial DNA copy number
was associated with obesity-related-type 2 diabetes (Dahlman et al., 2006).
56
Chapter II. Theoretical Background
VAT mitochondrial activity in the context of obesity, and to highlight the role of PE
as an effective strategy to counteract the deleterious impact of obesity and
associated disorders in VAT metabolism.
Conclusions
In summary, the pathological increase of visceral adiposity has been associated
with systemic and adipose tissue morphological and metabolic alterations, which
likely contribute to VAT dysfunction in the context of obesity. PE impacts a large
number of molecules/factors involved in the improvement of adipokine profile
secretion, metabolic and inflammatory response at both systemic and local
levels. It also influences tissue browning, mitochondrial biogenesis and activity,
which are determinant factors for improving metabolic function and energy
expenditure of the tissue. These effects may be due to the novel PE-induced
adipokines/myokines secreted by “trained” adipose tissue and skeletal muscle;
nevertheless the understanding of putative adipokines/myokines and their role in
the function of tissues need further research. Moreover, future studies are needed
to understand whether these hypothetical benefits of PE on adipose tissue, which
have been performed primarily in rodent models, occur in human subjects, thus
contributing for counteracting lifestyle-related diseases.
57
Chapter II. Theoretical Background
Legend: IL6, interleukin- 6; NEFA, non-esterified fatty acids; adipQ, adiponectin; HMW adipQ,
high molecular weight adiponectin; RBP4, retinol binding protein 4; TLR4, toll-like receptor 4;
MCP-1, macrophage/monocyte chemotactic protein; HIF-1α, hypoxia-inducible factor 1α; TNF-α,
tumor necrosis factor α; GLUT4, glucose transporter 4; PDK4, pyruvate dehydrogenase lipoamide
kinase isoenzyme 4. a, conflicting data in human studies; ?, conflicting data.
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CHAPTER III. Experimental Work
Chapter III. Experimental Work. Study I
Study I
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Abstract
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1. Introduction
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hormone profile has not been investigated so far. Moreover, as physical exercise
is an advised strategy to counteract obesity-related metabolic abnormalities in
clinical practice, we aimed to study the role of physical exercise as a preventive
and therapeutic strategy against adiposopathy and related endocrine responses
in rats submitted to an isocaloric Lieber-DeCarli pair feeding diet.
All the experiments were approved by the local Institutional Ethics Committee
and followed the guidelines for the care and use of laboratory animals in research
advised by the Federation of European Laboratory Animal Science Association
and Portuguese Act 129/92. Male Sprague-Dawley rats were purchased from
Charles River (L'Arbresle, France), housed in cages (with an enriched
environment) and maintained at controlled environment conditions, 21–22°C; 50–
60% humidity and on 12 h light/dark cycle. To induce an increased visceral
adiposity accumulation, animals (6 weeks of age and weighing 233.9±2.6 g) were
pair-fed the Lieber-DeCarli control/standard (35Kcal% fat, 47Kcal%
carbohydrates, and 18Kcal% protein) or high-fat diets (HFD, 71Kcal% fat,
11Kcal% carbohydrate, and 18Kcal% protein) over 17 weeks. The diets were
purchased from Dyets Inc. with catalog no. 710027 and 712031, respectively.
During the first week, the standard diet was given to all animals as an adaptation
to the liquid feeding. Afterwards, animals were randomly assigned into 4 groups
as follows: standard-diet sedentary (SS), standard-diet voluntary physical activity
(SVPA), high-fat diet sedentary (HS), and high-fat diet voluntary physical activity
(HVPA). After 9 weeks of dietary treatment, half of the SS and HS animals were
submitted to an 8-week ET program, standard-diet endurance training (SET) and
high-fat-diet endurance training (HET) groups, respectively (figure 1).
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VPA: Animals from SVPA and HVPA groups had free access to a running wheel
during 17 weeks and the running distance was monitored daily from a digital
counter between 08.00 and 10.00 hours.
ET: Animals from SET and HET groups were acclimated to the treadmill during
the first week at 15m min-1 and 0% grade for 30 minutes. Then, animals
performed 8 weeks of moderate-intensity ET, 5 days/week-1, 60 minutes/day-1 at
a starting speed of 15 m min-1, which was gradually increased over the training
program until 25m min-1 was reached, based on earlier reports (Magalhaes et al.,
2014). Animals from SS and HS groups were placed on a non-moving treadmill
5 days/week-1 for 60 minutes in order to expose the sedentary animals to the
same environmental conditions but without promoting any physical training
adaptation.
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Energy intake (kilocalories per day) was measured daily while body weight was
monitored once weekly during the study. The feed efficiency ratio was calculated
as [(body weight/energy consumed) x 100] and the Lee Index was calculated as
the cubed root of body weight/nose-anus length. To avoid hypothetical influence
of the last period of exercise, endurance-trained animals were euthanized after
48 hours from the last training session and voluntary exercised animals were
placed in a cage without access to wheel running. All animals were fasted
overnight for 12 hours with access to drinking water. The VAT around internal
organs, including mesenteric (mWAT), retroperitoneal (rWAT) and epididymal
(eWAT), were excised and weighed to calculate visceral adiposity normalized to
body weight. Plasma and eWAT were rapidly stored at -80ºC for further analysis.
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Circulating leptin and ghrelin content: Plasma samples were diluted (1:20) in Tris-
buffered saline and 100µl was slot-blotted into a nitrocellulose membrane. The
slot-blot membranes were blocked and incubated with the specific primary
antibodies anti-leptin (842, Santa Cruz Biotechnology) and anti-ghrelin (134978,
Abcam), followed by an incubation with a solution of horseradish-conjugated anti-
rabbit (2317, Santa Cruz Biotechnology).
Circulating total and HMW AdipQ content: The relative amounts of plasma total
AdipQ and HMW AdipQ were determined in plasma by reducing SDS-PAGE and
nonreducing native PAGE, respectively, by Western blotting. For determination
of total AdipQ concentration, one volume of plasma was mixed with one volume
of reducing sample buffer, heated to 100°C for 8 minutes and separated on a
10% gel under denaturing conditions. For HMW AdipQ determination, one
volume of plasma was mixed with one volume of native sample buffer at room
temperature and separated on an 8% gel under native conditions, as previously
reported (Asada et al., 2007).
Epididymal WAT protein content: eWAT was homogenized in an ice-cold RIPA
lysis buffer supplemented with protease inhibitors using a Polytron homogenizer
for 30 seconds. Sample homogenates were centrifuged (13,000 g for 10 minutes
at 4°C) and the supernatant was harvested. An aliquot of the tissue lysates was
prepared in accordance with Goncalves, Passos, Rocha-Rodrigues, Torrella, et
al. (2014).
Afterwards, proteins were electrophoretically transferred into polyvinyldifluoride
membranes (Millipore) in a tank buffer system and blotted with rabbit anti-
Ob(leptin) (842), rabbit anti-VEGF (152), and rabbit anti-HIF-1α (10790) from
Santa Cruz Biotechnology, and rabbit anti-AdipQ (ab62551) and rabbit anti-GHS-
R (85104) from Abcam. All membranes were stained with Ponceau S to evaluate
the quality of protein transfer. The original membranes containing eWAT proteins
were stripped and reblotted with β-actin (1616, Santa Cruz Biotechnology) as an
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2.7 Statistics
Values are presented as mean ± standard error of the mean (SEM). The normal
distribution was evaluated using the Kolmogorov–Smirnov test. The statistical
differences between groups were performed using a two-way (exercise and diet)
analysis of variance (ANOVA) followed by the Bonferroni multiple comparisons
post hoc tests. Pearson’s correlation coefficients were used to analyze possible
associations between variables. The level of significant difference was set at p <
0.05. Statistical analysis was performed using SPSS 21.0 for Windows (SPSS
Inc., Chicago IL, USA).
3. Results
3.1 Effects of diet and physical exercise on body weight, visceral adiposity and
adipocyte cell-size distribution
As seen in figure 2, the feed efficiency ratio and Lee index remained unchanged
at the end of the study. HFD and VPA did not induce changes in body weight;
however, HFD significantly increased the relative visceral adiposity (to body
weight) and the weight of each WAT depot in HS animals compared to the
standard diet-fed counterparts (SS). ET decreased body weight and visceral
adiposity in both standard and high-fat diet-fed groups compared to the sedentary
counterparts. VPA decreased mWAT weight in both diet types and decreased
rWAT and eWAT only in HFD-fed animals. ET decreased mWAT, rWAT and
eWAT weights in both diet regimens.
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Figure 2: Body weight (A), feed efficiency ratio (B), Lee index (C), visceral adiposity
(D) and relative weights of mWAT, rWAT and eWAT (E). Data are expressed as the
mean±SEM. SS, Standard diet sedentary; SVPA, Standard diet voluntary physical
activity; SET, standard diet endurance trained; HS, high-fat diet sedentary; HVPA, high-
fat diet voluntary physical activity; HET, high-fat diet endurance training. p≤0.05; DxE,
diet and exercise interaction; D, diet effect; E, exercise effect; NS, not significant; avs.SS;
b
vs.HS; cvs.SVPA; dvs.SET; evs.HVPA
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Chapter III. Experimental Work. Study I
3.2 Effects of diet and physical exercise on VEGF and HIF-1α protein
expression in eWAT
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Chapter III. Experimental Work. Study I
Figure 4: The adipose tissue hypoxia-related markers. The protein expression of HIF-
1α (A) and VEGF (B). Representative Western blot images for the indicated protein are
shown below the graph. Data are expressed as the mean±SEM. p≤0.05; DxE, diet and
exercise interaction; D, diet effect; E, exercise effect; NS, not significant, avs.SS; bvs.HS;
c
vs.SVPA;d vs. SET; e vs. HVPA
3.3 Effects of diet and physical exercise on leptin, ghrelin and growth hormone
secretagogue receptor protein expression
HFD increased eWAT leptin levels but did not alter plasma leptin and ghrelin
protein contents. Similarly, VPA did not induce alterations in plasma leptin and
ghrelin levels; however, VPA increased eWAT leptin and growth hormone
secretagogue receptor (GHS-R) protein contents in rats fed with HFD (HVPA
group). ET decreased plasma and eWAT leptin protein content and increased
plasma ghrelin and eWAT GHS-R protein content in both diet regimens (figure
5). Plasma leptin was positively correlated with visceral adiposity (r=0.59;
p=0.003) and negatively correlated with QUICKI (r=-0.587; r=0.003). Plasma
leptin did not correlate with eWAT leptin (p>0.05), and was positively correlated
with body weight (r=0.634; p=0.001), visceral adiposity (r=0.806; p=0.001), and
HOMA-IR (r=0.745; p<0.001) and negatively correlated with QUICKI (r=-0.601;
p=0.002).
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Chapter III. Experimental Work. Study I
3.4 Effects of diet and physical exercise on total and high molecular weight
adiponectin protein expression
As seen in figure 6, HFD decreased plasma total AdipQ and had no significant
impact on HMW protein content compared to standard diet-fed animals. VPA
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Chapter III. Experimental Work. Study I
increased the HMW/total AdipQ ratio and tended to increase HMW in standard
diet-fed animals. The ET increased plasma total AdipQ, HMW protein expression,
and the HMW/total AdipQ ratio in both diet regimens and increased eWAT AdipQ
expression only in the SET group.
