University of Porto - Repositório Aberto · University of Porto, according to the Law 74/2006 from March 24th. ... from the Spanish Instituto de Salud Carlos III, the Department

University of Porto

Faculty of Sport

Research Center in Physical Activity, Health and Leisure

PHYSICAL EXERCISE AND WHITE ADIPOSE TISSUE PLASTICITY IN THE

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.

Supervisor: Professor José Fernando Magalhães Pinto Pereira

Co-Supervisor: Professor António Alexandre Moreira Ribeiro de Ascensão

Sílvia Fernanda da Rocha Rodrigues

Porto, 2017

ii

Rocha-Rodrigues, S. (2017). Physical exercise and white adipose tissue

plasticity in the context of obesity. Dissertação de Doutoramento em Atividade

Física e Saúde apresentada à Faculdade de Desporto da Universidade do Porto.

PALAVRAS-CHAVE: EXERCISE, VISCERAL ADIPOSITY, MYOKINES,

BEIGING, ADIPOKINES

iii

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).

v

In memory of my dad, who I love and miss dearly.

vii

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.

To my colleagues from Laboratory of Metabolism and Exercise (Lametex): Inês

Gonçalves, Inês Aleixo, Emanuel dos Passos, Pedro Coxito, Mané, Diogo,

André, Jorge, Telma, João for all support during the experimental studies.

To colleagues from Department of Physiology (Faculty of Medicine, University of

Porto), Pedro for all dedication and help in the beginning of my journey in the lab

and also Ana Filipa and Nádia for all kindness and enjoyment that they conveyed

to me.

To Professor Maria João Martins, Jugal and Susana from Departament of

Biochemistry (Faculty of Medicine, University of Porto) for teaching me all about

gene expression and support during gene analyses. A special thanks to Raquel

by sharing knowledge, doubts and for always being available to help.

To Professor Alexandra Gouveia, Professor Adriana Rodrigues and Maria from

Departament of Experimental Biology (Faculty of Medicine, University of Porto)

for all dedication, simplicity and patience during my short visit to learn and

perform real-time PCR technique and scientific contribution for the research

papers.

viii

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.

To Professor Rosário Domingues, Ana Moreira and Elisabete Maciel from

Departament of Chemistry (University of Aveiro) for the contribution in lipidomics

studies, technical assistance in mass spectrometry and gas chromatrography-

mass spectrometry analysis.

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.

As Antoine Sain-Exupéry said “Those who pass by us do not go alone, do not

leave us alone, they leave a bit of themselves take a little of us” and I certainly

take a little of each of you with me.

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.

ix

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.

xi

Table of Contents

Acknowledgements ........................................................................................... vii

Table of Contents ............................................................................................... xi

List of Figures ................................................................................................... xiii

List of Tables ................................................................................................... xvii

Resumo ............................................................................................................ xix

Abstract ............................................................................................................ xxi

List of Abbreviations ....................................................................................... xxiii

CHAPTER I. General Introduction ...................................................................... 1

1. Introduction ............................................................................................ 3

2. Aims ..................................................................................................... 13

CHAPTER II. Theoretical Background ............................................................. 29

Review Article .......................................................................................... 31

CHAPTER III. Experimental Work .................................................................... 95

Study I ...................................................................................................... 97

Study II ................................................................................................... 123

Study III .................................................................................................. 149

Study IV ................................................................................................. 175

Study V .................................................................................................. 203

CHAPTER IV. General Discussion ................................................................. 225

xii

General Discussion ................................................................................ 227

CHAPTER V. Conclusions ............................................................................. 243

Conclusions ........................................................................................... 245

References ..................................................................................................... 247

References............................................................................................. 249

FACSIMILE .................................................................................................... ccci

xiii

List of Figures

CHAPTER II. Theoretical Background

Figure 1. Schematic view of hypothetical mechanisms underlying physical

exercise impact on white adipose tissue morphological, metabolic and

inflammatory features in the context of obesity…………………………………58

CHAPTER III. Experimental Work

Study I

Figure 1. Flow diagram outlining the experimental design……………….102

Figure 2. Body weight, feed efficiency ratio, Lee index, visceral adiposity to

body weight, and relative weights of mWAT, rWAT and eWAT...…………......106

Figure 3. Adipocyte cell-size distribution……………………………….…..107

Figure 4. The adipose tissue hypoxia-related markers. The protein

expression of HIF-1α and VEGF on eWAT……………………………………...108

Figure 5. Adipose and non-adipose-derived hormones. The protein

expression of leptin and ghrelin in plasma and leptin and GHS-R in eWAT.…109

Figure 6. Plasma and eWAT adipQ expression. Plasma total adipQ, HMW

adipQ form, HWM/total adipQ ratio and eWAT adipQ protein expression…...110

Figure 7. Plasma analysis and insulin sensitivity/resistance determination.

Plasma insulin, glucose levels, HOMA-IR and QUICKI……………………….111

Study II

Figure 1. Body weight, energy intake and relative fat depots weights…..133

Figure 2. Adipocyte-size profiling of adipocytes from WAT……………...134

xiv

Figure 3. Soleus, gastrocnemius muscle weights, total mass-body weight

ratio, and citrate synthase activity…………………………………………………135

Figure 4. Activation/phosphorylation of AMPK, IL-6, FNDC5 on skeletal

muscle and circulating irisin content…………………………………..…………..136

Figure 5. Beige and brown adipose-selective markers. Semi-quantitative

RT-qPCR analysis of Bmp7 mRNA, and expression of BMP7 protein. Quantitative

analysis for Tmem26, Prdm16 and Western Blot for UCP1 on

eWAT………………………………………………………………………………...138

Figure 6. The protein expression of PGC-1α, SIRT1, SIRT3, UCP2 and

FNDC5 on eWAT……………………………………………………………………139

Study III

Figure 1. Plasma glycerol and NEFA levels……………….……………….159

Figure 2. The mRNA and protein expression of lipid accumulation

regulators, AQP7 and FAT/CD36………………………………………………….160

Figure 3. The relative protein content of AMPK and ACC as well as their

phosphorylation at Thr 172 ans Ser 79, respectively……………………………..161

Figure 4. The protein expression of full-length and truncated SREBP1c and

the ratio between truncated and full-length SREBP1c……………………….…162

Figure 5. The protein expression of OXPHOS subunits………………….163

Figure 6. The protein expression of COX, TFAM, MFN1, MFN2 and

OPA1…………………………………………………………………………………164

xv

Study IV

Figure 1. Fatty acid relative content in eWAT triglycerides. The saturated

fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids…...188

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

Figure 4. The monocyte/macrophages infiltration and migration markers in

eWAT. The gene and protein expression of Ccl2/MCP1, F4/80 ……………..192

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

Figure 3. The expression of autophagy-related markers, Beclin-1, Lc3II,

p62………………………………………………………………………………...…215

Figure 4. The expression of apoptosis-related markers. Bcl-2, Bax, Beclin-

1/Bcl-2 ratio, Bcl-2/Bax ratio. Activity of initiator caspases, caspase-8 and

caspase-9, and effector caspase-3 ……………………………………………...216

CHAPTER III. General Discussion

Figure 1. Summary of the systemic, VAT and mitochondrial adaptations

induced by HFD and the preventive -VPA and therapeutic –ET- effect of

exercise……………………………………………………………………….233

xvii

List of Tables


Study II

Table 1. Primers sequences for RT-qPCR…………………………………134

Study III

Table 1. Animal characteristics and plasma analysis…………..…………160

Study IV

Table 1. Body weight, total energy intake, visceral adiposity, and adipocyte

size determinations…………………………………………………………………192

xix

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.

PALAVRAS-CHAVE: EXERCÍCIO, ADIPOSIDADE VISCERAL, MIOCINAS,

BEIGING, ADIPOCINAS

xxi

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.

KEYWORDS: EXERCISE, VISCERAL ADIPOSITY, MYOKINES, BEIGING,

ADIPOKINES

xxiii

List of Abbreviations

ACC Acetyl CoA

AdipQ Adiponectin

AMPK 5’AMP-activated protein kinase

AQP7 Aquaglyceroporin 7

ATG Autophagy-related genes

ATGL Adipose triglyceride lipase

ATP Adenosine triphosphate

BAT Brown adipose tissue

BDNF Brain-derived neurotrophic factor

BMI Body mass index

BMP7 Bone morphogenetic protein 7

COX Cytochrome c oxidase

CS Citrate synthase

DIO Diet-induced obesity

DLK1/PREF-1 Pre-adipocyte factor 1

ET Endurance training

eWAT Epididymal white adipose tissue

FA Fatty acid

FAT/CD36 Fatty acid translocase

FGF21 Fibroblast growth factor 21

xxiv

FNDC5 Fibronectin type III-domain containing 5

GH Growth hormone

GHS-R Growth hormone secretagogue receptor

GLUT4 Glucose transporter 4

HFD High-fat diet

HIF-1α Hypoxia-inducible factor 1 alpha

HMW AdipQ High molecular weight adiponectin

HOMA-IR Homeostasis model assessment of insulin resistance

HSL Hormone-sensitive lipase

IGF-I Insulin-like growth factor I

IKKβ IκB kinase beta

IL-10 Interleukin 10

IL-6 Interleukin 6

IR Insulin resistance

JNK c-Jun N-terminal kinases

LC3 Light chain 3

MDH Malate dehydrogenase

METs Metabolic equivalents

MFN1 Mitofusin 1

MFN2 Mitofusin 2

Mn-SOD Manganese superoxide dismutase

xxv

MUFA Monounsaturated fatty acids

mWAT Mesenteric white adipose tissue

NASH Nonalcoholic steatohepatitis

NEFA Non-esterified fatty acids

NF-κB Nuclear factor kappa B

OPA1 Optic atrophy 1

OXPHOS Oxidative phosphorylation

p38 MAPK p38 mitogen-activated protein kinase

PDK4 Pyruvate dehydrogenase lipoamide kinase isoenzyme 4

PE Physical exercise

PECK Phosphenolpyruvate carboxykinase

PGC-1α Peroxisome proliferator-activated receptor gamma coactivator 1-alpha

PPARγ Peroxisome proliferator-activated receptor gamma

PRDM16 PR domain containing 16

PUFA Polyunsaturated fatty acids

QUICKI Quantitative insulin sensitivity check index

RBP4 Retinol binding protein 4

RT Resistance training

RT-qPCR Reverse transcriptase real-time polymerase chain reaction

rWAT Retroperitoneal white adipose tissue

SFA Saturated fatty acids

xxvi

SIRT1 Sirtuin 1

SIRT3 Sirtuin 3

SNS Sympathetic nervous sympathetic

SREBP1 Sterol regulatory element-binding transcription factor 1

SVF Stromal vascular fraction

TFAM Mitochondrial biogenesis transcript factor A

TG Triglycerides

TNF-α Tumor necrosis factor alpha

U/S Unsaturated and saturated fatty acids ratio

UCP1 Uncoupling protein

VAT Visceral adipose tissue

VEGF Vascular endothelial growth factor

VO2 max Maximal oxygen consumption

VPA Voluntary physical activity

WAT White adipose tissue

CHAPTER I. General Introduction

Chapter I. General Introduction

3

1. Introduction

The prevalence of overweight and obesity, although preventable, are growing at

an alarming rate in developed and developing countries throughout the world

(Kelly et al., 2008). According to the World Health Organization, obesity has more

than doubled since 1980. Overall, more than one-third of children and

adolescents were overweight or obese in 2012, and an estimated 39% (more

than 1.9 billion) of adults were overweight and of these over 600 million were

obese, in 2014 (Organization, 2016). The high prevalence of overweighting and

obesity are risk factors for diabetes, cardiovascular and neurodegenerative

diseases, and cancer (Kelly et al., 2008). These life-long risks will affect both life

expectancy and life quality of the population besides imposing a huge economic

burden on health-care systems (Withrow & Alter, 2011).

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


4

mechanism that could be stimulated by distinct pharmacological and non-

pharmacological approaches to treat obesity and associated disorders.