Figure 6: Plasma and eWAT adipQ expression. Plasma total adipQ (A), HMW adipQ
form (B), HWM/total adipQ ratio (C) and eWAT adipQ (D) protein expression. Reduced
SDS-PAGE and nonreduced native PAGE plasma proteins were separated and
visualized by native PAGE and Western blotting and representative Western blot images
for the indicated protein are shown below the graph. Data are expressed as the
mean±SEM. p≤0.05; DxE, diet and exercise interaction; D, diet effect; E, exercise effect;
NS, not significant; avs.SS; bvs.HS; cvs.SVPA; d vs. SET; e vs. HVPA
Plasma total AdipQ was negatively correlated with visceral adiposity (r=-0.786;
p>0.001), HOMA-IR (r=-0.771; p<0.001), and insulin (r=-0.423; p=0.0032) and
positively correlated with QUICKI (r=0.731; p<0.001). Plasma HMW AdipQ was
negatively correlated with visceral adiposity (r=-0.540; p=0.004) and insulin (r=-
0.375; p=0.05), and positively correlated with QUICKI (r=0.503; p=0.009). Both
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Chapter III. Experimental Work. Study I
plasma AdipQ and HMW positively correlated with eWAT AdipQ (r=0.781;
p>0.001 and r=0.582; p=0.004, respectively). eWAT AdipQ negatively correlated
with visceral adiposity (r=-0.784; p<0.001).
3.5 Effects of diet and physical exercise on plasma insulin, glucose, HOMA-IR
and QUICKI
Plasma glucose levels remained unchanged after dietary and physical exercise
interventions. Seventeen weeks of HFD and VPA had no significant impact on
plasma insulin levels in standard and high-fat diet-fed animals. The ET reduced
plasma insulin levels in the HET group, although a tendency for decreases was
also observed in the SET group. HOMA-IR, a surrogate index of IR validated
against the clamp technique (Cacho et al., 2008), significantly increased in HS
animals compared to SS animals. In contrast, ET decreased HOMA-IR and
increased QUICKI, an insulin sensitivity index, in both SET and HET groups
compared to the sedentary counterparts (figure 7).
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Chapter III. Experimental Work. Study I
4. Discussion
The modified version of the Lieber-DeCarli diet based on high-fat corn oil was
developed to induce obesity-related pathological disorders, including NASH
(Goncalves, Passos, Rocha-Rodrigues, Torrella, et al., 2014; Li et al., 2015). As
scarce information is available regarding the effects of this Lieber-DeCarli diet
model on morphological and endocrine alterations associated with visceral
adiposopathy, our first goal was to characterize this diet model. When compared
to other diets, the Lieber-DeCarli liquid diet has the advantage of having all
ingredients in water, and thus, energy intake is easily monitored. As an
isoenergetic pair feeding, the total energy intake and the body weight gain can
be efficiently controlled, excluding any effects that may be related to these two
parameters. Studies reported that a pair-fed model with HF feeding did not
produce significant changes in body weight gain (Diaz-Rua et al., 2014; Li et al.,
2015), which is consistent with our findings. Accordingly, Betz et al (Betz et al.,
2012) showed that a pair feeding of low-carbohydrate/HF diets or HFD with
intermediate amounts of carbohydrates had no effect on body weight gain,
whereas the control diet (rich in carbohydrates and low in fat) induced an
increased body weight. In the present study, despite similar energy intake and
final body weight observed in HS animals compared to SS, the significantly
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Chapter III. Experimental Work. Study I
increased visceral adiposity (and each fat depot weight) promoted by the HFD
feeding was consistent with an increased number of large adipocytes. The
concept of adiposopathy or “sick” fat (Bays et al., 2008) emerged and has been
attributed to adipocyte hypertrophy, visceral adiposity accumulation, and/or
ectopic fat deposition, which results in adverse endocrine and immune
consequences leading to several obesity-related metabolic abnormalities
(Jacobs et al., 2016; Lopes et al., 2016). In this context, it has also been
demonstrated that an expansion of VAT can result in hypoxic tissue conditions
with a consequent induction of increased HIF-1α expression in obese rodents (Ye
et al., 2007; Yin et al., 2009) and humans (Virtanen et al., 2002). This affects the
production and secretion of endocrine hormones, such as AdipQ, leptin, ghrelin
(Ye et al., 2007), and peripheral IR (Yin et al., 2009). The adiposopathy observed
in the HS animals were consistent with an increased HIF-1α and leptin in VAT as
well as with a decreased AdipQ in plasma, which is in agreement with other HFD-
induced obesity models (Gollisch et al., 2009; Linden et al., 2014). As reduced
levels of plasma total AdipQ are related to insulin-resistant conditions, such as
obesity (Berg et al., 2002), signs of IR were observed in HS animals. Moreover,
when HOMA-IR values were normalized to plasma AdipQ levels (data not
shown), i.e. adjusted to the degree of adiposity (Vilela et al., 2016), HFD-fed rats
still exhibited higher HOMA-IR values compared to standard diet-fed
counterparts. Altogether, our data suggest that this modified version of the Lieber-
DeCarli pair feeding diet induces morphological and metabolic characteristics
typical of adiposopathy observed in other HFD-induced obesity models.
Physical exercise has been well recognized as a strategy to induce body weight
and visceral adiposity reduction as it lowers the energetic balance by increasing
energy expenditure (Gollisch et al., 2009; Linden et al., 2014; Reseland et al.,
2001). As reported by others (Gollisch et al., 2009; Miyazaki et al., 2010), our
data revealed that physical exercise prevented and reverted the large-sized
adipocytes under HFD feeding conditions. Furthermore, the effects of ET were
more evident than those prompted by the VPA, as the number of small- to
medium-sized adipocytes increased and the hypoxia-responsive markers
improved (i.e. decreased HIF-α and increased VEGF) in both SET and HET
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Chapter III. Experimental Work. Study I
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Chapter III. Experimental Work. Study I
The total plasma ghrelin levels are usually altered during chronic alteration of
energy status with low levels in “simple” obesity and high levels after weight loss
(Espelund et al., 2005; Leidy et al., 2004). Here, we confirmed previous findings
that reported an inverse correlation between ghrelin levels and body weight,
visceral adiposity, and adipocyte size (Purnell et al., 2003). So far, there is a
scarcity of knowledge regarding the mechanisms underlying the effects of ghrelin
on body weight and visceral adiposity (Mihalache et al., 2016). Moreover, ET
ameliorated ghrelin production, with a concomitant decrease in body weight and
adiposopathy, which are in accordance with Tiryaki-Sonmez et al. (Tiryaki-
Sonmez et al., 2013), although in contrast with others (Ghanbari-Niaki et al.,
2011; Mason et al., 2015). The ghrelin effects on growth hormone release and
appetite are mediated through its receptor, the GHS-R (Mihalache et al., 2016).
The reduced visceral adiposity observed in old Ghsr-null mice is due to energy
expenditure but not due to food intake or physical activity (Sun, 2015). However,
in the present study, GHS-R protein expression increased in response to VPA
and ET in HFD-fed animals, which seems to be associated with reduced
adiposopathy. Additional studies are clearly needed to further understand the
mechanistic involvement of GHS-R in this exercise-induced modulation of
obesity-related constraints.
5. Conclusions
Taken together, data from our study showed that physical exercise reverted HFD-
induced pathoanatomical features of adiposopathy (e.g., adipocyte hypertrophy
and visceral fat accumulation). Moreover, accompanying the improvement in
these anatomical manifestations of adiposopathy, an enhancement in adipocyte
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Chapter III. Experimental Work. Study I
and adipose tissue function was also found, as observed by increases in AdipQ,
HMW AdipQ, ghrelin, and GHS-R protein, along with a decrease in leptin, which
likely contributed to improved insulin sensitivity. From a clinical perspective, it can
be assumed that physical exercise potentially attenuates adiposopathic
alterations implicated in metabolic diseases. Nevertheless, additional research is
required to further clarify the implications of physical exercise in the obesity
context.
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Study II
doi: 10.1016/j.lfs.2016.09.023
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Abstract
browning, UCP1
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1. Introduction
White and brown adipocytes are two distinct types of fat cells with opposite
functions. White adipocytes are highly adapted to store excess energy, whereas
brown adipocytes utilize fatty acids for generating heat via mitochondrial
uncoupling protein 1 (UCP1) (Harms & Seale, 2013), thereby dropping the
availability of substrates for storage in WAT. Moreover, two populations of brown-
like fat cells with thermogenic properties have been identified, the “classical
brown” and “beige” adipocytes (Harms & Seale, 2013). Interestingly, the
abundance of these two brown cell types can be induced in response to
appropriate physiological stimuli, such as chronic cold exposure, irisin,
peroxisome proliferator-activated receptor γ (PPARγ) agonists or β-adrenergic
stimulation (Bostrӧm et al., 2012; Morton et al., 2016; Tiano et al., 2015; Wu et
al., 2014), increasing whole-body metabolic rate, and therefore preventing diet-
induced obesity (DIO) and related diseases (Harms & Seale, 2013; Wu et al.,
2014).
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2012; Weigert et al., 2014), including liver and visceral adipose tissue (VAT).
Accordingly, several studies have focused on myokines released from muscle
during and immediately after exercise, such as interleukins (IL)-6, -8 and -15,
leukemia inhibitory factor (LIF) and fibroblast growth factor 21 (FGF21) (for a
review see ref. Pedersen & Febbraio, 2012; Weigert et al., 2014). In addition, the
discovery of fibronectin type III-domain containing 5 (FNDC5) as a PGC-1α-
dependent myokine showed that its derived product secreted into circulation
(irisin) has the ability to drive a brown adipocyte-like phenotype in WAT (Bostrӧm
et al., 2012). In vitro undifferentiated white adipocytes treated with FNDC5
overexpressed UCP1-positive cells (Bostrӧm et al., 2012) and increased
mitochondrial density and gene expression, which led to increased oxygen
consumption, heat loss and greater energy expenditure (Wu et al., 2014).
In this context, a new role for myokines has emerged in the field of exercise-
related adaptations; however little is still known regarding the physiological
impact of myokines on the adaptive response to chronic exercise, and particularly
on the brown adipocyte-like phenotype stimulation of VAT in a rat model of DIO.
Thus, using this nutritional model of obesity in rats, the main goal of the present
study was to analyze the role of voluntary physical activity (VPA) and endurance
training (ET) as hypothetical modulators of an increase in brown-like phenotype
in WAT.
2.1 Reagents
Bradford reagent, Laemmli sample buffer and block reagent were purchased from
Bio-Rad (Hercules, CA, USA). Chemiluminescent reagent ECL-Plus™ from GE
Healthcare, Amersham Biosciences (Buckinghamshire, U.K.). Polyvinylidene
difluoride membranes (Immobilon-N) were purchased from Merck Millipore
Corporation (Bedford, MA, USA) and nitrocellulose membranes were obtained
from Whatman, Protan (Pittsburgh, PA, USA). Protease inhibitor and
phosphatase inhibitor cocktails were purchased from Sigma-Aldrich (Barcelona,
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Chapter III. Experimental Work. Study II
Energy intake (kilocalories per day) was measured daily while body weights were
monitored once weekly during the period of the present study.
The study was approved by the Institutional Ethics Committee of the Faculty of
Sport, University of Porto and followed the guidelines for the care and use of
laboratory animals in research advised by the Federation of European Laboratory
Animal Science Association (FELASA).
ET program: Eight weeks after the diet intervention, SET and HET animals were
submitted to a chronic ET program. Initially, animals were progressively
acclimated to the motor-driven treadmill (Le8700, Panlab Harvard Apparatus) for
5 days per week at 15 m min-1 and 0% grade during 30 min. Then, animals
performed a moderate intensity ET program (corresponding to 50-60% of VO2
max) consisted of 5 sessions per week and 60 min per day at a starting speed of
15 m min-1, which was progressively increased over the training program until 25
m min-1 was reached (Magalhaes et al., 2014; Sertie et al., 2013). In order to
understand the therapeutic effects of ET, animals started the ET program 8
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Chapter III. Experimental Work. Study II
weeks after the diet treatment since at this time point HFD-fed animals already
exhibited obesity-related metabolic features. Sedentary animals (SS and HS
groups) were placed on a non-moving treadmill during the training sessions to be
exposed to the same environmental conditions, but without promoting any
physical training adaptation. In order to eliminate acute effects of exercise, SET
and HET animals were sacrificed 48h after the last training session.
All animals were fasted overnight for 12 h with access to drinking water before
sacrifice. Animals were weighed at the day of sacrifice and anesthetized with
ketamine (90 mg.kg-1) and xylazine (10 mg.kg-1). Blood was collected and plasma
separated by centrifugation 3000 g for 15 min at 4 ºC. Afterwards, visceral WAT
depots (mesenteric, epididymal and retroperitoneal) and skeletal muscle
(gastrocnemius and soleus) were excised and weighted. Epididymal fat pad and
both gastrocnemius and soleus were processed for analysis as described below.
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Citrate synthase (CS) activity was measured in soleus homogenate using the
method proposed by Coore et al (1971). Briefly, CoA-SH released from the
reaction of acetyl-CoA with oxaloacetate was measured by its reaction with a
colorimetric agent [5, 5-dithiobis (2-nitrobenzoate)]. The change in color was
monitored spectrophotometrically at 412 nm.
Plasma samples were diluted (1:20) in Tris buffered saline (TBS; 100 mmol Tris,
1.5 mmol NaCl, pH 8.00) and 100 µl was slot-blotted into a nitrocellulose
membrane (Whatman, Protan). The slot-blot membranes were blocked with 5%
(w/v) dry nonfat milk in TBS with 0.05% Tween 20 (TBS-T) and then incubated
for 30 min with rabbit anti-monoclonal FNDC5 diluted 1:1,000 in 5% (w/v) nonfat
dry milk in TBS-T. Afterwards, membranes were incubated with a solution of
horseradish-conjugated anti-rabbit antibody diluted at 1:10,000. The blots were
detected by ChemiDoc™ XRS+System (Bio-Rad Laboratories, Inc., Hercules,
CA, USA) and band densities were quantified using Image Lab™ software 262
5.2.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Control for protein loading
was confirmed by Ponceau S staining.
The gastrocnemius muscle and epididymal white adipose tissue (eWAT) were
homogenized in ice-cold RIPA buffer containing 50 mmol/L TrisHCl (pH 7.40), 1
mmol/L EDTA, 0.2% sodium dodecyl sulfate, 0.2% deoxycholate and 1% Triton
X-100 supplemented with protease and phosphatase inhibitors using a Polytron
homogenizer for 30 sec. Samples homogenates were centrifuged (13,000 g for
10 min at 4°C) and the supernatant was harvested. An aliquot of the tissue lysates
was used to determine the concentration of protein in each sample by Bradford
method. Samples were prepared in 2×Laemmli buffer containing 710 mmol/L β-
mercaptoethanol and heated in a boiling water bath for 5 min. To determine the
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total-AMPK, pAMPK (62 kDa), IL-6 (28 kDa) and FNDC5 (25 kDa) contents in
gastrocnemius, aliquots containing 25 μg were subjected to SDS-PAGE and then
transferred to polyvinyldifluoride membranes (Millipore). To determine PGC-1α
(91 kDa), SIRT1 (75 kDa), SIRT3 (28 kDa), FNDC5 (25 kDa), UCP2 (33 kDa),
UCP1 (33 kDa), BMP7 (55 kDa) and β-actin (42 kDa) contents in eWAT, 50 μg
protein were loaded onto the gels. All primary antibodies were at 1:1,000 dilution.
The blots were detected by ChemiDoc™ XRS+System (Bio-Rad Laboratories,
Inc., Hercules, CA, USA) and band densities were quantified using Image Lab™
software 262 5.2.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes
were stripped and re-probed with an anti-β-actin antibody, as an internal control.
The values were obtained by dividing the density of the band of interest by that
of either β-actin (as indicate in the figure legends) from the same blot.
Total RNA of eWAT was isolated using TRIzol® reagent as described by the
manufacturer. The concentration and purity of RNA were assessed using
NanoDrop™ 1000 spectrophotometer by reading absorbance at 230 nm, 260 nm
and 280 nm (Thermo Scientific, CA, USA). One point five micrograms of total
RNA worked as template for cDNA production using the NZY First-strand cDNA
Synthesis Kit (MB12501), which included a combination of random hexamers and
oligo(dT) primers. Quantitative Real-time PCR (RT-qPCR) was conducted using
SYBR® Green PCR Master Mix on a Step One Plus thermocycler (Applied
Biosystems) using paired reverse and forward primers as shown in table 1. Each
sample was assayed in a 12 µL reaction in duplicate. If the duplicate contained a
cycle threshold (CT) standard deviation of >0.5, it was reassayed. All reactions
were performed under the same conditions 95 ºC for 3 min, 40 cycles of 95 ºC
for 15 sec and 60 ºC for 1 min. The data were analyzed using GAPDH as the
internal control with the cycle threshold (2-ΔΔCT) method as recommended by
Applied Biosystems.
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Values are presented as mean ± standard error of the mean (SEM). The effect of
diet and exercise was analysed by a two-way analysis of variance (ANOVA). The
Bonferroni post hoc test was applied for comparisons between groups and
differences were considered significant at p≤0.05. Pearson’s correlation
coefficients were used to analyze possible associations between variables.
Statistical analysis was performed using SPSS 15.0 for Windows (SPSS Inc.,
Chicago IL, USA).
3. Results
3.1 Effects of exercise and diet on body weight, energy intake and visceral
adiposity
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Chapter III. Experimental Work. Study II
Figure 1: Body weight, energy intake and relative fat depot weights. The mean body
weight gain (A) and cumulative energy intake, expressed as kcal per day (B), and the
relatives weights of eWAT (C), mWAT (D) and eWAT (E) fat depots. Data are expressed
as the mean ± SEM. DxE, diet and exercise interaction; D, diet effect; E, exercise effect;
NS, not significant; avs.SS; bvs.HS; cvs.SVPA; d, vs. SET; e vs. HVPA
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Figure 2: Adipocyte-size profiling of adipocytes from WAT (A and C). Bar graphs
representing the percentage (%) of adipocytes with area below 2000 and higher or equal
2000 μm2 (B) and, below 5000 and higher or equal 5000 μm2 (D) Adipocytes areas were
determined from 4 sections per rat. Representative images of haematoxylin and eosin
staining of visceral WAT (B). Data are expressed as the mean±SEM. DxE, diet and
exercise interaction; D, diet effect; E, exercise effect; NS, not significant; avs.SS; bvs.HS;
c
vs.SVPA; d, vs. SET; e vs. HVPA *significant between areas in the same group.
3.3 Effects of diet and physical exercise on skeletal muscle characteristics and
on circulating irisin
Long-term HFD did not affect neither soleus, gastrocnemius nor total muscle
mass/body weight ratio (figure 3). On the other hand, VPA increased the relative
gastrocnemius weight and total muscle mass in SVPA group. Neither HFD nor
VPA interventions have impact on muscle CS activity, a validated marker of
oxidative adaptation to ET program. In contrast, 8-week ET increased the relative
soleus, gastrocnemius and muscle mass weights, as well as the CS activity in
SET and HET groups when compared with their sedentary counterparts.
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Figure 3: Soleus (A) and gastrocnemius (B) muscle weight and the total muscle mass-
body weight ratio (C), and citrate synthase (CS) activity (D). Data are expressed as the
mean±SEM. DxE, diet and exercise interaction; D, diet effect; E, exercise effect; NS, not
significant; avs.SS; bvs.HS; cvs.SVPA; d, vs. SET; e vs. HVPA
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Chapter III. Experimental Work. Study II
Skeletal muscle FNDC5 correlated positively with muscle mass (r=0.48, p=0.02,)
and negatively with body weight (r=-0.70, p<0.001), total VAT depots (r=-0.64,
p=0.001). Circulating irisin protein content remained similar within groups. A
negative correlation between circulating irisin and total VAT depots (r=-0.54,
p=0.01) and eWAT (r=-0.49, p=0.023) was observed.
3.4 Effects of diet and physical exercise on eWAT beige and brown-specific
markers
As depicted in figure 5, long-term HFD per se and VPA interventions did not
affect eWAT bone morphogenetic protein (BMP7) gene or protein expression,
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Chapter III. Experimental Work. Study II
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Chapter III. Experimental Work. Study II
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Chapter III. Experimental Work. Study II
correlations between FNDC5 protein content and body weight (r=-0.71, p<0.001)
and total VAT depots (r=-0.72, p<0.001) were observed.
Figure 6: The protein expression of PGC-1α (A), SIRT1 (B), SIRT3 (C), UCP2 (D) and
FNDC5 (E) protein expression on eWAT and the representative Western Blot images
(F). Data are expressed as the mean±SEM. DxE, diet and exercise interaction; D, diet
effect; E, exercise effect; NS, not significant; avs.SS; bvs.HS; cvs.SVPA;
4. Discussion
The main findings of the present study suggest that rats submitted to a Lieber
DeCarli-isoenergetic diet developed several VAT morphological and metabolic
alterations, which strongly resemble the pathological features observed under
obesity-related scenarios (Lieber et al., 2004; Tiano et al., 2015; Wu et al., 2014).
Importantly, ET for 8 weeks was able to antagonize some of the VAT alterations
induced by the HFD by stimulating IL-6 and FNDC5 production by skeletal muscle
which may be involved in the “brown-like” phenotype of VAT from rats fed a HFD.