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


5

glycerol and hepatic glucose production, as well as adipocyte hypertrophy

(Catalán et al., 2008). Both FAT/CD36 and AQP7 are important intracellular lipid

accumulation mediators and their regulation provides important information

regarding the underlying mechanisms of adipocyte hypertrophy and visceral

adiposity accumulation in obesity. In fact, several studies suggest that an

increased adipocyte size is associated with cellular metabolic abnormalities

rather than adipocyte number (i.e. adipogenesis), which, in turn, occurs in small

cells with low fat storage capacity (Heinonen et al., 2014). The metabolic

abnormalities associated with adipocyte hypertrophy compromise mitochondrial

metabolism, including altered mitochondria structure (Cummins et al., 2014),

reduced mitochondrial function and activity (Heinonen et al., 2014; Laye et al.,

2009), and mitochondrial DNA copy number (Dahlman et al., 2006), which could

lead to reduced substrate oxidation and impaired metabolic capacity in VAT.

Recent evidence have also demonstrated a relationship between adipocytes

inflammation and mitochondrial function (Vieira-Potter, 2014). Indeed, “sick”

adipocytes are characterized by having dysfunctional mitochondria, dysregulated

lipolysis and by contain “M1” macrophages, which are recruited to the VAT and

perpetuate the tissue inflammatory scenario by secreting pro-inflammatory

cytokines (Vieira-Potter, 2014). Therefore, at some point, mitochondrial function

seems to be involved in the pro-inflammatory signaling observed in obesity

conditions. For example, mitochondrial dynamics referred as the regulation of

mitochondrial morphologic alterations and distribution throughout mitochondria

life-cycle, which are strictly dependent on fusion and fission events (Hahn et al.,

2014), seem to play an important role in VAT metabolism (Mishra & Chan, 2016).

In fact, decreased expression of proteins involved in mitochondrial fusion,

particularly mitofusin 2 (MFN2), has been reported to contribute for reduced

mitochondrial function in some tissues, including skeletal muscle and liver of rats

fed with diet-induced obesity (DIO) (Goncalves et al., 2016; Lionetti et al., 2014).

However, to our best knowledge, the role of dynamic-related mechanisms in VAT

mitochondrial dysfunction in obesity is poorly investigated so far.

In addition to the marked adipose tissue metabolic alterations characterizing

obesity, systemic changes involving the secretion of several hormones and


6

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”


7

polarization state to a pro-inflammatory “M1” polarization state (Kawanishi et al.,

2015; Kawanishi et al., 2010). Recently, studies reported that the inflammatory

process may be associated, at least in part, to the fatty acids profile of VAT

triglycerides (Chan et al., 2015; Finucane et al., 2015; Oliveira et al., 2015), such

as saturated fatty acids (SFA) (Choi et al., 2014; Tousoulis et al., 2010),

monounsaturated fatty acids (Esser et al., 2015) and n6-PUFA (Johnson &

Fritsche, 2012). In fact, a fat-rich diet in SFA likely enhances circulating

biomarkers of inflammation in health individuals (Tousoulis et al., 2010). In vitro

macrophages exposed to SFA showed increased pro-inflammatory gene levels

and cytokine secretion, such as TNF-α and IL-6, and the chemokine CXCL1/KC

(Choi et al., 2014). The various oxidized forms of linoleic acid (C18:2n6) seem to

contribute to stimulate inflammation (Johnson & Fritsche, 2012), at least in part,

due to their role as precursor of arachidonic acid-mediated eicosanoid

biosynthesis and by reducing synthesis of anti-inflammatory eicosanoids from

eicosapentaenoic acid and docosahexaenoic acid (Fritsche, 2015); however,

others reported no inflammatory effects (Vaughan et al., 2015). Another

determinant mechanism that underlies obesity-induced inflammation is the

hypoxia that results from the aggregation of hypertrophied adipocytes, i.e.,

adipocytes become more distant from the vasculature in expanding VAT

(Trayhurn, 2014). In fact, several studies reported an increased hypoxia-inducible

factor (HIF-1α) expression, a key regulator of hypoxia responses, (Goossens et

al., 2011; Hosogai et al., 2007; Virtanen et al., 2002; Ye et al., 2007; Yin et al.,

2009) and its relationship with the presence of macrophages, which in turn

migrate to the hypoxic regions and alter their profile toward to a pro-inflammatory

state (Fujisaka et al., 2013). In this context, the hypoxic environment together

with the pro-inflammatory state had been related to the activation of adipocyte

cell death- and/or remodeling/quality control- related mechanisms (Benbrook &

Long, 2012; Salminen et al., 2013), such as apoptosis and autophagy. Generally,

apoptosis contributes to cell death, while autophagy is a pro-survival mechanism

whereby damaged organelles and proteins are degraded by lysosomes to

maintain intracellular homeostasis (Salminen et al., 2013). An upregulation of

apoptotic markers in obese animals (Alkhouri et al., 2010; Feng et al., 2011) and


8

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


9

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


10

higher impact reducing circulating leptin levels independently of body fat

reduction (Reseland et al., 2001). Discrepancies exist regarding the effects of ET

on AdipQ mRNA levels in obese rats, with studies revealing increases (Krskova

et al., 2012) or no alterations (Gollisch et al., 2009). HMW AdipQ, its more active

form, is more associated with IR when decreased (Lara-Castro et al., 2006).

Circulating levels of adipQ increased after an ET program in severely obese

adults (Bruun et al., 2006) and adolescents (Balagopal et al., 2005), without

changes in body weight (Balagopal et al., 2005). Moreover, the circulating ghrelin

alterations in response to physical exercise resulted in conflicting data. Increased

circulating ghrelin levels were demonstrated after long-term ET program in obese

individuals (Mason et al., 2015); however, reductions (Broom et al., 2009;

Ghanbari-Niaki et al., 2011) or no alterations (Ebrahimi et al., 2013) in the

expression of this protein were also observed. Overall, given the small number of

studies dedicated to understand the role of physical exercise on the expression

of these hormones/peptides in the context of obesity, the underlying mechanisms

are still not fully understood. Moreover, the influence that physical exercise exerts

on adipokines and other peptides/hormones also significantly affects VAT lipid

metabolism and mitochondrial function (Choe et al., 2016). Previous studies

suggested reduced lipolysis in isolated rat adipocytes in response to ET, which

is reflected in reduced circulating NEFA and glycerol levels (Chapados et al.,

2008; Pistor et al., 2015). One approach to analyze such increased intracellular

lipid accumulation is the detection of mediator proteins of this process.

Nevertheless, variation in the expression of FAT/CD36 and AQP7, important

intracellular lipid accumulation mediators, with physical exercise have been

poorly investigated. A study conducted by Lebeck and coworkers (Lebeck et al.,

2012) reported an increase (in women) and a decrease (in men) in the AQP7

protein expression after 10-weeks of ET, while Trachta and coworkers (Trachta

et al., 2014) found no alterations. An increased mitochondrial oxidative capacity

in VAT could reduce the availability of NEFA for esterification (Sutherland et al.,

2009) and the utilization of glucose for the formation of glycerol in white

adipocytes (Pistor et al., 2015). However, the role of physical exercise regarding

NEFA oxidation and mitochondrial functions in white adipocytes is less studied.


11

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


12

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.

Another issue of particular concern is the effectiveness of exercise used as a

preventive or a therapeutic strategy in obesity. The prevention is focused in the

promotion of a health-related life-style that precludes the onset of the pathology,

while therapy refers to the promotion of metabolic conditions that offset the

progression of the disease. In light of this definition, most animal studies that

intended to analyze the effects of physical exercise on obesity-induced adipose

tissue dysfunction are designed in a preventive perspective, as exercise


13

protocols are implemented in parallel with the progression of obesity. In addition,

in the context of the studies presented in this dissertation, we attempt to analyze

the effectiveness of physical exercise as a preventive and therapeutic tool in the

control and modulation of obesity-mediated mechanisms.

2. Aims

Considering the relevance of the obesity management in the context of a global

pandemic, as well as the systemic and VAT-related mechanisms involved in this

pathological condition, and the recognition of physical exercise as a non-

pharmacological strategy able to attenuate several health-deleterious conditions,

the main purpose of this dissertation was to analyze the impact of two physical

exercise models, VPA and ET, against high-fat diet (HFD)-induced VAT adverse

metabolic and endocrine consequences in rodents.

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

To analyze the role of physical exercise against adiposopathy and related

endocrine responses in rats submitted to a HFD. In particular, to investigate the

effects of VPA and ET on:

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.


14

Study 2

To analyse the impact of physical exercise on myokines and its hypothetical

modulator effect on brown-like phenotype in WAT of HFD feeding rats.

Specifically, to study the effects of VPA and ET on:

i) myokines (IL-6 and FNDC5) in skeletal muscle and circulating irisin

content;

ii) beige (Tmem26) and brown (BMP7, Cidea, Prdm16 and UCP1)

adipose-selective markers in eWAT;

iii) browning-related molecules [PGC-1α, Sirtuins 1 and 3, uncoupling 2

(UCP2) and FNDC5] in eWAT.

Study 3

To investigate the effects of physical exercise on lipid accumulation regulators

and mitochondrial content and biogenesis in VAT of rats submitted to a HFD. In

particular, the effects of VPA and ET on:

i) plasma glycerol and NEFA levels;

ii) lipid accumulation mediators (AQP7 and FAT/CD36) in eWAT;

iii) mitochondrial oxidative phosphorylation (OXPHOS) subunits,

mitochondrial biogenesis and fusion-related proteins in eWAT.

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:

i) fatty acids profile in eWAT TG;

ii) circulating and eWAT pro- and anti-inflammatory cytokines (IL-6, TNF-

α and IL-10);


15

iii) macrophage migration and infiltration markers (MCP-1 and F4/80) in

eWAT.

Study 5

To analyse the effects of ET on autophagy and apoptotic signaling in VAT of HFD-

fed rats. In particular, the effects of ET on:

i) autophagy-related markers [Beclin-1, light-chain 3 II (LC3II) and p62],

in eWAT;

ii) pro- and anti-apoptotic molecules (Bax and Bcl-2);

iii) caspases 3, 8 and 9-like activities;

iv) adipocyte differentiation.

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CHAPTER II. Theoretical Background

Chapter II. Theoretical Background

31

Review Article

The effects of physical exercise on white adipose tissue: browning and

metabolic adaptations

Sílvia Rocha-Rodrigues1, António Ascensão1, José Magalhães1

1 CIAFEL - Research Centre in Physical Activity, Health and Leisure, Faculty of

Sport, University of Porto, Porto, Portugal;

Submitted


32

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.

KEYWORDS: endurance training, visceral adiposity, adipocyte turnover


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Introduction

Physical exercise (PE) is a well-recognized non-pharmacological strategy to

prevent diet-induced fat mass gain by increasing energy expenditure (Bajer et al.,

2015; Way et al., 2016). Moreover, PE provides several positive adaptations in

skeletal muscle function, including improvements on insulin sensitivity, utilization

of metabolic substrates, protection against oxidative insults (Bajer et al., 2015;

Murphy et al., 2014), as well as signaling to other organs, such as white adipose

tissue (WAT). Recently, numerous studies reported that PE-induced myokines

act locally in the skeletal muscle in an autocrine/paracrine manner, but also are

released into circulating as endocrine factors, mediating the acute and chronic

physiological benefits of PE in distant organs (Rodríguez et al., 2016). The

recently proposed myokine has gained considerable attention as a potential

therapeutic agent for the treatment of obesity. Irisin is produced from the cleavage

of fibronectin type III domain-containing protein 5 (FNDC5) secreted by skeletal

muscle in response to PE, and has the ability to induce a brown adipocyte-like

phenotype in WAT (Bostrӧm et al., 2012), which lead to heat loss and

consequently increase energy expenditure (Bostrӧm et al., 2012; Wu et al.,

2014). Furthermore, distinct PE modalities have been suggested to promote

acute and/or chronic adaptations on adipokines, adipogenesis, lipid and glucose

metabolism, mitochondrial content and biogenesis and apoptotic signaling (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), highlighting the relevance of WAT in

whole-body adaptions induced by PE and a promising target to treat obesity and

its comorbidities.

1. White adipose tissue

1.1 Body fat distribution: implications for obesity

The WAT adipocytes are specialized in the storage of energy as triglycerides

(TG). Since the discovery of leptin and other adipose-derived factors, collectively

designed as adipokines, WAT not only is considered as an energy reserve tissue

but also as an endocrine organ, which affects the inflammation and immune


34

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).

Although genetic factors may contribute to the variance of visceral adiposity

accumulation, in most cases the combination of positive caloric intake and

sedentary lifestyle is the primary cause and leads to an adipocyte hypertrophy

and visceral adiposity accumulation (Lopes et al., 2016; Ye et al., 2007). In this

context, pathological increased visceral adiposity, also referred as adiposopathy

or “sick fat” (Bays et al., 2008), typically results in adverse metabolic and

endocrine consequences (Jacobs et al., 2016; Lopes et al., 2016). Generally,

lipolysis in “sick fat” is higher than in “healthy fat”, leading to elevated NEFA levels

in obese individuals with adiposopathy (Rodriguez et al., 2014). The excess of

circulating NEFA will reach other glucose-metabolizing tissues, such as

pancreas, skeletal muscle and liver, supporting that adiposopathy, but not simple

obesity, is linked to the progression of obesity-associated complications, such as

type 2 diabetes (Bays, 2012). In fact, it is now recognized that adiposopathy is

associated with a local inflammatory state, which becomes systemic through the

release of numerous pro-inflammatory mediators, including interleukin 6 (IL-6),

and tumor necrosis factor (TNF)-α, into the bloodstream (Lopategi et al., 2016).

The pro-inflammatory mediators are initially secreted by the “sick fat”, but also by

macrophages infiltrating adipose tissue (Kawanishi et al., 2013; Kawanishi et al.,

2015). In VAT, diet-induced obesity (DIO) promoted a phenotypic switch of


35

macrophages from the M2 polarization state, which inhibits secretion of anti-

inflammatory cytokines, into the M1 polarization state that produces large

amounts of pro-inflammatory cytokines (Kawanishi et al., 2015; Kawanishi et al.,

2010).

2. Physical exercise-induced adaptations on visceral adipose tissue mass

2.1 Adipocyte turnover

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


36

type I myofibers, was able to modulate adipose progenitor activity, supporting a

link between skeletal muscle and adipose progenitors (Zeve et al., 2016). In

chronically exercised skeletal muscle, when circulating nutrients are provided by

mature white adipocytes, there is no need for newly adipocytes formation, and

thus adipose progenitors may remain in a quiescent state (Zeve et al., 2016). In

fact, it seems that muscle, in a non-autonomous manner, regulates adipose

progenitor homeostasis. These findings support a humoral link between skeletal

muscle and adipose progenitors and highlight a role for muscle-derived secreted

factors in the manipulation of adipose stem cell function (Zeve et al., 2016).

Regarding WAT-related quality control and apoptotic signaling, 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). An increase of anti-apoptotic Bcl-xl expression in

mature epididymal adipocytes in response to high-intensity ET (Sakurai et al.,

2005) and the occurrence of apoptotic nuclei in fat cells were reduced after 12-

wks of ET were reported (Sertie et al., 2013). In line, unpublished data from our

laboratory revealed that 8-wks of ET reverted HFD-induced apoptosis signaling

in VAT (Rocha-Rodrigues et al.). Moreover, one study showed that 9 wks of ET

increased autophagy-related markers, such as microtubule-associated protein

1A/1B-light chain 3 (LC3) and autophagy-related gene (ATG)7, in WAT and SVF

cells of lean rats (Tanaka et al., 2015). In context of obesity, an increased

accumulation of autophagosomes, reduced mechanistic target of rapamycin

(mTOR) signaling, and an improved autophagy-related markers, such as ATG5,

ATG7, ATG12, beclin-1 and LC3 were observed in obese individuals (Kosacka

et al., 2015; Kovsan et al., 2011; Mikami et al., 2012; Rodríguez et al., 2012).

Markers of autophagy are correlated with whole body adiposity, visceral adiposity

and adipocyte hypertrophy (Kovsan et al., 2011; Rodríguez et al., 2012). Some

mechanisms have been described to be involved in the obesity-induced altered

autophagy: i) insulin represents a major regulator of autophagy, being insulin

resistance a potential activator of this process; ii) ghrelin also act as negative


37

regulator of autophagy in human adipocytes through the downregulation of ATG5

and ATG7 (Purnell et al., 2003); and iii) the inflammatory response stimulated by

the TNF-α and c-Jun N-terminal kinases (JNK)-dependent endoplasmic reticulum

(ER) stress induces adipocyte autophagy, being both processes upregulated in

obesity (Rodríguez et al., 2012; Yin et al., 2015). However, data from our group

(unpublished) demonstrated that 8 wks of ET increased p62 protein expression

in eWAT in HFD-fed animals, suggesting that ET suppressed autophagy (Rocha-

Rodrigues et al.). Furthermore, the increased autophagic adapter p62 in

response to ET might provide mechanistic insight regarding PE-induced positive

adaptions in VAT (Ahn & Kim, 2014), as p62 inhibits extracellular regulated

protein kinase (ERKs) activity and function by direct interaction (Rodríguez,

Duran, et al., 2006). These findings suggest that suppressed autophagy activity

induced by PE may constitute a mechanism by which exercise potentiate positive

adaptations in VAT in order to prevent adipocyte hypertrophy and inflammation

in the context of obesity.

Angiogenesis, a complex process that involves the participation of multiple

angiogenic factors, also play an important role in adipogenesis (Corvera &

Gealekman, 2014). An upregulation of vascular endothelial growth factor (VEGF)

gene expression and its receptor have been reported in adipocytes and SVF cells

of exercised rats (Baynard et al., 2012; De Matteis et al., 2013; Hatano et al.,

2011; Stanford et al., 2015). However, data from Hatano and co-workers (Hatano

et al., 2011) suggest that VAT angiogenesis might not be dependent on adipocyte

size and/or number as an increase of endothelial cells number per adipocyte and

a decrease of adipocyte size and number were described after 9-wks of ET.

Altogether, these findings suggest that PE modulates adipocyte turnover, which

results in decreased visceral adiposity through an inhibition of adipogenesis of

adipocyte precursor cells, but without compromising angiogenesis.


38

3. Physical exercise-induced adaptations on adipokines secretion

3.1 Adiponectin (adipQ)

As an important secretory organ, WAT synthesizes and secretes numerous

hormones, cytokines and other factors that play critical roles in several

physiological processes, such as energy expenditure and metabolism,

inflammation, insulin sensitivity and immunity (Harms & Seale, 2013; Mather et

al., 2008; Rodríguez, Becerril, et al., 2015). In the context of obesity, reduced

levels of circulating adipQ in obese mice (Fukao et al., 2010) and individuals

(Lara-Castro et al., 2006) are inversely correlated with BMI, visceral adiposity,

and indices of IR (Choe et al., 2016). In contrast, circulating adipQ is elevated in

healthy individuals in response to a high-intensity resistance training - RT (Davis

et al., 2015), ET (Bluher, Williams, et al., 2007; Huang et al., 2015) and a program

using both ET and RT (Markofski et al., 2014). However, conflicting data have

been published regarding the impact of distinct models and intensities of exercise

on adipQ levels. For instance, regarding RT effects, some data revealed an

increased adipQ levels after moderate and high-intensity, but not after low-

intensity sessions (Fatouros et al., 2005). Moreover, a 6-mo ET regimen (~45min

at 65-80% peak O2 consumption) increased insulin sensitivity despite no changes

in plasma adipQ (Hulver et al., 2002) and no variations on body weight or fat

mass were observed. In line, some studies demonstrated that low or moderate-

intensity acute exercise (<65% VO2 max) had no impact on circulating adipQ

levels (Ferguson et al., 2004; Jamurtas et al., 2006; Punyadeera et al., 2005),

which suggests that high-intensity seems to be require for the modulation of the

adipokine levels. Surprisingly, a study with obese subjects reported an increase

in adipQ concentrations 30 minutes after acute bouts of exercise at high (75%

VO2 peak), but also at low (50% VO2 peak) intensities (Saunders et al., 2012).

Accordingly, other studies reported increased circulating adipQ in impaired

glucose tolerance and type 2 diabetic individuals (Bluher, Williams, et al., 2007;

Oberbach et al., 2006), as well as in severely obese adults (Bruun et al., 2006)

and adolescents (Balagopal et al., 2005), without changes in body weight

(Balagopal et al., 2005) after a chronic ET program. However, others studies

observed no variations on adipQ in response to ET (Christiansen et al., 2010;


39

Nassis et al., 2005; O'Leary et al., 2006) in obese individuals. In an attempt to

clarify this intriguing issue, some studies support the hypothesis that the high

molecular weight (HMW) adipQ is the more active form of this protein (Hada et

al., 2007; Waki et al., 2003), thus with particular relevance in insulin sensitivity

and obesity. Furthermore, some studies did not reveal significant changes on

circulating HMW levels after acute exercise in young (Numao et al., 2008), older

insulin-resistant (O'Leary et al., 2007) and obese individuals (Numao et al., 2011);

while others observed an increase in its levels in subjects with normal glucose

tolerance (Numao et al., 2011).

Regarding adipose tissue, the gene expression of adipQ was elevated in

response to chronic ET in SAT of obese individuals (Bruun et al., 2006;

Christiansen et al., 2010; Moghadasi et al., 2013) and in retroperitoneal (WAT) of

Zucker rats (Krskova et al., 2012), while no changes were reported in epididymal

(Krskova et al., 2012) and mesenteric WAT depots (Svidnicki et al., 2015). Taken

together, data suggest that PE seems to modulate adipQ secretion by adipose

tissue in a VAT depot-dependent manner. The PE-induced increase of adipQ

levels has been linked to an improved inflammatory response as this adipokine

exerts several anti-inflammatory effects, including the inhibition of endothelial

nuclear factor kappa B (NF-kB), suppression of phagocytic activity, and TNF-α

production in macrophages (Lim et al., 2014), but also the increased production

of anti-inflammatory cytokines, such as IL-10 (Wolf et al., 2004). Of note is that

adipQ synthesis and secretion are partly controlled by other hormones, including

insulin and cathecolamines (Blumer et al., 2008; Fasshauer et al., 2003), and

negatively influenced by pro-inflammatory cytokines, such as IL-6 (Fasshauer et

al., 2003) and TNF-α (Bruun et al., 2003).

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.,


40

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).

Regarding exercise, a study performed in rowers showed a decline in circulating

leptin concentrations immediately after maximal rowing (Jurimae & Jurimae,

2005). Moreover, reduced leptin levels were observed after one bout of long-term

ET session (>60 min) that stimulates NEFA release (Desgorces et al., 2004;

Duclos et al., 1999), or after exercise with significant levels of energy expenditure

(higher than 800 kcal) (Leal-Cerro et al., 1998; Zaccaria et al., 2002); however,

several others studies reported no effects of acute exercise in the levels of leptin

of healthy trained or untrained individuals (Ferguson et al., 2004; Olive & Miller,

2001; Perusse et al., 1997; Weltman et al., 2000). Regarding chronic exercise,

both ET (Hickey et al., 1997; Perusse et al., 1997) and RT (Fatouros et al., 2005)

programs seem to result in reduced fat mass accompanied by lower leptin

concentrations in healthy individuals. Nevertheless, even though several VPA

and ET interventions have been described as effective in decreasing leptin levels

in plasma and VAT of distinct obese models (Bradley et al., 2008; Chapados et

al., 2008; Gollisch et al., 2009; Huang et al., 2010; Jenkins et al., 2012; Linden et

al., 2014; Zachwieja et al., 1997), other studies reported no changes (Baynard et

al., 2012; Jenkins et al., 2012; Krskova et al., 2012; Vieira, Valentine, Wilund,

Antao, et al., 2009; Vieira, Valentine, Wilund, & Woods, 2009). Eventually, the

intensity of the exercise programs and the efficacy concerning changes in body

weight and fat loss might explain, at least in part, some of the discrepancies. In

fact, a 6-mo RT program in obese individuals decreased circulating leptin levels

in all exercise sessions compared to pre-training levels, being the magnitude of

the decrease dependent on the exercise intensity (Fatouros et al., 2005). In

addition, reduced leptin levels have been related to fat mass loss after ET

programs (Halle et al., 1999; Hsieh & Wang, 2005; Miyatake et al., 2004; Okazaki

et al., 1999; Polak et al., 2006; Reseland et al., 2001); however, other studies

also demonstrated that lower plasma leptin levels was independent of body

weight, fat mass or adipose tissue mRNA expression of leptin after a RT program


41

(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

Similarly to other adipokines, ghrelin (non-adipose-derived hormone) has been

suggested to have an important role in the regulation of energy expenditure,

dietary behavior, and insulin sensitivity (Choe et al., 2016; Flores et al., 2006). In

fact, this secretagogue peptide hormone that stimulates growth hormone (GH)

release has been described to function as an adiposity signal since its circulating

levels inversely correlate with body mass index (Shiiya et al., 2002) and were

increased during weight loss (Espelund et al., 2005; Leidy et al., 2004). However,

in obesity conditions, the ghrelin production has been reported to be markedly

suppressed in obese individuals (Oner-Iyidogan et al., 2007; Tschop et al., 2001)

and in animals submitted to DIO (Handjieva-Darlenska & Boyadjieva, 2009).