Skeletal muscle has been considered a major endocrine organ in the human
body. In fact, besides its local paracrine-mediated impact on several signaling
pathways involved in muscle structure and metabolism, it has been postulated
that skeletal muscle also produces and releases myokines, which work in an
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Chapter III. Experimental Work. Study II
During chronic HFD feeding adipocytes expand in size and in number altering
whole VAT distribution and function, accordingly with our findings, which was
prevented and attenuated (VPA and ET, respectively) after physical activity
interventions, also reported by others (Sertie et al., 2013; Wu et al., 2014). A
small-sized adipocyte may positively influence the secretion of appropriated
levels of adipokines, thus affecting the whole-body metabolism and
neuroendocrine control of behavior that are related to feeding (for ref. see
Guilherme et al., 2008).
The IL-6 is considered the prototype myokine (Knudsen et al., 2015; Wallenius et
al., 2002; Weigert et al., 2014), with its circulating levels being acutely elevated
in response to exercise. IL-6 has the ability to promote metabolic alterations in
skeletal muscle itself and in other organs, including WAT, in an endocrine manner
(Pedersen & Febbraio, 2012). Due to the pleiotropic properties of IL-6 (Weigert
et al., 2014), data demonstrated that whole-body Il6-knockout mice are more
prone to develop obesity (Wallenius et al., 2002) and that endogenous IL-6
seems to play an important role preventing HFD-induced insulin resistance in
mice (Knudsen et al., 2015). The beneficial endocrine effects of increased IL-6
levels in response to exercise on distinct organs, such as WAT may be related to
the increases in AMPK activity (Bijland et al., 2013; Knudsen et al., 2015;
Ruderman et al., 2006). In fact, AMPK is a fuel-sensing enzyme that among other
actions increases its activity in cellular depressed energy states. Considering that
the increase in IL-6 concentration correlates temporally with increases in AMPK
activity, some studies suggest that IL-6 is involved in AMPK activation during the
later stage of exercise when the energetic state of skeletal muscle is down
(Bijland et al., 2013; Ruderman et al., 2006). In accordance with (Wu et al., 2014),
data from the present study show that HFD feeding decreased AMPK activity.
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Chapter III. Experimental Work. Study II
Recently, the role of the myokine FNDC5/irisin has also been a matter of intense
debate. This myokine production and secretion seems to induce exercise-based
adaptations in muscle metabolism apparently through AMPK–PGC-1α signaling
cascade (Huh et al., 2014). In accordance with our findings, some studies (Huh
et al., 2014; Wu et al., 2014) have demonstrated that physical activity increased
muscle FNDC5 expression. Moreover, chronic VPA did not alter FNDC5 protein
expression in both diets, which suggests that similarly to AMPK modulation,
exercise distinctly modulated FNDC5 expression. Our findings are also
consistent with other studies (Liu et al., 2015; Norheim et al., 2014; Vosselman
et al., 2015; Zhou et al., 2015) reporting an increase in muscle FNDC5 expression
although no alterations in circulating irisin levels under exercising conditions,
which seems to be related to the acutely increase of irisin levels during exercise.
In fact, Norheim and coworkers (Norheim et al., 2014) found a peak
concentrations of irisin immediately after 45 min ergometer cycling (~1.2-fold).
This acute increase was independent of an increase in FNDC5 mRNA,
suggesting that the observed increase of irisin in plasma is caused by increased
translation of FNDC5 mRNA in skeletal muscle. Thus, it seems that the irisin
generated by endurance exercise training might be used to initiate exercise-
based adaptations in skeletal muscle.
The myokine FNDC5/irisin has received particular interest due its potential to
mediate the browning effects in WAT (Bostrӧm et al., 2012; Wu et al., 2014).
Emerging evidence suggests that a specific beige and/or brown adipocytes
accumulation within WAT might be triggered by skeletal muscle-derived FNDC5
in response to exercise, which produces subsequently positive effects on
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Chapter III. Experimental Work. Study II
Given the pleiotropic effects of IL-6, the participation of this myokine in the
browning process has also been suggested (Knudsen et al., 2014; Li et al., 2002).
In fact, the overexpression of the Il6 gene seems to increase the expression of
thermogenic gene and elevate the protein levels of UCP1 in rat BAT (Li et al.,
2002) and WAT (Knudsen et al., 2014). Accordingly, ET-induced skeletal muscle
IL-6 and FNDC5 expression was associated with the increased transcript levels
of brown genes in non-obese and obese conditions. This ET-induced browning
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Chapter III. Experimental Work. Study II
Previous studies shown that WAT is also able to produce FNDC5 in human and
animals (Moreno-Navarrete et al., 2013; Roca-Rivada et al., 2013; Wu et al.,
2014), supporting the cross-talk between skeletal muscle and adipose tissue.
This cross-talk is of particular interest on obesity conditions as a dysregulation of
secretion and production of adipokines and myokines might contribute to
increased visceral adiposity and consequent obesity-related diseases (Roca-
Rivada et al., 2013). In fact, reduced VAT FNDC5 gene levels were observed in
obese patients with or without type 2 diabetes (Moreno-Navarrete et al., 2013),
suggesting that this adipokine could underlie the obesity-associated lower
amounts of brown or beige adipocytes in human adipose tissue (van Marken
Lichtenbelt et al., 2009). Interestingly, few studies have shown that FNDC5 in
adipose tissue was upregulated after exercise (Roca-Rivada et al., 2013; Wu et
al., 2014), which are in agreement with our findings. However, other reports
shown that FNDC5 seems to be differentially regulated in skeletal muscle and
WAT in response to leptin and fasting (Roca-Rivada et al., 2013; Rodríguez,
Becerril, et al., 2015).
5. Conclusions
Taken together, data suggest that both VPA and ET induced improvements in
obesity-related features, such as reductions in body weight, visceral WAT depots
and adipocyte size, whereas only ET was able to induce myokine production as
well as brown-like morphology and function in eWAT.
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partially via the PGC-1alpha pathway. Mol Cell Endocrinol, 412, 349-357.
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Study III
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Abstract
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1. Introduction
The obesity epidemic and related chronic metabolic pathologies are often
attributed to Western lifestyles, characterized by the consumption of fat-rich diets
and low physical activity levels. In this context, the over-accumulation of visceral
adipose tissue (VAT) mass relies on hypertrophy rather than hyperplasia
(McLaughlin et al., 2016), which has been associated with lipid metabolism
dysregulation (Frühbeck et al., 2014; McLaughlin et al., 2016), and thus leads to
obesity-associated disorders. Moreover, at certain point, the expansion of VAT
results in an increase of basal adipocyte lipolysis and subsequent nonesterified
fatty acids (NEFA) and glycerol release (Frühbeck et al., 2014). Studies have
shown that glycerol effluxes out of adipocytes mainly via aquaglyceroporin 7
(AQP7) (Rodríguez, Catalan, Gomez-Ambrosi, & Frühbeck, 2011) in a process
that contributes to the regulation of lipid accumulation in VAT (Rodríguez,
Catalan, et al., 2006). Therefore, the expression of VAT AQP7 was elevated in
obese individuals, which may be associated with an increased lipolysis and
adipocyte hypertrophy (Catalán et al., 2008). On the other hand, the NEFA
transport across the membrane is facilitated by several membrane proteins,
including fatty acid translocase (FAT/CD36) (Frühbeck et al., 2014), which has
been described as a key player in fatty acids uptake and thus, intracellular lipid
metabolism (Zhou et al., 2012). However, the involvement of FAT/CD36 in the
regulation of lipid accumulation in VAT has been poorly investigated, particularly
in the obesity context.
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has been correlated with the level of obesity (Heinonen et al., 2015), while a lower
mitochondrial DNA copy number was associated with type 2 diabetes (Dahlman
et al., 2006). Moreover, as previously demonstrated by our group (Goncalves,
Passos, Rocha-Rodrigues, Diogo, et al., 2014) and others (Lionetti et al., 2014),
the fusion-related proteins were downregulated in liver mitochondria of high-fat
diet (HFD)-induced obese rats. To our best knowledge, the mitochondrial fusion
processes in the regulation of lipid accumulation in VAT are not fully understood.
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2. Methods
Sixty male Sprague-Dawley rats (6-wks old and weighed 233.9±2.6 g) were
purchased from Charles River Laboratories, allocated in standard cages (two per
cage) and maintained in a pathogen-free room (12-h light/dark cycle), constant
temperature (21-22 °C) and humidity (50-60%) with unrestricted access to water
and food (provided in a liquid state). Rats were fed ad libitum a nutritionally
adequate isoenergetic, isoproteic standard (composed by 35 kcal% fat, 47 kcal%
carbohydrates, and 18 kcal% protein) or a high-fat (HFD, composed by 71 kcal%
fat, 11 kcal% carbohydrates, and 18 kcal% protein) diet purchased from Dyets
Inc. (catalog no. 710027 and 712031, respectively) over a 17-week period. The
main source of fat in HFD was corn oil (40 g). This diet has been shown to induce
visceral adipose tissue accumulation in experimental animals, as we previously
reported (Goncalves, Passos, Rocha-Rodrigues, Diogo, et al., 2014). The
standard diet was given to all animals as an adaptation to the liquid feeding during
the first week. Afterwards, animals were divided into six groups (n=10/group):
standard-diet sedentary (SS), standard-diet voluntary physical activity (SVPA),
standard-diet endurance-trained (SET), high-fat diet sedentary (HS), high-fat diet
voluntary physical activity (HVPA) and high-fat diet endurance-trained (HET).
Energy intake (kilocalories per day) and body weight (g) were recorded daily and
weekly, respectively, during the 17 weeks of the experiment. The feed efficiency
ratio was calculated as follows [(body weight /energy consumed x 100].
VPA intervention: SVPA and HVPA animals were allocated in cages equipped
with a 365-mm (diameter) running wheel coupled to turn counter (type 304
stainless steel, Tecniplast). The wheel revolutions were daily recorded from a
digital counter between 08.00 and 10.00 h.
ET intervention: After a 9-week dietary intervention, SET and HET groups were
gradually submitted to a continuous running protocol on a motor-driven rodent
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treadmill (Le8700, Panlab Harvard Apparatus), 5 days per week for 8 wks.
Exercise intensity was progressively increased from 15 min per day at 15 m min-
1 up to 60 m min-1 at 25 m min-1 in the last 4 wks of the program. In order to
understand the therapeutic effects of ET, animals began the ET program after 9-
wks of dietary treatment since at this time point HFD-fed animals already
exhibited obesity-related metabolic features, according to our previous study
(Goncalves, Passos, Rocha-Rodrigues, Torrella, et al., 2014). Sedentary animals
were placed on a non-moving treadmill to be exposed to the same environmental
conditions but without promoting any physical training adaptation. The study was
approved by local Ethics Committee and followed the guidelines of the Federation
of Laboratory Animal Science Associations (FELASA).
Animals were fasted overnight for 12 h with free access to drinking water. To
avoid acute effects of exercise, SET and HET groups were sacrificed 48 h after
the last training session. At sacrifice, blood was collected, centrifuged to separate
plasma (3000 g for 15 min at 4 ºC) and stored at -80 ºC for biochemical analyses.
Afterwards, all visceral adipose depots around internal organs were excised and
weighted to calculate relative white adiposity as [(sum of fat pad weights)/(body
weight)x100]. Epididymal white adipose tissue (eWAT) was quickly removed and
stored at -80 ºC until RT-qPCR and Western Blot analyses were performed.