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).

The PE-induced alterations in adipokines and ghrelin in obesity conditions may

also be dependent on changes in fat-free mass and on some specific

hormones/factors, such as cathecolamines and insulin. In fact, several studies

reported small increases in fat-free mass following ET programs (Markofski et al.,


42

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.

4. Effects of physical exercise on lipid and glucose homeostasis

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,


43

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).


44

5. Physical exercise-induced adaptations on inflammatory-related

mechanisms

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).

Regarding the impact of PE in this chronic low-grade inflammatory state, few

studies have suggested that exercise attenuates obesity-induced activation of the

IKKβ/NF-kB pathway. Rats fed with a HFD and submitted to acute (Oliveira et al.,

2011) or chronic (da Luz et al., 2011) exercise reduced the activation of the NF-

kB pathway in epididymal WAT along with an attenuation of the ER stress and an

improvement of the insulin sensitivity. Also, both acute and chronic swimming

exercise mitigated obesity-induced adipose tissue activation of toll-like receptor

4 (TLR4), which is an activator of both IKKβ and JNK. These findings suggest

that PE-induced VAT adaptations involve the suppression of NEFA-induced

activation of TLR4 signaling as TLR4 is activated by NEFA (Rodriguez et al.,

2014). The reduced TLR4 expression in adipose tissue is likely the consequence


45

of the PE-induced suppression of M1 macrophage infiltration (Ito et al., 2008),

and/or of the phenotype switching from pro-inflammatory M1 macrophages to

anti-inflammatory M2 macrophages (Bradley et al., 2008; Christiansen et al.,

2010; Haczeyni et al., 2015; Kawanishi et al., 2015; Kawanishi et al., 2010;

Linden et al., 2014; Vieira, Valentine, Wilund, Antao, et al., 2009; Vieira,

Valentine, Wilund, & Woods, 2009; Wainright et al., 2015). The resident or M2

(“alternatively activated”) macrophages are characterized by the expression of

the chitinase-like protein Ym1, arginase and the production of anti-inflammatory

cytokines (e.g. IL-10, IL-1Ra), while the recruited or M1 (“classically activated”)

macrophages express the CD11 surface marker, iNOS and produce high levels

of pro-inflammatory cytokines (e.g. TNF-α, IL-6) (Hotamisligil, 2006). In contrast,

a shift to the activation state of adipose tissue macrophages from an M2-polarized

state to an M1 pro-inflammatory state (Haczeyni et al., 2015; Kawanishi et al.,

2015; Vieira, Valentine, Wilund, Antao, et al., 2009) has been observed in DIO

animals. In humans, macrophage infiltration is correlated with both increased

adipocyte size and BMI (Weisberg et al., 2003). Moreover, the majority of

macrophages in obese adipose tissue aggregate in “crown-like structures”

completely surrounding dead (necrotic-like) adipocytes and scavenging

adipocyte debris (Weisberg et al., 2003), which has been demonstrated to be

reverted by VPA in obese mice (Haczeyni et al., 2015; Wainright et al., 2015).

Kawanishi and co-workers (Kawanishi et al., 2013) found that CD8+ T cells, which

have a critical role in adipose tissue inflammation and are abundant in adipose

tissue of DIO animals, were suppressed by 12-wks of ET. Moreover, exercise

regimens reduced both RANTES and C-C chemokine receptor type 5 in the

adipose tissue of obese individuals (Baturcam et al., 2014), and decreased

circulating pro-inflammatory markers, such as IL-6 (Baturcam et al., 2014; Bruun

et al., 2006; Donges et al., 2013; Kohut et al., 2006; Leggate et al., 2012;

Oberbach et al., 2008), TNF-α (Bruun et al., 2006; Donges et al., 2013; Kohut et

al., 2006; Straczkowski et al., 2001) and pJNK (Baturcam et al., 2014).


46

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

Regarding the effects of PE on TNF-α concentrations, an important

pathophysiological player in obesity, the literature is also rather inconsistent.

Plasma TNF-α levels increased immediately after an acute bout of moderate

exercise in healthy males and females (Ferguson et al., 2004; Hojbjerre et al.,

2007), which may be related to the lipolytic effects of TNF-α in adipose tissue

(Kawakami et al., 1987; van Hall et al., 2003). In fact, an increased TNF-α


47

concentration may help to provide lipids, possibility to be up taken by skeletal

muscle and replenish energy stores after a bout of acute exercise (Gollisch et al.,

2009). However, the combination of both ET and RT sessions did not affect TNF-

α concentrations in healthy women although increases in total energy

expenditure and activity-related energy expenditure were higher in these women

compared to other groups engaged in lighter sessions (Hunter et al., 2013).

In obesity, macrophage-infiltrated VAT is the main site of TNF-α production and

plays a crucial role in the pathogenesis of obesity and associated disorders

(Hotamisligil, 2006). Some studies reported that increased TNF-α expression in

VAT from DIO animals was suppressed by VPA (Bradley et al., 2008; Huang et

al., 2010) and ET (Kawanishi et al., 2015; Kawanishi et al., 2010; Linden et al.,

2014; Speretta et al., 2012) interventions, whereas others reported no changes

(Baynard et al., 2012; Krskova et al., 2012; Marcinko et al., 2015; Shen et al.,

2015; Svidnicki et al., 2015). However, a contrasting increase on TNF-α levels

after chronic ET and VPA in the mesenteric fat depot of insulin-resistant rats

(Nara et al., 1999) and in peritoneal macrophages of obese Zucker rats (Martin-

Cordero et al., 2011) was also found, suggesting a compensatory mechanism

induced by PE that may control metabolic homeostasis in order to modulate

hyperinsulinemic state (Martin-Cordero et al., 2011; Nara et al., 1999).

Interestingly, this effect was not observed when the acute exercise was

performed by the previously trained animals, which suggest a chronic exercise-

induced adaptation to respond to an acute bout of exercise (Martin-Cordero et

al., 2011). Regarding the effects of RT-related exercise in DIO animals, a

decreased Tnfα gene expression in VAT after 8 weeks of climbing exercise was

observed (Speretta et al., 2012), while other study showed no changes on this

adipocytokine expression after isometric strength training (Mardare et al., 2016).

Likewise, studies using obese subjects reported conflicting data regarding the

impact of distinct exercise regimens on TNF-α expression. For example, the Tnfα

gene expression in adipose tissue of overweight and obese subjects remained

unchanged after an acute bout of exercise (Christiansen et al., 2013; Hojbjerre et

al., 2007). Moreover, some studies using programs of ET reported a decrease

(Donges et al., 2013; Kohut et al., 2006; Kondo et al., 2006; Straczkowski et al.,


48

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).

Under obesity conditions, an increased reactive oxygen species production in

adipocytes may also be associated with the dysregulated expression of

inflammatory-related adipokines (Sakurai et al., 2013). As acute exercise

momently elevates oxidative stress, the chronic adaptation of VAT against PE-

induced oxidative stress may be mediated by increases in antioxidant system via

increases in the antioxidant enzyme manganese superoxide dismutase (Mn-

SOD) and possibly decreases in the pro-inflammatory adipokines. In fact, studies

have shown that ET significantly lower levels of lipid peroxidation in VAT of lean

(Sakurai et al., 2005) and in DIO rats (Farias et al., 2012) and elevated protein

levels of Mn-SOD in the epididymal WAT of ET group as well in lean (Sakurai et

al., 2009; Sakurai et al., 2005) and DIO rats (Farias et al., 2012). In those studies,

the protein content of TNF-α, MCP-1 as well as the phosphorylation of ERK,

which is activated by reactive oxygen species and is important for MCP-1

expression, were significantly reduced in the epididymal WAT of the ET rats.

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.


49

5.4 Hypoxia

Adipose tissue hypoxia is a determinant mechanism that underlie obesity-

induced inflammatory alterations (Trayhurn, 2014) and has been associated with

increases in the expression of the hypoxia-inducible factor (HIF)-1α in obese

animals (Ahn & Kim, 2014; Hosogai et al., 2007; Linden et al., 2014; Ye et al.,

2007; Yin et al., 2009) and humans (Goossens et al., 2011; Virtanen et al., 2002).

In fact, the reduced blood flow per vessel rather than cell size appears to be the

major player for VAT hypoxia in obesity (Trayhurn, 2014), most apparent in

capillaries containing adherent leucocytes and platelet aggregates (Trayhurn,

2014). Regarding the effects of PE on the HIF-1α expression on VAT , some

studies showed that ET reduced HIF-1α in DIO animals (Ahn & Kim, 2014; Linden

et al., 2014) and others showed an increase in the gene expression of Vegf and

its receptor in adipocytes and SVF cells of lean rats (Baynard et al., 2012; De

Matteis et al., 2013; Hatano et al., 2011; Stanford et al., 2015) and DIO animals

(Disanzo & You, 2014; Stanford et al., 2015; Yan et al., 2012), which is consistent

with a metabolic and morphological attempt to reduce hypoxia and ameliorate the

oxygen flux to adipose tissue. More studies are yet needed to further elucidate

the molecular mechanisms involved in the process.

5.5 Fatty acids

Recent studies have reported that obesity-related inflammation may be

associated with the dietary FA composition, e.g., that saturated or unsaturated

FA may have an important influence on the inflammatory and macrophage

polarization states in the context of obesity (Oliveira et al., 2015; Vaughan et al.,

2015). In vitro studies showed that macrophages exposed to saturated fatty acids

(SFA) increased pro-inflammatory gene expression and cytokine secretion, such

as TNF-α, IL-6, and the chemokine CXCL1/KC (Choi et al., 2014). In contrast,

n3-polyunsaturated fatty acids (PUFA) suppressed these inflammatory effects on

monocytes/macrophages (de Sa et al., 2016a; Ghadge et al., 2016; Monk et al.,

2015; Oliveira et al., 2015). Moreover, the effects of the palmitoleic acid


50

(C16:1n7) in the regulation of systemic metabolic homeostasis has been explored

(Cao et al., 2008); however the findings are controversial. Chan et al (Chan et al.,

2015) reported that palmitoleic acid reversed the pro-inflammatory gene

expression and cytokine secretion in bone marrow-derived macrophages from

DIO mice, and also increased anti-inflammatory genes, such as Il10,

characteristic of M2 macrophages. However, other studies (Guo et al., 2012)

showed that this recently considered adipokine involved in the lipid-mediated

endocrine network, increased the hepatic deposition of triglycerides (TG).

Regarding the impact of exercise on TG composition, acute swimming exercise

induced significant increases in palmitoleic (C16:1n7) and linoleic (C18:2n6) FA

and U/S ratio, as well as significant decreases in palmitic (C16:0), oleic (C18:1n9)

and arachidonic (C20:4n6) FA of rat SAT (Dvorakova & Bass, 1970). However,

TG from SAT did not change during 30 min of cycling or after 15 min of recovery

(Petridou & Mougios, 2002). On the other hand, chronic ET decreased adipose

tissue monounsaturated fatty acids (MUFA) in humans (Danner et al., 1984;

Sutherland et al., 1981) and rats (Bailey et al., 1993; Wirth et al., 1980), and

increased the percentage of long chain and PUFA (Bailey et al., 1993; Petridou

et al., 2005; Thorling & Overvad, 1994; Wirth et al., 1980), suggesting a

stimulation of mechanisms related with FA chain elongation, polydesaturation

and/or depressed monodesaturation (Petridou et al., 2005; Thorling & Overvad,

1994; Wirth et al., 1980). Nevertheless, data are still scarce and the findings are

not consistent (Rocquelin & Juaneda, 1981; Thorling & Overvad, 1994). Mardare

and co-workers (Mardare et al., 2016) showed that 10-wks of ET or RT did not

induce alterations in FA profile in standard chow-fed animals, whereas both RT

and ET programs affected fatty acid profile in adipose tissue from obese rats. In

these obese animals, the ET and RT regimens decreased n6-PUFA (C18:2n6

and C20:4n6) and palmitic (C16:0) FA. The vaccenic (C18:1n7) acid, an isomer

of oleic acid increased in response to RT but remained unchanged after an ET

program.