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Values are presented as mean ± standard error of the mean (SEM). The effect of
diet and exercise was analysed by a two-way analysis of variance (ANOVA). The
Bonferroni post hoc test was applied for post hoc comparisons between groups
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3. Results
3.1 Effects of diet and exercise on body weight, food-efficiency ratio, visceral
adiposity and adipocyte area
The initial body weight of the animals after one week of adaptation was 233.9±2.6
g. The HS animals showed similar body weight, but increased visceral adiposity
and adipocyte hypertrophy compared to that of SS group (table 1). HFD did not
affect relative muscle mass weight. Food-efficiency ratio did not change among
studied groups. Regarding the impact of physical activity, VPA had no impact on
body weight, while 8-wks of ET reduced body weight in both diet types. The HFD-
induced increases in visceral adiposity and adipocyte area were reversed by both
VPA and ET interventions. HFD did not change plasma insulin and glucose
levels. Although no alterations were seen in glucose levels, ET decreased insulin
levels in HFD-fed animals. HFD increased HOMA-IR while ET decreased in both
standard and HFD-fed animals.
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Table 1: Animal characteristics and plasma analysis
Muscle mass (%) 0.71±0.01 0.87±0.03a 1.00±0.03a,c 0.66±0.02 0.65±0.01c 0.85±0.02b,d,e DxE, D, E
158
Plasma
Data are expressed as the mean±SEM. a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA.
Chapter III. Experimental Work. Study III
Chapter III. Experimental Work. Study III
3.2 Effects of diet and exercise on AQP7 and FAT/CD36 expression in eWAT
Figure 1: Plasma glycerol (A) and NEFA (B) levels. Data are expressed as the
mean±SEM. DxE, diet and exercise interaction; D, diet effect; E, exercise effect; NS, not
significant; a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA
To gain further insight into the molecular mechanisms involved in the rise of
circulating glycerol and NEFA induced by exercise, we next analysed the mRNA
and protein expression of AQP7 and FAT/CD36, two glycerol and NEFA
transporters in adipocytes (Rodríguez, Catalan, et al., 2006). Long-term HFD per
se did not induce changes in both AQP7 gene and protein expression, whereas
ET decreased AQP7 mRNA and protein in both SET and HET groups compared
to their sedentary counterparts. VPA significantly decreased Aqp7 gene and
tended to decrease AQP7 protein content in SVPA animals (vs. SS group).
Moreover, a positive and strong correlation was found between AQP7 protein
expression and plasma glycerol (r=0.66, p=0.001,) and visceral adiposity (r=0.73,
p<0.001). Regarding FAT/CD36 expression, HFD tended to increase both gene
and protein expression in all groups, although with no statistical significance. VPA
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Figure 2: The mRNA and protein expression of lipid accumulation regulators, AQP7 and
FAT/CD36, respectively (A-D). Data are expressed as the mean±SEM. DxE, diet and
exercise interaction; D, diet effect; E, exercise effect; NS, not significant; a vs. SS; b vs.
HS; c vs. SVPA; d vs. SET; e vs. HVPA
3.3 Effects of diet and exercise on factors involved in the control of fatty acid
oxidation and lipogenesis in eWAT
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Figure 3: Total protein content of AMPK (A) and ACC (C) as well as their phosphorylation
at Thr172 and Ser79, respectively (B and D). Data are expressed as the mean±SEM.
DxE, diet and exercise interaction; D, diet effect; E, exercise effect; NS, not
significant; a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA
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Figure 4: The protein expression of full-length and truncated SREBP1c (A) and the ratio
between truncated and full length SREBP1c (B). Data are expressed as the
mean±SEM. DxE, diet and exercise interaction; D, diet effect; E, exercise effect;
NS, not significant; a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA;
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increased MFN2 protein expression only in SET animals and COX, TFAM, MFN1
and OPA1 protein expression in both SET and HET groups (figure 5 and 6).
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Figure 6: Protein expression of COX (A), TFAM (B), MFN1 (C), MFN2 (D) and OPA1
(E). Data are expressed as the mean±SEM. DxE, diet and exercise interaction;
D, diet effect; E, exercise effect; NS, not significant; a vs. SS; b vs. HS; c vs. SVPA;
d vs. SET; e vs. HVPA
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4. Discussion
The main findings of the present study suggest that rats submitted to a high-fat
Lieber DeCarli-isoenergetic diet developed distinct eWAT morphological and
metabolic alterations, which strongly resemble the pathological features
observed under the obesity context. In fact, with the high-fat regimen, eWAT and
its correspondent mitochondrial metabolism suffered several constrains
prompting lipid accumulation instead of lipid oxidation. On the other hand,
physical activity, in particular the ET program attenuated or, at least in part,
reverted some of the metabolic impairments imposed by obesity in eWAT. Data
from the present study demonstrated that ET i) reduced VAT lipolysis along with
lower levels of NEFA and glycerol in plasma, ii) increased mitochondrial-related
content and biogenesis (OXPHOS and TFAM) and fusion proteins (MFN1 and
OPA1), and iii) reduced lipogenic markers (ACC and SREBP1c), which positively
remodel important lipid accumulation regulators in both VAT and in mitochondria
of HFD-induced obese rats. As previously reported by our group (Goncalves,
Passos, Rocha-Rodrigues, Diogo, et al., 2014), the Lieber-DeCarli diet
represents a suitable HFD model to study obesity and related metabolic disorders
in rats, including NAFLD/NASH. One of the major mechanisms related to the
development of obesity-related NAFLD/NASH is the increased VAT lipolysis,
leading to NEFA and glycerol release (Frühbeck et al., 2014; Rodríguez, Catalan,
Gomez-Ambrosi, Garcia-Navarro, et al., 2011; Rodríguez, Ezquerro, et al., 2015).
The positive outcomes exerted by physical exercise against NASH-related
deleterious consequences were previously demonstrated by our group
(Goncalves et al., 2016) and others (Haczeyni et al., 2015). In the present study,
we further demonstrate that ET impact was evident on eWAT lipolysis, which
were observed by reduced plasma NEFA and glycerol levels. This is in
agreement with an inhibitory exercise effect on the adipocyte lipolytic activity of
obese rats (Chapados et al., 2008; Pistor et al., 2015). However, until now and to
our best knowledge, limited information is available regarding the effects of
chronic exercise on NEFA and glycerol transporters in eWAT in the context of an
HFD-induced obesity. The AQP7 is considered the main gateway for the delivery
of lipolysis-derived glycerol from WAT (Rodríguez, Catalan, et al., 2006;
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FAT/CD36 is highly expressed in metabolic tissues and play a key role in lipid
uptake and fatty acids homeostasis (Frühbeck et al., 2014; Xu et al., 2013;
Yoshida et al., 2013). Although the role of FAT/CD36 in VAT is still under
investigation, some studies have reported that an overexpression of FAT/CD36
resulted in decreased visceral adiposity and low circulating levels of NEFA,
triglycerides (TG) and cholesterol (Coburn et al., 2000; Vroegrijk et al., 2013;
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Zhou et al., 2012). Consistently, we observed that the gene and protein
expression FAT/CD36 increased in endurance-trained animals, which are in line
with decreases in plasma NEFA, TG and visceral adiposity found in the present
study. To date, the effects of FAT/CD36 have been extensively investigated in
skeletal muscle. Studies have shown that ET increased FAT/CD36 expression in
skeletal muscle, possibly to support the increased need for fatty acids to support
the ET-induced skeletal muscle fatty acid oxidation (for ref. see Yoshida et al.,
2013).
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increased visceral adiposity and adipocyte hypertrophy. Data from other studies
(Bach et al., 2003; Lionetti et al., 2014) suggest that reduced mitofusin-related
protein content is critical for the maintenance of the mitochondrial network and
metabolism, helping to understand some of the metabolic alterations associated
to obesity. Unexpectedly and, in contrast with other studies (Cummins et al.,
2014; Goncalves et al., 2016; Lionetti et al., 2014), no alterations on mitofusin-
related proteins were observed in HFD-fed animals. However, the decrease in
both eWAT mass and adipocyte size induced by exercise and a concomitant
decreased plasma NEFA levels might have contributed to the improvements of
mitochondrial content and biogenesis, which are in agreement with some studies
(Laye et al., 2009; Pistor et al., 2015). In liver mitochondria, ET positively
modulated fusion-related proteins in HFD-induced obese animals (Goncalves et
al., 2016; Lionetti et al., 2014). To our knowledge, the effects of exercise on
mitochondrial dynamics in VAT, particularly in the obesity context, have not yet
been studied.
5. Conclusions
Generally, our data suggest that daily physical activity (mimicked by VPA) is not
fully effective to protect against obesity-related cellular disorders. In contrast, an
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Study IV
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Abstract
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1. Introduction
Fatty acids (FA) are energy-rich molecules that play important physiological
roles in several organs, including white adipose tissue (WAT) (Oliveira et al.,
2015; Vaughan et al., 2015). In humans, the most abundant FA esterified to
triglycerides are oleic (C18:1n9), palmitic (C16:0) and linoleic (C18:2n6) that
account for over 85% of FA in WAT triglycerides (Hodson et al., 2008). Some
FA have been suggested to be involved as direct regulators in inflammation
(Oliveira et al., 2015; Vaughan et al., 2015), a complex and tightly regulated
biological process that is triggered by obesity (Kawanishi et al., 2015; Vieira,
Valentine, Wilund, Antao, et al., 2009). Obesity is the result of a positive energy
intake with low levels of physical activity, interacting or not with genetic factors,
which explain at least in part the excess of visceral adiposity accumulation
observed in large proportions worldwide (Bray & Bellanger, 2006). Obesity-
associated inflammation is locally observed in the expanded VAT (Lumeng et al.,
2007), but then becomes systemic through the release of several pro-
inflammatory mediators, including interleukin (IL)-6 and tumor necrosis factor
(TNF)-α, among others (Lopategi et al., 2016; Lumeng et al., 2007). Therefore,
this inflammatory state triggers the secretion of macrophage chemoattractant
protein (MCP)-1 by macrophages, which can either sustain a macrophage-like
phenotype in undifferentiated precursor cells or diminish the ability of adipocytes
to further expand and store lipids, thus supporting a deleterious “vicious cycle”
(Lopategi et al., 2016; Lumeng et al., 2007). Some recent studies reported that
specific fatty acids, such as saturated fatty acids (SFA) (Choi et al., 2014;
Tousoulis et al., 2010), monounsaturated fatty acids (Esser et al., 2015) and n6-
polyunsaturated fatty acids (PUFA) (Johnson & Fritsche, 2012) are involved in
the pathophysiology of inflammation during obesity (Chan et al., 2015; Finucane
et al., 2015; Oliveira et al., 2015; Vaughan et al., 2015). In fact, a fat diet rich in
SFA likely enhance circulating biomarkers of (pro) inflammation in health
individuals (Tousoulis et al., 2010), and in vitro studies showed that macrophages
exposed to SFA increased pro-inflammatory gene expression and cytokine
secretion, such as TNF-α and IL-6, and the chemokine CXCL1/KC (Choi et al.,
2014). A MUFA-enriched high-fat diet (HFD) reverted inflammation-mediated
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insulin resistance and adipose tissue dysfunction when compared with SFA-fed
mice (Finucane et al., 2015). On the other hand, MUFA intake induced an
upregulation of several pro-inflammatory genes in obese individuals (Esser et al.,
2015), possibly because the unsaturated double bond of MUFA turns these FA
more susceptible to oxidation, and therefore to the activation of a pro-
inflammatory stress response (Calder, 2006). Moreover, the various oxidized
forms of linoleic acid (C18:2n6) contribute to stimulate inflammation (Johnson &
Fritsche, 2012), mainly due to its role as a precursor of arachidonic acid-mediated
eicosanoid biosynthesis and in the reduction of the synthesis of anti-inflammatory
eicosanoids from eicosapentaenoic acid and docosahexaenoic acid (Fritsche,
2015).