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


51

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).

6. Effects of physical exercise on white adipose tissue “brown” signaling

6.1 “Brown”-like white adipose tissue

Recent studies demonstrated that in response to proper physiological stimuli,

such as chronic cold exposure (Petrovic et al., 2010), PE (Bostrӧm et al., 2012;

Fain et al., 2013; Tiano et al., 2015; Wu et al., 2014), peroxisome proliferator-

activated receptors γ (PPARγ) agonists or β-adrenergic stimulation (Ishibashi &

Seale, 2010), a brown adipocyte-like phenotype may be aroused in classical

white adipocytes, being the increased abundance of these type of cells a process

designed as “browning” or “beiging”.

Regarding PE, animal studies data showed that browning-associated genes,

such as uncoupling protein 1 (Ucp1) and PR domain containing 16 (Prdm16),

increased its expression in both SAT and VAT in response to an acute bout of

exercise (Bostrӧm et al., 2012), ET (Tiano et al., 2015; Wu et al., 2014) or VPA

(Cao et al., 2011; Haczeyni et al., 2015; Tiano et al., 2015). Moreover,

morphological alterations in SAT, such as increased UCP1 immunofluorescence

with multilocular cells, were also observed after 11 days of VPA in HFD-fed mice

over 6 weeks (Stanford et al., 2015). Accordingly, a humans-based study found

that the combination of ET and RT over 12 weeks promoted a mild SAT increased

expression of the “beige”- and “brown”-associated genes, transmembrane protein

26 (Tmem26) and Ucp1, respectively (Norheim et al., 2014). However, so far,

most studies performed in humans (Besse-Patin et al., 2014; Norheim et al.,

2014; Ronn et al., 2014) failed to clearly demonstrate the browning phenomenon

in response to PE. Distinct important factors, such as type, duration and intensity


52

of exercise, ambient temperature, genetic factors, age, the pre-training body

mass index (BMI), and the initial cardiorespiratory fitness levels of the individuals

could, at least in part, explain the discrepant data between animals and human

studies.

As an important transcriptional coactivator involved in energy metabolism (Canto

& Auwerx, 2009), the peroxisome proliferator-activated receptor gamma

coactivator 1α (PGC-1α) has been associated to the increased mitochondrial and

fatty acid oxidation in both SAT and VAT, and also to browning, preventing body

weight gain and improving glucose tolerance in HFD-fed mice (Stanford et al.,

2015; Tiano et al., 2015). Another metabolic sensor closely involved in regulation

of the process is the sirtuin 1 (SIRT1), a NAD+-dependent type III deacetylase

(Canto & Auwerx, 2009). SIRT1 activates PGC-1α and deacetylates two critical

lysine residues on PPARγ in adipocytes, which contributes to increase energy

expenditure and seems to promote brown adipocyte-like phenotype in VAT

(Qiang et al., 2012). Data from our group showed that ET-induced increases in

PGC-1α and SIRT1 protein content in VAT were associated with the increased

expression of “browning” markers and their regulators, such as bone

morphogenetic protein 7 (BMP7) in conditions of pre-exiting obesity (Rocha-

Rodrigues, Rodriguez, et al., 2016a). The BMP7 activates a full program of

brown-related adipogenesis, including the induction of mitochondrial biogenesis

via p38 mitogen-activated protein kinase (p38 MAPK) and PGC-1α-dependent

pathways, increase the expression of the early regulator of brown fat fate

PRDM16, brown-specific marker UCP1, and adipogenic transcription factors

PPARγ and CCAAT/ enhancer-binding proteins [28]. Clearly more studies are still

needed to unravel the inconsistencies found in literature as well as the precise

regulatory mechanisms regarding the potential impact of physical exercise in

human’s white adipose tissue “brown” signaling.

6.2 Myokines

Notwithstanding the gaps regarding the effects of physical exercise on brown

adipocyte-like phenotype response, some hypotheses have been proposed for


53

the underlying molecular mechanisms. An increased number of studies suggest

that besides some exercise-induced myokines that act locally in the muscle

through autocrine/paracrine mechanisms, others, such as brain-derived

neurotrophic factor (BDNF) (Cao et al., 2011), IL-6 (Knudsen et al., 2014),

meterion-like (Rao et al., 2014) or irisin (Bostrӧm et al., 2012) are released into

circulation as endocrine factors and may drive important stimuli ultimately leading

to a brown adipocyte-like phenotype in WAT. In fact, an overexpression of the Il6

gene mediated by exercise increased the UCP1 gene and protein expression in

rat brown adipose tissue (BAT) (Li et al., 2002) and SAT (Knudsen et al., 2014).

Moreover, the ET-induced increase in muscle IL-6 levels was strongly correlated

with brown adipocyte-like phenotype markers expression and regulators in HFD-

fed rats (Rocha-Rodrigues, Rodriguez, et al., 2016a). Nevertheless, among the

myokine the potentially regulate browning adipocyte-like phenotype, irisin is the

one that has received more attention in literature and can be seen in the following

subsection.

6.3 Irisin

Fibronectin type III domain-containing protein 5 (FNDC5) is produced by skeletal

muscle, proteolytically cleaved and released into circulation as irisin. It was

demonstrated that irisin is elevated after 3-wks of VPA (Bostrӧm et al., 2012;

Roca-Rivada et al., 2013), acute exercise (Brenmoehl et al., 2014; Liu et al.,

2015), and chronic ET and RT programs (Kim et al., 2015; Rocha-Rodrigues,

Rodriguez, et al., 2016b; Tiano et al., 2015; Wu et al., 2014; Zhou et al., 2015).

In fact, studies with rodents revealed that circulating irisin levels were acutely

overexpressed immediately after one bout of strenuous exercise, and associated

with increased oxidative stress markers (Brenmoehl et al., 2014). As acute

vigorous exercise is related to a significant increase in oxygen consumption, an

increased production of free radicals has consistently been reported (Bachur et

al., 2007). Oxidative stress is known to stimulate p38 MAPK and the ERK

(Sanchis-Gomar & Perez-Quilis, 2014; Zhang et al., 2014), which in turn activates

PGC-1α in skeletal muscle, an important regulator of FNDC5, but also of the


54

browning signaling process (Bostrӧm et al., 2012). In exercised animals, skeletal

muscle FNDC5 and circulating irisin levels were positively associated with the up-

regulation of browning-related markers, including PGC-1α and its downstream

target UCP1, Cidea and Dio2 genes, and PRDM16, a transcriptional regulator of

BAT differentiation, among others (Bostrӧm et al., 2012; De Matteis et al., 2013;

Stanford et al., 2015; Sutherland et al., 2009; Tiano et al., 2015; Wu et al., 2014;

Xu et al., 2011). Accordingly, this augmented signaling was also coupled with

reductions in adipocyte hypertrophy, visceral adiposity as well as with

improvements in glucose tolerance in HFD-fed animals (Wu & Spiegelman,

2014).

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 VO2 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.


55

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 VO2max 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).

Collectively, data suggest that circulating irisin levels seems to be increased in

response to acute intense exercise in humans; however new insights into irisin

regulation during PE are clearly needed. Of note is that some inconsistencies

may be related to interactions between irisin and other cytokines and hormones,

such as IL-6, BDNF, adipQ (Comassi et al., 2015; Pedersen & Febbraio, 2012;

Wrann et al., 2013). Moreover, other tissues, such as cardiomyocytes and

purkinje cells of cerebellum (Raschke & Eckel, 2013; Roca-Rivada et al., 2013;

Wrann et al., 2013), have been described to interfere with irisin

metabolism/regulation, and should also be considered in future studies.

6.4 Mitochondrial activity

Studies suggest that adipocyte mitochondrial function might play an important

role in the development of obesity due to its relevance in the efficiency of


56

oxidative phosphorylation (OXPHOS), impairment of lipogenesis and lipolysis,

regulation of adipocyte differentiation, production of oxygen radicals, apoptotic

signaling, and also regulation of brown adipocyte-like phenotype (Boudina &

Graham, 2014). It is known that PE increases adipocyte oxygen consumption

(Stanford et al., 2015), mitochondrial number and content (Ringholm et al., 2013;

Xu et al., 2011), as well as seems to elevate UCP1 content (Ringholm et al.,

2013; Xu et al., 2011), an important protein for triggering mitochondrial

uncoupling respiration. Moreover, the expression of the Pgc1α, a critical player

in mitochondrial biogenesis regulation (Conley, 2016) and the expression of

mitochondrial transcription factor (Tfam) are upregulated in VAT after both acute

(Sutherland et al., 2009) and chronic ET (Castellani et al., 2014; Sutherland et

al., 2009). Accordingly, some studies (Hashimoto et al., 2013; Stallknecht et al.,

1993; Sutherland et al., 2009) showed that mitochondrial activity, measured by

the activities of cytochrome c oxidase (COX) and malate dehydrogenase (MDH),

increased in VAT from ET lean rats.

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).

Regarding the impact of exercise in the context of obesity, the information

provided by the literature is still scarce. Nevertheless, oxidative phosphorylation

(OXPHOS) subunits genes, such as mt-Nd5 and mt-Cytb, are overexpressed

after 16 wks of VPA in adipocytes of HFD-induced obese mice (Sae-Tan et al.,

2015). Furthermore, few studies demonstrated that PE induced positive

mitochondrial alterations, including COX, cytochrome C oxidase subunit IV

(COXIV) and Pgc1α expression in HFD-fed mice (Xu et al., 2011) and in

hyperphagic, obese rats (Laye et al., 2009). Therefore, further studies are

required to better understand the mechanisms underlying the impact of PE on


57

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.


58

Figure 1. Schematic view of hypothetical mechanisms underlying PE impact on white

adipose tissue morphological, metabolic and inflammatory features in the context of

obesity.

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. Study I

97

Study I

Physical exercise mitigates high fat diet-induced adiposopathy and

related endocrine alterations

Sílvia Rocha-Rodrigues1, Inês O. Gonçalves1, Jorge Beleza1;António

Ascensão1, and José Magalhães1

1CIAFEL - Research Centre in Physical Activity, Health and Leisure, Faculty of

Sport, University of Porto, Porto, Portugal

Submitted for publication on Nutrition Journal (Elsevier)


98

Abstract

Purpose: The dysregulation of adipokines secretion owing to adiposopathy can

contribute to the pathogenesis of obesity-related disorders. Therefore, we aimed

to analyze the role of physical exercise as a preventive and therapeutic strategy

against high-fat diet (HFD)-induced adiposopathy and related adipokines and

ghrelin alterations.

Methods: Sprague-Dawley rats were assigned into sedentary (S) and voluntary

physical activity (VPA) groups and fed with an isocaloric standard (SS and SVPA)

or high-fat (HS and HVPA) diet for 17 weeks. After 9 weeks, half of SS and HS

animals were submitted to 8 weeks of endurance treadmill training (SET and

HET) maintaining the respective diets.

Results: Although there were no changes in body weight, HFD increased visceral

adiposity, percentage of large adipocytes, hypoxia inducible factor (HIF)-1α, and

leptin contents in epididymal adipose tissue (eWAT) and decreased plasma

content of adiponectin (AdipQ). Both VPA and ET decreased visceral adiposity

and percentage of large adipocytes (HVPA and HET), but ET also increased the

percentage of small- to medium-sized adipocytes (SET and HET). VPA increased

plasma high molecular weight (HMW)/AdipQ ratio (SVPA) and growth hormone

secretagogue receptor (GHS-R), and decreased leptin protein (HVPA). ET

decreased plasma insulin (HET) and leptin levels, and adipocyte HIF-1α and

leptin expression (SET and HET). Moreover, ET improved insulin sensitivity,

plasma HMW, AdipQ and ghrelin levels (SET and HET), and increased eWAT

AdipQ (SET) and GHS-R expression (SET and HET).

Conclusions: Our data suggest that physical exercise, particularly ET, reverted

adiposopathy and related endocrine alterations induced by an isocaloric HFD

pair-fed diet.

Keywords: high-molecular weight adiponectin, adiposopathy, exercise, ghrelin,

leptin


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1. Introduction

Obesity is a worldwide epidemic with the prevalence of patients dramatically

increasing in developed Western countries (Bray & Bellanger, 2006). An

imbalance between energy intake and expenditure, mainly due to low physical

activity levels and excessive intake of energy-dense foods, may result in

adipocyte hypertrophy and increased visceral adipose tissue (VAT) (Lopes et al.,

2016; Ye et al., 2007). Both represent the typical pathoanatomical features of

adiposopathy or “sick fat” (Bays et al., 2008), which most likely results in tissue

hypoxia and endocrine dysregulation (Gollisch et al., 2009; Lara-Castro et al.,

2006). Adipose tissue has been recognized as an active endocrine organ

affecting body homeostasis (Bays et al., 2008). Some circulating adipose tissue-

driven peptide-hormones, including adiponectin (AdipQ), leptin, and ghrelin, can

selectively regulate energy expenditure, dietary behavior and insulin sensitivity

(Flores et al., 2006). Circulating AdipQ levels are reduced in individuals with

obesity (Lara-Castro et al., 2006) and are inversely correlated with the degree of

adiposity and insulin resistance (IR). On the other hand, leptin levels are elevated

in obesity, but hypothalamic leptin resistance aggravates obesity status through

appetite control inhibition and lipid oxidation, and is positively correlated with body

fat and adipocyte size (Gollisch et al., 2009; Linden et al., 2014). Circulating levels

of ghrelin, a growth hormone (GH) released by the gastrointestinal track that

functions as a neuropeptide in the central nervous system, fluctuate in response

to alterations in energy status (Sun, 2015). Low ghrelin levels were observed in

individuals with obesity and high levels in individuals after weight loss (Espelund

et al., 2005; Leidy et al., 2004). Physical exercise is among the most efficient non-

pharmacological interventions used to reduce body weight and visceral adiposity,

as well as to improve non- and adipose-derived endocrine hormone secretion

and production profile, thus positively impacting whole body metabolism (Gollisch

et al., 2009; Linden et al., 2014; Reseland et al., 2001). Several studies have

investigated the effects of physical exercise on AdipQ, leptin and ghrelin levels in

distinct obese models; however, the underlying mechanisms associated with

those positive effects are not fully understood. The high molecular weight (HMW)

AdipQ has been recognized as the active form of this protein (Lara-Castro et al.,

http://topics.sciencedirect.com/topics/page/Growth_hormone

http://topics.sciencedirect.com/topics/page/Growth_hormone


100

2006) and a better predictor of insulin sensitivity and other obesity-related

disorders (Lara-Castro et al., 2006). Some studies reported an increase in HMW

AdipQ after acute exercise (Bluher, Brennan, et al., 2007), while others

suggested no changes despite an improved body fat and insulin sensitivity in

older insulin-resistant subjects (O'Leary et al., 2007). Data showed that a short-

term endurance training (ET)-induced decrease in leptin levels was associated

with fat mass loss (Miyatake et al., 2004), while others (Reseland et al., 2001)

demonstrated that a long-term ET regimen (more than 12 weeks) had higher

impact on reducing circulating leptin levels independently of body fat reduction

(Reseland et al., 2001). An increase in circulating ghrelin levels was observed

after a long-term ET program in individuals with obesity (Mason et al., 2015), with

decreases (Ghanbari-Niaki et al., 2011) or absence of alterations (Ebrahimi et al.,

2013) also reported. White adipose tissue (WAT) plays several key roles in

mammalian physiology, including energy storage, inflammatory processes, and

glucose homeostasis (Trayhurn and Beat-tie, 2001). WAT is a major energy

storage organ which stores energy in the form of triglycerides. It also functions

as an endocrine organ, controlling energy homeostasis and metabolism through

secretion of adipokines such as adiponectin and leptin (Otto and Lane, 2005;

Rosen, 2005). WAT develops in distinct intra-abdominal depots, that is, visceral

fat, including epididymal (EWAT), mesenteric, and perirenal depots, and in the

subcutaneous layer (SWAT) (Seale et al., 2011). Published data showed that

subcutaneous and visceral WAT express unique gene signatures and have

distinct metabolic effects (Fox et al., 2007; Gesta et al., 2006; Miyazaki et al.,

2002; Porter et al., 2009; Rexrode et a In the context of the laboratory animal-

based research, the modified version of the Lieber-DeCarli diet is one of the

nutritional-induced obesity models used to promote obesity-related pathological

disorders. Originally, the Lieber-DeCarli diet with ethanol was developed to

induce liver damage (Lieber & DeCarli, 1989), but a modified version of this diet

based on high-fat corn oil was established to induce non-alcoholic steatohepatitis

(NASH) (Goncalves, Passos, Rocha-Rodrigues, Torrella, et al., 2014). However,

to our best knowledge, whether this diet model induces alterations on

adiposopathy-related features as well as on non- and adipose-derived endocrine


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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.

2. Material and methods

2.1 Animals and diets

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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2.2 Physical exercise protocols

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.

Figure 1: Flow diagram outlining the experimental design


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2.3 Body weight, energy intake and visceral adiposity assessments

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.

2.4 Adipose cell-size distribution

A portion of WAT was fixed in a 10% formalin solution, embedded in paraffin,

sectioned, and stained with hematoxylin and eosin. The digital images were

acquired using a microscope (Zeiss AX10 imager A.1, Oberkochen, Germany)

under x40 magnification. The adipocyte area cell surface area was determined,

removing any objects below an area of 350 μm2, as these cells may be a mixture

of adipocytes and stromal vascular cells, and quantified using the Image J

software (National Institutes of Health, Bethesda, MD, USA). The distribution of

frequencies of adipocyte areas were quantified from 4 sections per rat and 4 rats

per group (> 1500 adipocytes counted per group).

2.5 Plasma analysis and insulin resistance/sensitivity determinations

Plasma glucose concentration was quantified using a standardized method for

an automated clinical chemistry analyzer (Olympus AU54001). Plasma insulin

(10-1137-01, Mercodia) was determined using enzymatic methods with

commercial kits. Insulin resistance/sensitivity was calculated as follows:

homeostasis model assessment of IR (HOMA-IR) = [fasting plasma insulin X

fasting plasma glucose/2.43]; and quantitative insulin sensitivity check index


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(QUICKI) = 1/log(fasting plasma insulin)+log(fasting plasma glucose) (Cacho et

al., 2008).

2.6 Protein analysis

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


105

internal loading control. Chemiluminescent detection was performed with

horseradish peroxidase-conjugated secondary antibodies [anti-rabbit (2317),

anti-mouse (2317), and anti-goat (2020)] in ChemiDoc™ XRS+System and band

densities were quantified using Image Lab™ software 262 5.2.1 (Bio-Rad

Laboratories, Inc.).

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;

bvs.HS; cvs.SVPA; dvs.SET; evs.HVPA

Both VPA and ET reverted an HFD-induced higher percentage of large

adipocytes (higher than 9000 μm2). Moreover, ET increased the percentage of

smaller adipocytes (below 999 μm2 and between 1000-2999 μm2) and decreased

adipocytes in the 3000-8999 μm2 size range in standard and high-fat diet-fed

groups. (figure 3) .


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Figure 3: Adipocyte cell-size distribution. Bar graphs representing the percentage of

adipocytes size below 999 μm2, 1000-2999 μm2, 3000-5999 μm2, 6000-8999 μm2 and

higher than 9000 μm2 in each group (A). Adipocytes areas were determined from 4

sections per rat. Representative images of hematoxylin and eosin-stained adipose tissue

(B). 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

3.2 Effects of diet and physical exercise on VEGF and HIF-1α protein

expression in eWAT

As seen in figure. 4, HFD increased hypoxia-inducible factor 1α (HIF-1α)

expression, while no impact on vascular endothelial growth factor (VEGF) protein

expression was observed with HFD. Both HIF-1α and VEGF expression

remained unchanged after VPA. ET decreased HIF-1α and increased VEGF

protein expression in both standard and high-fat diet-fed animals.


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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;

cvs.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).

Plasma ghrelin negatively correlated with visceral adiposity (r=-0.630; p=0.002),

HOMA-IR (r=-0.640; p=0.001), and insulin (r=-0.661; p=0.001) and positively

correlated with QUICKI (r=0.660; p=0.001). Moreover, plasma ghrelin correlated


109

moderately with eWAT GHS-R (r=0.575; p=0.006). Plasma ghrelin negatively

correlated with body weight (r=-0.663; p=0.001), visceral adiposity (r=-0.630;

p=0.002), HOMA-IR (r=-0.640; p=0.001) and insulin (r=-0.661; p=0.001) and

positively correlated with QUICKI (r=0.660; p=0.001). Moreover, plasma ghrelin

correlated moderately with eWAT GHS-R (r=0.575; p=0.006).

Figure 5: Adipose and non-adipose-derived hormones (A-D). The protein expression of

leptin and ghrelin in plasma (A and C) and leptin and GHS-R in eWAT (B and D).

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

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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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


111

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).

Figure 7: Plasma analysis and insulin sensitivity/resistance determination. Plasma

insulin (A), glucose (B) levels, HOMA-IR and QUICKI (C and D). 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


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4. Discussion

It is known that physical exercise promotes adaptations in adipose tissue,

highlighting the relevance of this tissue to whole-body adaptions to physical

exercise and as a promising target to treat obesity and its comorbidities (Holland

et al., 2016; Stanford et al., 2015). Based on previous data from our group,

suggesting that physical exercise improved lipid content in the liver (Goncalves,

Passos, Rocha-Rodrigues, Torrella, et al., 2014), we here aimed to analyze the

role of VPA and ET in adiposopathy and related endocrine response in rats

submitted to an isocaloric Liber-DeCarli pair feeding diet. The main findings of

the present study revealed that physical exercise promoted weight loss, which

was accompanied by a reduction in visceral adiposity and adipocyte size.

Moreover, ET reverted an HFD-induced increase in HIF-1α expression and

increased AdipQ, HMW AdipQ, ghrelin, and GHS-R protein. It also decreased

leptin content, which likely contributed to improved insulin sensitivity.

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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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


114

groups. A small-sized adipocyte has been associated with a positive impact on

non- and adipose-derived hormone production and secretion, thus regulating

energy balance and body weight through centrally mediated actions (Flores et

al., 2006). Two important satiety signals are insulin (released from the pancreas)

and leptin (from adipose tissue), which assist in long-term regulation of energy

balance (Hopkins & Blundell, 2016). The decrease of plasma insulin level in the

HET group is in accordance with other observations (Gollisch et al., 2009) and

seems to be related to exercise intensity, as reported by others (Kang et al.,

1996). This alteration must be related to elevated AdipQ as its insulin sensitizing

effects are well documented (Numao et al., 2011). It is well established that HFD-

fed rats exhibited resistance to the anorexigenic effects of leptin (Gollisch et al.,

2009; Linden et al., 2014), which contribute to body weight gain and visceral

adiposity accumulation (Linden et al., 2014), corroborating our findings. The

effects of leptin on energy intake and expenditure (Hopkins & Blundell, 2016;

Nakhuda et al., 2016) could be mediated via hypothalamic regulation (Flores et

al., 2006) or via direct action on peripheral tissues, including skeletal muscle. The

ET-induced reduction in plasma leptin expression of HFD-fed animals is in

accordance with other studies (Gollisch et al., 2009; Jenkins et al., 2012).

Moreover, a physical exercise-induced decrease in VAT leptin expression in

HFD-fed animals seems to be related more to reduced adipocyte size than to

visceral adiposity or body weight, which corroborates data from other reports

(Gollisch et al., 2009; Linden et al., 2014; Miyazaki et al., 2010).

Discrepancies exist regarding the effects of ET on AdipQ mRNA levels in obese

rats, with studies revealing increases (Krskova et al., 2012) or no alterations

(Gollisch et al., 2009; Svidnicki et al., 2015). Nevertheless, the decrease of its

more active form, the HMW AdipQ, is more associated with IR (Lara-Castro et

al., 2006). In the present study, plasma total AdipQ, but not HMW, was reduced

in response to HFD feeding for 17 weeks, and negatively associated with body

weight, visceral adiposity and insulin levels. Some studies demonstrated no

changes on HMW despite decreased body fat and improved insulin sensitivity

after acute exercise in patients with insulin-resistance (O'Leary et al., 2007) and

with obesity (Numao et al., 2011), while others observed increased HMW levels


115

in subjects with normal glucose tolerance (Bluher, Brennan, et al., 2007). As

expected, ET-induced increases in plasma AdipQ and HMW were consistent with

improved QUICKI in ET, but not in VPA animals, which suggests that ET

promoted more pronounced effects in the improvement of insulin sensitivity,

possibly due to its action via AdipQ receptors in skeletal muscle (Klimcakova et

al., 2006).