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study we aimed to analyze the impact of two distinct physical exercise regimens
(voluntary physical activity - VPA and endurance training - ET) on VAT fatty acids
profile in response to a high-fat diet (HFD) and to ascertain whether these
exercise-induced changes in specific FA have significant repercussions on the
inflammatory response.
The study was approved by local Institutional Ethics Committee and followed the
guidelines for the care and use of laboratory animals in research advised by the
Federation of European Laboratory Animal Science Association (FELASA) and
Portuguese Act 129/92. Male Sprague-Dawley rats (6 wks old) were purchased
from Charles River (L'Arbresle, France) and housed (2 rats per cage) in a
controlled environment (12-h light/dark cycle), constant temperature (21-22°C)
and humidity (50-60%) with free access to water and food (provided in a liquid
state). Rats with initial body weight 233.9±2.6 grams were fed a nutritionally
adequate isoenergetic standard (35Kcal% fat, 47Kcal% carbohydrates, and
18Kcal% protein) or a high-fat (HFD, 71Kcal% fat, 11Kcal% carbohydrates, and
18Kcal% protein)-liquid diets purchased from Dyets Inc. (catalog no. 710027 and
712031, respectively) over 17 weeks. The two diets differed in the amount of corn
oil in each other, 40 grams packed separately and then mixed into the diet to
obtain HFD (for a detailed description of diets see table 1). The standard diet was
given to all animals as an adaptation to the liquid feeding during the first week.
Afterwards, animals were divided into four groups (n=7-8/group): standard-diet
sedentary (SS), standard-diet voluntary physical activity (SVPA), high-fat diet
sedentary (HS) and high-fat diet voluntary physical activity (HVPA). After 9-weeks
of the beginning of the protocol, half of SS and HS animals were divided into
standard-diet endurance training (SET) and high-fat diet endurance training
(HET) groups. Energy intake (kilocalories) and body weight (g) were recorded
daily and weekly, respectively, during the 17 weeks of the experiment.
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Table 1: Fatty acid compositionof standard and high-fat diets
Safflower Olive oil Corn oil Total Safflower Olive oil Corn oil Total
180
C18:3n3 - 0.6 0.6 1.2 - 0.6 1.2 1.8
The standard and high-fat diets were purchased to Diets Inc., catalog #710027 and 7120131. The two diets
differed in the amount of corn oil each other, 40 g of was added to obtain high-fat diet. Standard diet contain 12%,
39 and 49% of saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids, respectively.
The high-fat diet contain 12%, 35% and 51% of saturated fatty acids, monounsaturated fatty acids and
polyunsaturated fatty acids, respectively
Chapter III. Experimental Work. Study IV
Chapter III. Experimental Work. Study IV
Voluntary physical activity: animals from SVPA and HVPA groups were
individually housed in cages equipped with 365-mm (diameter) running wheel
coupled to turn counter (type 304 stainless steel). The wheel revolutions were
recorded daily from a digital counter between 08.00 and 10.00 h.
Endurance Training: animals from SET and HET groups were submitted to a
continuous aerobic exercise on treadmill, 5 days week -1 for 8 weeks. Exercise
intensity was progressively increased from 15 min per day at 15 m min-1 up to 60
min per day at 25 m min-1 for the last 4 weeks of the program. Sedentary animals
were placed on a non-moving treadmill to be exposed to the same environmental
conditions but without promoting any physical training adaptation.
At the end of the study, all animals were fasted overnight for about 12h with free
access to drinking water. To eliminate acute effects of physical exercise, animals
from ET groups were sacrificed 48h after the last training session. At sacrifice,
blood was collected from the left ventricle and plasma centrifuged at 3000 g for
15 min at 4ºC, and stored at -80ºC for later biochemical analyses. All visceral
adipose deposits around internal organs were excised and weighed to calculate
relative visceral adiposity as [(sum of fat pad weights)/(body weight)x100)].
Epididymal white adipose tissue (eWAT) was rapidly excised and stored for
further analysis described below.
After fixation in 10% formalin, the adipose tissue samples were dehydrated in
absolute ethanol, cleared in xylene, and then embedded in paraffin. The paraffin
was cut into 5-μm sections that were stained with hematoxylin, counterstained
with eosin, and then evaluated using light microscopy (Zeiss AX10 imager A.1,
Oberkochen, Germany) under X40 magnification. Any objects below an area of
350 μm2 were excluded as these may be a mixture of adipocytes and stromal
vascular cells [14]. The distribution of adipocyte areas and the adipocyte area
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mean were determined from 4 sections per rat and 4 rats per group (>1500
adipocytes counted per group).
Fatty acids composition analysis: Esterified fatty acids from triglycerides fraction
were analysed by gas chromatography- mass spectrometry (GC-MS). For each
sample, an amount equivalent at 5 µL of triglycerides was transesterified. Fatty
acids methyl esters (FAMEs) were prepared using a methanolic solution of
potassium hydroxide (2 M), according to (Aued-Pimentel et al., 2004). FAMEs
were resuspended in 200 μL n-hexane, and 1 μL of each n-hexane solution was
used for GC-MS analysis (GCMS-QP2010 Ultra, Shimadzu, Kyoto, Japan)
equipped with an autosampler and a DB-FFAP column with 30 m of length, 0.32
mm of internal diameter, and 0.25 μm of film thickness (J&W Scientific, Folsom,
CA). Each sample was injected in split mode (split ratio of 2) at 250°C. The oven
temperature was programmed from an initial temperature of 80°C, standing at
this temperature for 3 min, a linear increase to 160°C at 25°C min−1, followed by
linear increase to 210°C at 2°C min−1, and another linear increase to 250°C at
30°C min−1, and then it was maintained at 250ºC during 10 min. Helium was used
as carrier gas at a linear velocity of 43.6 cm s−1. The ion source and interface
temperatures were 200°C and 250°C, respectively. Full scan mass spectra (m/z
35–700) were acquired in 0.3 s cycles. The relative amounts of FA were
calculated by the percent area method with proper normalization considering the
sum of all areas of the identified FA. FAMEs identification was performed by
comparing their retention time and mass spectrum, with mass spectra of
commercial FAMEs standards (Supelco 37 Component FAME Mix) and
confirmed by comparison with the spectral library “The AOCS Lipid Library”
(Christie, 2012).
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Plasma samples were diluted (1:20) in Tris-buffered saline and 100µL was slot-
blotted into a nitrocellulose membrane. The slot-blot membranes were blocked
and then incubated with specific primary antibodies; anti-IL-6 (ab6672), anti-TNF-
α (ab80039), anti-IL-10 (ab9969) from Abcam (Cambridge, UK), followed with
incubation with a solution of horseradish-conjugated anti-rabbit (sc2317) antibody
from Santa Cruz Biotechnology (Dallas, TX, USA). The blots were detected by
ChemiDoc™ XRS+System and band densities were quantified using Image
Lab™ software 262 5.2.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Control for protein loading was confirmed by Ponceau S staining.
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Values are presented as mean ± standard error of the mean (SEM). All data were
compared with two-analysis of variance (ANOVA) and Bonferroni’s post hoc test
was applied for comparisons between groups. Pearson’s correlation was used to
describe the linear association between variables. The differences were
considered significant at p≤0.05. Statistical analyses were performed using SPSS
21.0 for Windows (SPSS Inc.).
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Chapter III. Experimental Work. Study IV
3. Results
3.1 Effects of diet and physical exercise on body weight, energy intake, visceral
adiposity and adipocyte size
As seen in table 2, HFD and VPA did not affect the initial body weight and after
9 weeks of the study. At the end of the study, HFD increased visceral adiposity,
but did not induce alterations in body weight in HS group. Both VPA and ET
interventions significantly decreased visceral adiposity in both diet types, but only
ET was able to reduce body weight in SET and HET groups. No alterations were
found in total energy intake among groups.
Regarding adipocyte size, the HFD regimen increased adipocyte area mean as
well as the percentage of adipocytes sized greater than 5000μm 2. The adipocyte
area mean decreased after VPA and ET interventions in HFD-fed animals.
Moreover, both VPA and ET increased the percentage of adipocytes sized below
5000 μm2 and decreased those that are greater than 5000 in HFD-fed animals
(table 2).
185
Table 2: Body weight, total energy intake, visceral adiposity, and adipocyte size determinations
186
Visceral 9.01±0.21 7.49±0.46a 4.76±0.46a,c 11.76±0.37a 8.92±0.37b 6.77±0.145b,d D, E
adiposity (%)
Adipocyte area
Data are expressed as the mean±SEM. DxE, diet and exercise interaction; D, diet effect; E, exercise effect; NS, not
significant; a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA
Chapter III. Experimental Work. Study IV
Chapter III. Experimental Work. Study IV
3.2 Effects of diet and physical exercise on fatty acid profile in eWAT
triglycerides
The most abundant FA esterified into triglyceride are depicted in figure 1. The
long-term HFD regimen decreased the relative content of SFA (C16:0 and C18:0)
and MUFA (C16:1n7; C18:1n7; C18:1n9) while increased C18:2n6 in eWAT
triglycerides. Moreover, VPA decreased MUFA (C16:1n7; C18:1n9) and
increased PUFA (C18:2n6) only in HFD-fed animals. Eight-weeks of ET
decreased the relative content of SFA (16:0) and MUFA (C18:1n9) in standard
diet-fed animals, MUFA (C16:1n7; C18:1n9) in both diet types, and increased
C18:2n6 in eWAT triglycerides of HFD-fed animals.
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Figure 1: Fatty acid relative content in eWAT triglycerides. The saturated fatty acids,
palmitic and stearic acid (A and C); the monounsaturated fatty acids, palmitoleic,
vaccenic and oleic (B, D and E); polyunsaturated fatty acids, linoleic acid. Data are
expressed as the mean±SEM. DxE, diet and exercise interaction; D, diet effect; E,
exercise effect; NS, not significant; a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA
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Chapter III. Experimental Work. Study IV
The circulating TNF-α content increased after the HFD intervention (HS vs. SS).