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


116

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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Chapter III. Experimental Work. Study II

123

Study II

Effects of physical exercise on myokines expression and brown adipose-

like phenotype modulation in rats fed a high-fat diet

Sílvia Rocha-Rodrigues1, Amaia Rodríguez2, Alexandra M. Gouveia3, Inês O.

Gonçalves1, Sara Becerril2, Beatriz Ramírez2, Jorge Beleza1, Gema

Frühbeck2,4, António Ascensão1, José Magalhães1


Sport, University of Porto, Porto, Portugal;

2Metabolic Research Laboratory, Clínica Universidad de Navarra; Obesity &

Adipobiology Group, Instituto de Investigación Sanitario de Navarra (IdiSNA);

CIBEROBN, Instituto de Salud Carlos III, Pamplona, Spain;

3Department of Experimental Biology, Faculty of Medicine, University of Porto,

Porto, Portugal; Instituto de Investigação e Inovação em Saúde, Institute for

Molecular and Cell Biology (IBMC), University of Porto, Porto, Portugal;

4Department of Endocrinology & Nutrition, Clínica Universidad de Navarra,

Pamplona, Spain;

Published on: Life Sciences 165: 100–108 (2016)

doi: 10.1016/j.lfs.2016.09.023


124

Abstract

Purpose: Exercise-stimulated myokine secretion into circulation may be related

with browning in white adipose tissue (WAT), representing a positive metabolic

effect on whole-body fat mass. However, limited information is yet available

regarding the impact of exercise on myokine-related modulation of adipocyte

phenotype in WAT from obese rats.

Methods: Sprague-Dawley rats (n=60) were divided into sedentary and voluntary

physical activity (VPA) groups and fed with standard (35 kcal% fat) or high-fat

(HFD, 71 kcal% fat)-isoenergetic diets. The VPA-groups had unrestricted access

to wheel running throughout the protocol. After-9 weeks, half of sedentary

standard (SS) and sedentary HFD (HS)-fed animals were exercised on treadmill

(endurance training, ET) for 8-weeks while maintaining the dietary treatments.

Results: The adipocyte hypertrophy induced by HFD were attenuated by VPA

and ET. HFD decreased 5' AMP-activated protein kinase (AMPK) activity in

muscle as well as peroxisome proliferator-activated receptor gamma coactivator-

1α (PGC-1α) and uncoupling protein 1 (UCP1) proteins in eWAT, while not

affecting circulating irisin. VPA increased eWAT Tmem26 mRNA levels in the

standard diet-fed group, whereas ET increased AMPK, interleukin 6 (IL-6) and

fibronectin type III domain-containing protein 5 (FNDC5) protein expression in

muscle, but had no impact on circulating irisin protein content. In eWAT, ET

increased bone morphogenetic protein 7 (Bmp7), Cidea and PGC-1α in both diet-

fed animals, whereas BMP7, Prdm16, UCP1 and FNDC5 only in standard diet-

fed group.

Conclusions: Data suggest that ET-induced myokine production seems to

contribute, at least in part, to the “brown-like” phenotype in WAT from rats fed a

HFD.

Keywords: endurance training, skeletal muscle, adiposity, FNDC5/irisin, IL-6,

browning, UCP1


125

1. Introduction

Overweight and obesity are highly prevalent conditions in industrialized countries.

Although genetic factors contribute to the variance of adiposity, in most cases,

obesity results from an imbalance between energy intake and expenditure due to

a high consumption of hypercaloric food and/or reduced levels of physical activity.

The white adipose tissue (WAT) constitutes the main store of lipids, buffering

daily dietary fat entering from the circulation (Guilherme et al., 2008).

Nonetheless, obesity-induced WAT dysfunction can lead to ectopic fat

accumulation in other tissues, which have been associated with obesity-related

metabolic diseases (Goncalves, Passos, Rocha-Rodrigues, Torrella, et al., 2014;

Guilherme et al., 2008; Sertie et al., 2013).

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).

The metabolic alterations induced by physical exercise have pleiotropic effects.

Previous studies have shown that regular exercise affects not only the exercised

skeletal muscle, but also other non-contractile organs over time (Bostrӧm et al.,

2012; Norheim et al., 2014; Wu et al., 2014). One potential mechanism underlying

this crosstalk is the secretion of proteins that mediate communication between

muscle and other tissues through endocrine mechanisms (Pedersen & Febbraio,


126

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,


127

Spain). Goat anti-BMP7 (sc9305), goat anti-β-actin (sc1616) and secondary

peroxidase-conjugated antibodies (anti-rabbit, sc-2317, anti-mouse, sc-2317 and

anti-goat, sc-2020) were obtained from Santa Cruz Biotechnology (Dallas, TX,

USA). Rabbit anti-phospho[Thr172]AMPKα (2531) and rabbit anti-SIRT3

(C73E3) were obtained from Cell Signalling Tecnhology, Inc. (Danvers, MA,

USA). Goat anti-PGC-1α (ab106814), rabbit anti-IL-6 (ab6672), mouse anti-

AMPK (ab80039), rabbit monoclonal anti-FNDC5 (ab174833), rabbit anti-UCP1

(ab23841) were acquired from Abcam (Cambridge, UK). Goat anti-UCP2

(Sab2501087) was obtained from Sigma Aldrich and rabbit anti-SIRT1 (13161-1-

AP) from Proteintech Group, Inc. (Chicago, IL, USA). TRIzol® reagent and

SYBR® Green PCR Master Mix were purchased from Life Technologies

(Carlsbad, USA), NZY First-strand cDNA Synthesis Kit (NZYTech, Lisbon,

Portugal). All other chemicals were purchased from Sigma-Aldrich (Barcelona,

Spain).


Six-week-old male Sprague-Dawley rats (n=60, Charles River, L'Arbresle,

France) were kept in a pathogen-free room, constant temperature (21-22 °C) and

humidity (50-60%) with a fixed 12-h light/12-h dark cycle. Rats (initial body weight

233.9±2.6 g) were fed ad libitum during 17 weeks 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 carbohydrate, and 18% kcal protein)-liquid diet purchased from

Dyets Inc. (catalog no. 710027 and 712031, respectively). This DIO model has

been commonly used to induce visceral adipose tissue accumulation and related

metabolic disorders in experimental animals (Goncalves, Passos, Rocha-

Rodrigues, Torrella, et al., 2014; Lieber et al., 2004). All animals had free access

to water and food (provided in the liquid state) throughout the entire protocol.

During the first week, the standard diet was given to all animals as an adaptation

to the liquid feeding. Afterwards, animals were divided into six groups

(n=10/group): standard-diet sedentary (SS), standard-diet voluntary physical


128

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), as previously described (Goncalves, Passos, Rocha-Rodrigues,

Torrella, et al., 2014).

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).

2.3 Voluntary physical activity and endurance training interventions

VPA intervention: voluntary-exercised animals (SVPA and HVPA) were assigned

in cages equipped with 365-mm (diameter) running wheels. Based on the

evidence that VPA may be an effective strategy to prevent the progress of, at

least, some obesity-associated metabolic abnormalities (Goncalves, Passos,

Rocha-Rodrigues, Torrella, et al., 2014), animals had unrestricted access to the

running wheels during the 17 weeks of the feeding protocol. Wheel revolutions

were daily recorded from a digital counter between 08.00 and 10.00 h. SVPA and

HVPA animals were moved to standard cages for one night before sacrifice to

avoid interference of exercise-related acute effects.

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


129

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.

2.4 Visceral adipose tissue and blood collection

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.

2.5 Adipocyte-size profiling

Morphological analyses were performed using light microscopy as previously

described (Parlee et al., 2014). Briefly, visceral WAT was fixed in 4%

formaldehyde solution (Sigma Aldrich) embedded in paraffin blocks, sectioned,

and stained with hematoxylin and eosin. Stained samples were visualized using

a microscope (Zeiss AX10 imager A.1, Oberkochen, Germany) under x40

magnification. Average of adipocyte area was determined, removing any objects

below an area of 350 μm2, as these cells may be a mixture of adipocytes and

stromal vascular cells (Parlee et al., 2014). The average of adipocyte area and

the frequency of distribution were quantified from 4 sections per rat and 4 rats per

group (> 1500 adipocytes counted per group).


130

2.6 Citrate synthase activity in skeletal muscle

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.

2.7 Circulating irisin content

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.

2.8 Western Blot studies

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


131

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.

2.9 RT-qPCR analyses

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.


132

Table 1. Primer sequences used for RT-qPCR.

2.10 Statistical analysis

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

As illustrated in figure 1, body weight of HS animals remained similar to that of

SS group. Moreover, VPA had no impact on body weight while ET for 8 weeks

significantly reduced body weight in both exercised groups compared with

sedentary counterparts. No significant alterations were observed in cumulative

energy intake (kcal) between studied groups, as expected in an isoenergetic pair-

feeding diet. Nevertheless, HFD induced an increase in the relative weights of

GenBank Accession no. Primer sequence (5’-3’)

Tmem26 NM_001107623 F-CCGAGGCTACAAATGGCTTTC

R-ACTGGTTTCCATGGTGCATTTC

Bmp7 NM_001191856 F-CCTGCAAGAAACACGAGCTGTAT

R-AGGCACACTCTCCCTCACAGTAGT

Cidea NM_001170467 F-TGACATTCATGGGGTTGCAGA

R-GGCCAGTTGTGATGACCAAGA

Prdm16 XM_008764418 F-CTCCGAGATCCGAAACTT

R-CTCAGGCCGTTTGTCCAT

Gapdh NM_017008 F-GGTGAAGGTCGGAGTCAACG

R-CAAAGTTGTCATGGATGACC


133

mesenteric, epididymal and retroperitoneal fat depots, which were reverted by

VPA in mesenteric fat depot, epididymal and retroperitoneal fat pads. In addition,

ET reduced all WAT depots in SET and HET groups compared with their

sedentary counterparts.

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

3.2 Effects of diet and exercise on adipocyte-area profiling visceral WAT

Animals from HS group presented a higher percentage of larger adipocytes

(≥5000 μm2) when compared to SS group (figure 2). VPA did not alter the

percentage of adipocytes below 5000 μm2 in both SVPA and HVPA groups;

however, VPA reduced the percentage of larger adipocytes in HVPA group

compared to sedentary counterpart. Moreover, ET significantly increased the

percentage of smaller adipocytes (<2000 μm2) and reduced the number of larger

adipocytes (≥ 5000 μm2) in both endurance-trained groups.


134

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; cvs.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.


135

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

As shown in figure 4, HS animals exhibited diminished skeletal muscle 5’ AMP-

activated protein kinase (AMPK) activation (pAMPK/tAMPK ratio), as

quantification of AMPK Thr(172) phosphorylation by immunoblotting is a

recognized surrogate for AMPK activity (Lim et al., 2012), compared to SS group.

Concerning exercise interventions, VPA had no effect in any parameter, but ET

significantly increased pAMPK/tAMPK ratio in both endurance-trained groups

compared to their sedentary and voluntary-exercised counterparts. The HFD

regimen per se had no impact on skeletal muscle IL-6 expression, while VPA

tended to increase and ET significantly increased its expression in both standard

and HFD-fed animals compared to sedentary and voluntary-exercised

counterparts. Moreover, skeletal muscle IL-6 positively correlated with muscle

mass (r=0.71, p<0.001) and negatively correlated with body weight (r=-0.65,

p=0.001) and total VAT depots (r=-0.79, p<0.001). Although without statistical

significance, HFD intervention tended to reduce skeletal muscle FNDC5 protein

expression in HS group. Despite the absence of any effect prompt by VPA, ET

for 8 weeks increased FNDC5 protein content in both endurance-trained groups.


136

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.