The VPA tended to increase plasma IL-10 and decrease TNF-α protein content
despite no statistical significance. Eight-weeks of ET decreased plasma IL-6 and
TNF-α protein content as well as increased IL-10 in both SET and HET groups.
Moreover, ET increased IL-10/TNF-α ratio in both standard and high-fat diets-fed
animals (figure 2).
Figure 2: Impact of diet and physical exercise on plasma cytokines. The protein content
of IL-6 (A), TNF-α (B), IL-10 (C) and IL-10/TNF-α ratio (D) and representative image blots
(E). Data are expressed as the mean±SEM. DxE, diet and exercise interaction; D, diet
effect; E, exercise effect; NS, not significant; a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e
vs. HVPA
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Chapter III. Experimental Work. Study IV
A positive correlation was observed between plasma IL-6 protein content and
adipocyte size (p<0.01, r=0.51) and visceral adiposity (p=0.001, r=0.641), as well
as between TNF-α and adipocyte area mean (p<0.001, r=0.69) and visceral
adiposity (p<0.001, r=0.843). A negative correlation was found between plasma
IL-10 protein and visceral adiposity (p<0.001, r=0.843).
As illustrated in figure 3, the HFD had no impact on neither IL-6 nor TNF-α gene
and protein expression, despite a tendency to increase TNF-α expression in HS
group. No alterations were observed for these cytokines after the VPA
intervention. Eight-weeks of ET decreased Il6 gene expression (only in standard
diet-fed animals), IL-6 and TNF-α protein expression in both diet types. The IL-
10 protein content remained unchanged after HFD and VPA interventions while
ET program increased significantly this anti-inflammatory cytokine in both diet
types. The IL-10/TNF-α ratio was increased in both SET and HET groups.
Visceral adiposity was positively correlated with IL-6 (p<0.002, r=0.601) and TNF-
α (p<0.001, r=0.675) and negatively correlated with IL-10 (p=0.002, r=-0,603).
Adipocyte area mean was positively correlated with IL-6 (p<0.001, r=0.640) and
(p=0.003, r=0.594) and negatively correlated with IL-10 (p=0.002, r=-0.603).
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Chapter III. Experimental Work. Study IV
Figure 3: The pro- and anti-inflammatory cytokines in eWAT. The gene and protein
expression of IL-6 (A and B), TNF-α (C and D), protein expression of IL-10 (E), IL-
10/TNF-α ratio (F) and representative blots images (G). Data are expressed as the
mean±SEM. DxE, diet and exercise interaction; D, diet effect; E, exercise effect; NS, not
significant; a vs. SS; b vs. HS; c vs. SVPA; d vs. SET; e vs. HVPA
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Chapter III. Experimental Work. Study IV
The gene and protein expression of MCP-1 were significantly increased after
HFD (HS vs. SS). Conversely, VPA decreased MCP-1 gene and protein
expression only in HFD-fed animals although a tendency was observed in
standard diet-fed animals. The protein expression of F4/80 remained unchanged
after HFD and VPA interventions. Moreover, eight-weeks of ET decreased either
MCP-1 or F4/80 protein expression in both diet types (figure 4). A positive and
strong correlation between visceral adiposity and Mcp1 gene expression
(p<0.001, r=0.794), MCP-1 (p<0.001, r=0.856) and F4/80 protein content
(p<0.001, r=0.786) was found. Also, adipocyte area mean positively correlated
with Mcp1 gene expression (p<0.001, r=0.857), MCP1 (p=0.001, r=0.651) and
F4/80 protein content (p<0.001, r=0.717).
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4. Discussion
The main findings of the present study suggest that physical exercise induced FA
profile-specific changes in eWAT triglycerides, independently of dietary FA
composition, and that ET, but not VPA, attenuated the inflammatory response in
VAT of HFD-fed animals. Moreover, the physical exercise-induced FA profile
changes did not correlate with the modulation of the inflammatory response.
The isoenergetic pair-feeding diet used in the present study was chosen to
ensure equal caloric intake between studied groups excluding any effects that
could potentially be attributed to differences in energy intake. Our data show that
the intake of an isoenergetic pair feeding HFD, despite causing no overweight,
led to increased visceral adiposity accumulation, adipocyte size and other
adverse metabolic and inflammatory alterations, already described by our group
(Goncalves, Passos, Rocha-Rodrigues, Torrella, et al., 2014; Rocha-Rodrigues,
Rodriguez, Becerril, et al., 2016) and consistent with other findings (Diaz-Rua et
al., 2014; Estrany et al., 2011; Gollisch et al., 2009; Li et al., 2015). Of note is that
the visceral adiposity accumulation, rather than the excess body weight, has been
associated with increased risk of developing metabolic and inflammatory
diseases in obesity (Jacobs et al., 2016; Lopes et al., 2016). On the other hand,
physical exercise has been recognized as an effective non-pharmacological
strategy to reduce body weight and visceral adiposity accumulation in distinct
models of obesity (Jenkins et al., 2012; Vieira, Valentine, Wilund, Antao, et al.,
2009), which are in agreement with our findings. In the current study, these
alterations were not related with changes in energy intake as there were no
significant differences in energy intake between studied groups.
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Another goal of the present study was to analyze the effects of VPA and ET, as
preventive and therapeutic interventions, respectively, on the VAT inflammatory
response associated to the HFD. It is well established that under HFD feeding,
the hypertrophied stressed white adipocytes exhibit an increased release of pro-
inflammatory adipokines and chemokines, which attract immune cells into the
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Chapter III. Experimental Work. Study IV
VAT (Jenkins et al., 2012; Kawanishi et al., 2015; Lumeng et al., 2007). Our
findings are in line with these data as higher levels of plasma TNF-α and MCP-1,
one of the key chemokines that regulate the monocytes/macrophage migration
and infiltration, were found in sedentary HFD-fed animals. On the other hand, ET
was effective as a therapeutic intervention and reverted the moderate pro-
inflammatory phenotype imposed by the HFD, which is consistent with the well-
described anti-inflammatory effects of exercise (Gollisch et al., 2009; Kawanishi
et al., 2013; Kawanishi et al., 2015; Kawanishi et al., 2010).
Several studies reported that specific FA, including SFA, MUFA and linoleic acid
are implicated in the development of the inflammatory process during obesity,
(Chan et al., 2015; Finucane et al., 2015; Oliveira et al., 2015; Vaughan et al.,
2015). Accumulating evidence showed that SFA or their metabolites activate
inflammation by toll-like receptors dependent mechanisms (Nguyen et al., 2005),
which in turn leads to an up-regulation of the ceramide biosynthesis pathway
(Ussher et al., 2010). Moreover, the pro-inflammatory effects of C18:2n6 have
been associated to the arachidonic acid-mediated eicosanoid biosynthesis (for
ref. see Naughton et al., 2016). Therefore, we also intended to analyze whether
or not physical exercise-induced FA-specific changes have significant
repercussions on the VAT inflammatory response of HFD-fed animals. However,
data revealed that alterations on FA profile (namely the decreases in palmitoleic
acid, palmitic acid and increases in linoleic acid) did not correlate with the anti-
inflammatory phenotype induced by physical exercise, which suggest that other
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Chapter III. Experimental Work. Study IV
regulatory pathways are certainly involved in this cross-talk These findings are
consistent with data published by Vaughan and coworkers (Vaughan et al.,
2015), in which no alterations in pro-inflammatory markers was found after
consumption of a linoleic acid-rich diet.
5. Conclusions
Altogether, data from the present study suggest that VPA and ET interventions
had similar impact on FA-specific changes independently of diet composition.
However, only ET, but not VPA, was effective in attenuating the HFD-induced
inflammatory state. Finally, the exercise-induced FA profile changes did not
correlate with the modulation of the inflammatory response.
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Study V
Magalhães1
203
Chapter III. Experimental Work. Study V
Abstract
Purpose: Autophagy and apoptosis play critical roles in both development and
tissue homeostasis in response to pathological and physiological stimuli, such
as high-fat diet (HFD) and endurance training (ET). Therefore, we aimed to
investigate how ET modulates autophagy and apoptotic-related signaling in
VAT of long-standing HFD-fed rats.
Methods: The study was conducted over a 17-wk period on Sprague-Dawley
rats, which were divided into 4 groups (n= 8 per group): standard diet
sedentary (STD+SED), high-fat diet sedentary (HFD+SED), standard diet
endurance training (STD+ET) and high-fat diet endurance training (HFD+ET).
After 9-wks of HFD feeding, ET groups were trained for 8-wks on motor-driven
treadmill (5d/wk at 25m/min for 60min/day), while maintaining dietary
regimens. Autophagy and apoptotic signaling markers in epididymal white
adipose tissue (eWAT) were determined using RT-qPCR, Western blot and
spectrometry techniques.
Results: The ET reduced body weight, visceral fat mass and HOMA-IR in both
standard and high-fat diets-fed animals. Moreover, ET reverted the HFD-
induced increases in the percentage of larger adipocytes and also reduced the
percentage of smaller adipocytes. The HFD decreased pre-adipocyte factor 1
gene expression and increased the pro-apoptotic markers (Bax protein and
caspase 3-like activity), while had no impact on autophagy markers. On the
other hand, the ET increased the expression of pre-adipocyte factor 1 and Bcl-
2 in both diet types, while decreased the pro-apoptotic Bax and caspases 9, 8
and 3-like activities in HFD feeding rats. In addition, the protein expression of
Beclin-1 and p62 significantly increased in ET groups of both diet types.
Conclusions: Data demonstrate that 8 wks of ET was effective in attenuating
apoptotic-related signaling in long-standing HFD-fed rats. Moreover, HFD and
ET had no impact on VAT autophagy markers.
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1. Introduction
Apoptosis and autophagy are two critical processes in the development and
homeostasis of the organisms, but also have been linked to obesity-related
disorders (Benbrook & Long, 2012; Salminen et al., 2013). Under obesity
conditions, an increased synthesis and degradation of cellular components in
metabolically relevant tissues, such as VAT, and a failure in autophagy may
affect organelle function, cellular homeostasis (Sarparanta et al., 2016), and,
ultimately, lead to programmed cell death (Kang et al., 2011; Salminen et al.,
2013; Sarparanta et al., 2016). In the context of obesity, some studies have
found contradictory results, showing increased autophagy activity in obese
individuals (Haim et al., 2015; Kovsan et al., 2011), in genetic and dietary
models of obesity (Yang et al., 2010) and in obese mice lacking leptin (Lepob)
(Jansen et al., 2012). However, others studies (Yang et al., 2010)
demonstrated that restoration of Atg7 improved hepatic insulin action and
systemic glucose tolerance in obese mice. Few studies have been focused on
apoptosis in VAT; however it was proposed that its dysregulation contributes
to the development of obesity-related pathologies (Sun et al., 2011). Reports
showed that adipocyte apoptosis is prominent in both obese animals (Alkhouri
et al., 2010; Feng et al., 2011) and humans (Alkhouri et al., 2010). These
studies suggest that the regulation of both autophagy and apoptotic processes
and their connection is relevant in metabolic dysfunction of VAT and an
unexplored research field. In contrast, few studies suggest that exercise-
induced autophagy activation may have important implications in metabolic
tissues, thus protecting against HFD-induced metabolic disease in rodents
(Cui et al., 2013; Greene et al., 2015; He et al., 2012). Other studies reported
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Chapter III. Experimental Work. Study V
The study was approved by local Institutional Ethics Committee and followed
the guidelines for the care and use of laboratory animals in research advised
by the Federation of European Laboratory Animal Science Association
(FELASA) and Portuguese Act 129/92.
Energy intake (kilocalories) and body weight (g) were recorded daily and
weekly, respectively, during the 17 weeks of the experiment. Energy efficiency
was calculated as the ratio between body weight and energy consumed. At the
end of the study, all animals were fasted overnight for about 12h with free
access to drinking water. Blood was collected from the left ventricle and
plasma centrifuged at 3000 g for 15 min at 4ºC, and stored at -80ºC for later
biochemical analyses. All visceral adipose depots around internal organs were
excised and weighted to calculate total visceral fat mass. An aliquot of
epididymal white adipose tissue (eWAT) was rapidly removed and stored at -
80ºC for further analyses, described below.
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levels were determined using enzymatic methods with commercial kits (10-
1137-01, Mercodia). The homeostasis model assessment of insulin resistance
(HOMA-IR) was measured as follows, fasting plasma insulin X fasting plasma
glucose/2.43 (Cacho et al., 2008).
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209
Chapter III. Experimental Work. Study V
Data are expressed as mean ± standard error of the mean (SEM). Multiple
comparisons between groups were performed using two-way analysis of
variance (ANOVA). The Bonferroni post hoc test was applied for post hoc
comparisons between groups and differences were considered significant at
p≤0.05. Statistical analysis was performed using SPSS 15.0 for Windows
(SPSS Inc., Chicago IL, USA).
3. Results
3.1 Body weight, energy efficiency, visceral fat mass and HOMA-IR:
As seen in figure 1, the energy efficiency was similar between studied groups.
Although with no changes on body weight, HFD significantly increased visceral
fat mass and HOMA-IR, a surrogate index of IR validated against the clamp
technique, compared to standard diet group. ET for 8 weeks decreased body
weight, visceral fat mass and HOMA-IR in both diet types compared to
sedentary counterparts.
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Chapter III. Experimental Work. Study V
Figure 1: Body weight over a period of 17 weeks (A), final body weight (B), energy
efficiency (C), visceral fat mass (D) and HOMA-IR (E) Data are expressed as the
mean±SEM. DxE, diet and exercise interaction; D, diet effect; E, exercise effect;
NS, not significant *p<0.05 vs. STD+SED; **p<0.01 vs. STD+SED; ***p<0.001 vs.
STD+SED; # # #vs. p<0.001
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Figure 2: The frequency distribution of adipocyte size (A) and gene and protein
expression of DLK1/PREF1 (B and C). The representative images blots below. Data
are expressed as the mean±SEM. DxE, diet and exercise interaction; D, diet
effect; E, exercise effect; NS, not significant; *p<0.05 vs. STD+SED; **p<0.01 vs.
STD+SED; ***p<0.001 vs. STD+SED; # # p<0.01 vs. HFD+SED; # # #vs. p<0.001
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Figure 3: The expression of autophagy proteins. Beclin-1 (A), Lc3II (B), p62 (C) and
representative images blots of each protein (D). Levels were normalized to β-actin for
each sample. Data are expressed as the mean±SEM. DxE, diet and exercise
interaction; D, diet effect; E, exercise effect; NS, not significant. *p<0.05 vs.
STD+SED; **p<0.01 vs. STD+SED; ***p<0.001 vs. STD+SED; #p<0.05 vs.
HFD+SED; ## p<0.01 vs. HFD+SED; ###vs. p<0.001
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Chapter III. Experimental Work. Study V
4. Discussion
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Chapter III. Experimental Work. Study V
Originally, the Lieber DeCarli diet was established to induce liver damage
(Lieber et al., 2004), but the modified version of this diet was developed to
induce obesity-related diseases, such as non-alcoholic steatohepatitis
(Goncalves, Passos, Rocha-Rodrigues, Torrella, et al., 2014; Li et al., 2015).
Although with no alterations in body weight, the isoenergetic pair feeding HFD
caused a significant increase of total visceral fat mass, larger adipocytes and
HOMA-IR, a surrogate indicator of IR that has been linked to adverse
metabolic disturbances, as previously described elsewhere (Diaz-Rua et al.,
2014; Estrany et al., 2011; Gollisch et al., 2009; Li et al., 2015). Accumulating
evidence indicates that an excessive visceral adiposity accumulation, rather
than overweight, has been associated to a higher risk of developing obesity-
related disorders (Jacobs et al., 2016; Lopes et al., 2016). In the present study,
animals fed with a HFD during 9-wks were submitted to an ET program in order
to evaluate the therapeutic effects of ET against adverse cellular
consequences imposed by a HFD. In accordance with others (Gollisch et al.,
2009; Linden et al., 2014), data revealed that 8-wks of ET program was
effective in attenuating obesity-related features, reducing body weight, visceral
fat mass and adipocyte size.
Apoptosis (type I) and autophagy (type II) are considered the two major forms
of programed cell death (Sarparanta et al., 2016). Nevertheless, in some
conditions and till a certain degree of metabolic disorder, both can also function
as pro-survival mechanisms in order to maintain cellular homeostasis and
metabolism (Benbrook & Long, 2012; Salminen et al., 2013). These are
integrated processes as several autophagy-related proteins, such as Beclin-1
(Kang et al., 2011), are also substrates for caspases during apoptosis
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216
Chapter III. Experimental Work. Study V
5. Conclusions
Data from the present study demonstrate that despite neither HFD nor ET had
any significant impact on VAT autophagy markers, 8 wks of ET was effective
in attenuating apoptotic-related signaling in long-standing HFD-fed rats.
Therefore, a better understanding of the molecular mechanisms underlying
ET-induced adipose tissue autophagy and apoptotic adaptations are still
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Chapter III. Experimental Work. Study V
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CHAPTER IV. General Discussion
Chapter IV. General Discussion
General Discussion
In the context of obesity, a chronic excessive energy storage in the VAT initiates
pathological remodeling, including adipocyte hypertrophy, tissue hypoxia
(Goossens et al., 2011; Hosogai et al., 2007; Virtanen et al., 2002; Ye et al., 2007;
Yin et al., 2009), dysregulation of adipokines/hormones production and secretion,
adipocyte death (Ye, 2009), macrophage/monocytes infiltration (Kawanishi et al.,
2013; Kawanishi et al., 2010), and elevated pro-inflammatory cytokines
production (Azizian et al., 2016; de Sa et al., 2016b). In the present work, we
used a modified version of Lieber DeCarli pair-feeding diet that has been
described as a suitable model to induce obesity (Lieber et al., 2004). Data from
our studies (I-V) showed that this HFD induced the typical obesity-related
morphological, metabolic, and inflammatory and apoptotic features observed in
other HFD-induced obesity models (Gollisch et al., 2009; Linden et al., 2014;
Vieira, Valentine, Wilund, & Woods, 2009). Despite no alterations in final body
weight, an increased visceral adiposity and hypertrophic adipocytes, features of
the emerging concept “adiposopathy”, were induced by the HFD used in the
present work. In this context, an upregulation of the expression of HIF-1α,
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Chapter IV. General Discussion
228
Chapter IV. General Discussion
229
Chapter IV. General Discussion
Considered the major endocrine organ in the human body, skeletal muscle
secretes several myokines in response to physical exercise, which mediate
autocrine and paracrine effects in the muscle structure and metabolism
(Pedersen, 2011). However, these myokines also work in an endocrine manner,
some acting on white adipocytes has positive regulators of brown-like phenotype
in WAT (Bostrӧm et al., 2012; Cao et al., 2011; Knudsen et al., 2014; Rao et al.,
2014; Shan et al., 2013). In fact, myokines likely provide a conceptual basis to
understand how skeletal muscle “talks” with WAT, which is of particular interest
in the context of obesity. This established axis might be, at least in part, related
to VAT plasticity, a phenomenon enabling dynamic modulation of the VAT
phenotype in response to environmental and physiological stimulus, such as
physical exercise. In the present work, we focused on IL-6 and FNDC5/irisin given
its potential to induce brown-like phenotype development in response to physical
exercise (Bostrӧm et al., 2012; Knudsen et al., 2014). In accordance with others
(Bostrӧm et al., 2012; Knudsen et al., 2014; Tiano et al., 2015; Wu et al., 2014),
data from study II showed an increased IL-6 and FNDC5 in the skeletal muscle
of ET animals, which might had contributed to the increase of brown adipocyte-
like phenotype markers (Bmp7, Cidea, Prdm16, UCP1) in VAT in this sub-set of
exercised animals. In fact, ET, but not VPA, was associated with a “brown”-like
phenotype. This finding might be related to exercise intensity as AMPK activation
230
Chapter IV. General Discussion
231
Chapter IV. General Discussion
induction of early regulators of the brown fat fate PRDM16 and PGC-1α,
increased expression of the brown-specific marker UCP1, and stimulation of
mitochondrial biogenesis [28]. Data from studies II and III demonstrated that ET
stimulated mitochondrial OXPHOS subunits content and mitochondrial
biogenesis (PGC-1α and TFAM), mitofusin proteins (MFN1 and OPA1), brown-
related markers (Bmp7 and Cidea) in HFD-fed animals. Together with a tendency
to increase the expression of UCP1 (increased 25% vs. HS animals), our data
showed that ET induced a brown-like phenotype in VAT of obese rats. These
findings suggest that physical exercise-mediated effects on mitochondria content
and biogenesis have impact on the overall VAT metabolism in the context of
obesity.
Ultimately, findings from the present dissertation suggest that further studies are
needed to better understand the molecular mechanisms associated with the
effects of physical exercise on VAT plasticity and remodeling in the context of
obesity. Moreover, the potential cross-tolerance effects of some exercise
approaches and combinations as preventive and therapeutic tools for the
treatment of obesity are clear and should be further investigated.
232
Figure 1: Summary of the systemic and adipose tissue adaptations induced by HFD and the preventive – VPA and therapeutic –
ET effect of exercise.
233
Chapter IV. General Discussion
Chapter IV. General Discussion
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242
CHAPTER V. Conclusions
Chapter V. Conclusions
Conclusions
Based on the results that emerged from the studies comprised in this dissertation,
the main conclusions are summarized below:
iv) the VPA and ET interventions had similar impact on fatty acids-specific
changes by decreasing palmitoleic acid, palmitic acid and increasing linoleic
acid in VAT of HFD-fed rats;
vi) Physical exercise-induced fatty acids alterations did not correlate with
the anti-inflammatory phenotype also induced by physical exercise;
Generally, our data suggest that daily physical activity (mimicked by VPA) is not
fully effective to protect against obesity-related VAT disorders. In contrast, an ET
program with specific characteristics (intensity, duration and frequency) mitigates
several adverse metabolic consequences induced by obesity in VAT. These
245
Chapter V. Conclusions
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