Figure 4: Activation/phosphorylation of AMPK (A), IL-6 (B), FNDC5 (C) on skeletal

muscle and circulating irisin content (D). Representative blots of each protein (E). Values

of protein levels are expressed as % AU (arbitrary units), where SS group was set as

100%. 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

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,


137

whereas 8-week-ET significantly increased both BMP7 mRNA and protein

expression in SET group and gene expression in HET group. BMP7 protein

negatively correlated with body weight (r=-0.61, p=0.002) and total VAT depots

(r=-0.64, p=0.001). Regarding eWAT, Tmem26, mRNA levels remained

unaltered with both HFD and ET interventions, but were significantly increased in

SVPA animals compared to sedentary counterparts. HFD feeding had no impact

on either Cidea or Prdm16 mRNA levels in eWAT. Moreover, no significant

alterations were observed in the expression of both genes after VPA intervention

with standard or HFD-fed animals. In contrast, ET for 8 weeks increased Cidea

mRNA levels in both endurance-trained groups and Prdm16 mRNA levels in SET

animals.

A negative correlation was observed between Cidea (r=-0.79, p<0.001) and

Prdm16 (r=-0.67, p=0.001) and total VAT depots. The HFD regimen significantly

decreased eWAT UCP1 protein content compared to standard-fed counterpart,

while VPA did not induce any significant alteration in both diet regimens.

Moreover, ET increased UCP1 protein levels in standard-fed animals and tended

to attenuate the decreased expression in HFD-fed animals (figure 5). A strong

correlation between UCP1 and body weight (r=-0.77, p<0.001) and total VAT

depots (r=0.74, p<0.001). A positive correlation between skeletal muscle FNDC5

and brown-specific markers, including BMP7 (r=0.74, p<0.001), Cidea (r=0.70,

p=0.001) and UCP1 (r=0.63, p=0.001) was observed. Also, skeletal muscle IL-6

positively correlated with brown-specific markers, including BMP7 (r=0.72,

p<0.001), Cidea (r=0.67, p=0.002), Prdm16 (r=0.67, p=0.002) and UCP1 (r=0.75,

p<0.001).


138

Figure 5: Expression of beige and brown adipose-selective markers. Semi quantitative

RT-qPCR analysis of Bmp7 mRNA (A) and expression of BMP7 protein (B). Quantitative

RT-qPCR analysis for Tmem26 (C), Cidea (D), Prdm16 (E) and Western Blot for UCP1

(F) on eWAT. Representative images of Western Blot (G). Values of protein and gene

levels are expressed as % AU (arbitrary units), where SS group was set as 100%. 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

As illustrated in figure 6, animals fed a HFD reduced eWAT PGC-1α protein

expression compared to standard diet counterparts. The VPA intervention was

unable to revert the down-regulation induced by HFD; however, ET increased

PGC-1α protein content in both endurance-trained groups compared to sedentary

counterparts. Concerning sirtuin 1 (SIRT1) levels, the HFD had no impact per se,

but the ET increased SIRT1 protein expression in HET group compared to SET,

HS and HVPA groups. A significant negative correlation between SIRT1

expression and body weight (r=-0.55, p=0.01) and total VAT depots (r=-0.53,

p=0.01) was observed. No significant alterations were observed on eWAT sirtuin

3 (SIRT3) protein content after the HFD regimen or both the exercise

interventions, although a tendency in endurance-trained animals was noted. A

negative correlation was found between SIRT3 and total VAT depots (r=-0.47,

p=0.02). Regarding UCP2 protein content, both HFD and exercise interventions

failed to induce any significant alterations. With exception of the increased eWAT

FNDC5 protein expression observed in SET group (vs. SS and SVPA), while no

other significant alterations were found between groups. Strong negative


139

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


140

endocrine fashion, acting on other organs and tissues (Pedersen, 2011).

Therefore, myokines likely provide a conceptual basis to understand how skeletal

muscle “talks” with other organs and tissues, such as WAT, which is of particular

interest in context of obesity. However, to our best knowledge, the role of

exercise-induced myokines release and its potential signaling influence on WAT

metabolism is still a matter of debate.

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.


141

However, the phosphorylation/activation of AMPK was higher after ET in animals

fed with both diet type conditions but not after VPA, which is in accordance with

others (Horowitz, 2003), reporting that AMPK is activated by exercise in an

intensity-dependent manner. It is particularly noteworthy that an 8-week-program

of ET is able to significantly increase AMPK and IL-6 content even in obesity

context as both factors have been related with reduced body weight, VAT

accumulation and small-sized adipocyte in HFD-induced obese animals

(Knudsen et al., 2015).

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


142

systemic metabolism and whole-body fat mass (Moreno-Navarrete et al., 2013).

Therefore, an important goal of the present study was to understand whether or

not two distinct programs of physical activity (VPA and ET) were able to modulate

eWAT structure and key metabolic genes and proteins, particularly inducing a

“brown-like” phenotype in WAT from animals fed a HFD. In this regard, a new

role for BMP7 in brown adipogenesis and consequent increased energy

expenditure has been suggested (Tseng et al., 2008). Altogether, data suggest

that BMP7 activates a full program of brown adipogenesis including the induction

of early regulators of brown fat fate PRDM16 and PGC-1α, increased expression

of the brown-specific marker UCP1 and adipogenic transcription factors PPARγ

and CCAAT/enhancer-binding proteins, and induction of mitochondrial

biogenesis via p38 mitogen-activated protein kinase and PGC-1α-dependent

pathways (Tseng et al., 2008). However, data regarding the role of exercise in

the modulation of BMP7 gene and protein expression in VAT is still scarce. In

accordance with others (Bostrӧm et al., 2012; Morton et al., 2016; Norheim et al.,

2014; Tiano et al., 2015; Wu et al., 2014), data from the present study revealed

that ET increased the transcript levels of Bmp7 gene and others brown adipocyte-

specific markers (Cidea, Prdm16 and UCP1), but did not modulate the native

beige adipocyte-specific marker Tmem26. As state above, SIRT1 may also play

a role on the induction of brown-like phenotype in WAT by deacetylating two

critical lysine residues on PPARγ (Chang & Guarente, 2014). In accordance with

Chalkiadaki and coworkers (Chalkiadaki & Guarente, 2012), the full-length SIRT1

protein was overexpressed in our HET animals, which suggest that ET plays an

important role in the modulation of eWAT phenotype via SIRT1, thus contributing

to improve the browning-adipocyte like phenotype in VAT.

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


143

effect was consistent with the amelioration of several obesity-related outcomes,

such as reductions on body weight, VAT accumulation, adipocyte size, and

hepatic fat accumulation in HFD-induced NASH, as previously demonstrated by

our group (Goncalves, Passos, Rocha-Rodrigues, Torrella, et al., 2014).

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.


144

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34140.




Chapter III. Experimental Work. Study III

149

Study III

Physical exercise remodels visceral adipose tissue and mitochondrial

lipid metabolism in rats fed a high-fat diet

Sílvia Rocha-Rodrigues1; Amaia Rodríguez2; Sara Becerril2; Beatriz Ramírez2;

Inês O. Gonçalves1; Jorge Beleza1; Gema Frühbeck2,3; António Ascensão1;

José Magalhães1





CIBEROBN, Instituto de Salud Carlos III, Pamplona, Spain.


Pamplona, Spain

Published on: Clin Exp Pharmacol Physiol doi: 10.1111/1440-1681.12706.

[Epub ahead of print


150

Abstract

Purpose: We aimed to investigate the effects of two physical exercise models,

voluntary physical activity (VPA) and endurance training (ET) as preventive and

therapeutic strategies, respectively, on lipid accumulation regulators and

mitochondrial content in VAT of rats fed a high-fat diet (HFD).

Methods: Sprague Dawley rats (6 wks old, n=60) were assigned into sedentary

and VPA groups fed isoenergetic diets: standard (S, 35 kcal% fat) or HFD (71

kcal% fat). The VPA groups had free access to wheel running during the entire

protocol. After 9-wks, half of sedentary animals were exercised on a treadmill

while maintaining the dietary treatments.

Results: HFD induced no changes in plasma non-esterified fatty acids (NEFA)

and glycerol levels and decreased oxidative phosphorylation (OXPHOS) subunit

IV and increased truncated/full-length sterol regulatory element-binding

transcription factor 1c (SREBP1c) ratio in epididymal white adipose tissue

(eWAT). VPA decreased plasma glycerol levels, aquaglyceroporin 7 (AQP7) and

increased subunit I of cytochrome c oxidase (COX) protein, in standard fed

animals. Eight-wks of ET decreased body weight, visceral adiposity and

adipocyte size and plasma NEFA and glycerol levels, as well as AQP7 protein

expression in eWAT. ET increased fatty acid translocase (FAT/CD36),

mitochondrial content of complexes IV and V subunits, mitochondrial biogenesis

and dynamic (mitofusins and optic atrophy 1)-related proteins. Moreover,

lipogenesis-related markers (SREBP1c and acetyl CoA carboxylase) were

reduced after 8-wks of ET.

Conclusions: ET-induced alterations reflect a positive effect on mitochondrial

function and the overall VAT metabolism of HFD-induced obese rats.


151

Keywords: endurance training, lipolysis, lipogenesis, mitochondria, visceral

adiposity

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.

Being mitochondria one of the main regulators of lipid metabolism, impairments

on this organelle lead to reduced substrate oxidation and impaired metabolic

capacity in VAT (Heinonen et al., 2015). In fact, studies conducted in distinct

animals models of obesity reported that white adipose tissue (WAT) mitochondria

exhibited altered morphology (Cummins et al., 2014) and reduced mitochondrial

content and activity (Laye et al., 2009; Rong et al., 2007; Sutherland et al., 2008).

In humans, a reduced oxidative phosphorylation (OXPHOS) content and activity


152

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.

On the other hand, physical exercise has been recognized as an important

component of a healthy lifestyle with positive effects on both adipose tissue and

mitochondrial metabolism (for ref. see Ogasawara et al., 2015). However,

discrepant data regarding the effects of chronic exercise on lipolytic capacity in

adipocytes have been reported. Some studies described an increased lipolysis

(Ogasawara et al., 2012; Suda et al., 1993), while others suggested a reduced

lipolysis in isolated rat adipocytes (Chapados et al., 2008; Pistor et al., 2015) or

unaltered condition when assessed in situ by microdialysis technique in humans

(Stallknecht et al., 1995) in response to ET. Moreover, it has been reported that

an increased mitochondrial oxidative capacity in VAT could reduce the availability

of NEFA for esterification (Sutherland et al., 2009) and the utilization of glucose

for the formation of glycerol in white adipocytes (Pistor et al., 2015). Few studies

have reported that exercise induces positive alterations on VAT mitochondrial

metabolism, including increases on mitochondrial content and activity (Laye et

al., 2009; Sutherland et al., 2009; Xu et al., 2011) and on critical factors involved

in the regulation of mitochondrial biogenesis, such as peroxisome proliferator-

activated receptor gamma coactivator 1-alpha (PGC-1α) (Ruderman et al., 2003;

Sutherland et al., 2009; Xu et al., 2011). However, the impact of chronic exercise

on mitochondrial metabolism in obesity context remains poorly understood.

Therefore, the aim of the present study was to investigate the effects of two

physical exercise models, voluntary physical activity (VPA) and endurance

training (ET) as preventive and therapeutic strategies, respectively, on lipid

accumulation regulators and mitochondrial content in VAT of rats submitted to a

HFD.


153

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].

2.2 Voluntary physical activity and endurance training interventions

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


154

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).

2.3 Blood sampling and visceral adipose tissue collections

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.

2.4 Plasma analysis

Plasma glucose concentration was quantified using a standardized method for

an automated clinical chemistry analyser (Olympus AU54001). Plasma insulin

(10-1137-01, Mercodia), glycerol (F6428, Sigma Aldrich) and NEFA (434-91795,

WAKO) levels were determined using enzymatic methods with commercial kits.

Homeostasis model assessment of insulin resistance (HOMA-IR) was calculate

as [(fasting plasma insulin X fasting plasma glucose/2.43].


155

2.5 Adipocyte area determination

After fixation in 4% formaldehyde, 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) under x40 magnification. Average of adipocyte area was

determined from 4 sections per rat and 4 rats per group (> 1500 adipocytes

counted per group).

2.6 Protein analysis by Western Blot

The eWAT was homogenized in ice-cold RIPA buffer supplemented with

protease and phosphatase inhibitors (Sigma Aldrich) using a Polytron

homogenizer for 30 sec. The homogenates were centrifuged at 13000 g for 10

min at 4 ºC to remove insoluble material. The infranatant was harvested and used

for protein quantification by Bradford method (Bradford, 1976), then proteins were

boiled for 5 min in 2×Laemmli buffer containing 710 mmol/L β-mercaptoethanol.

Afterwards, 50 μg of protein extracts obtained from each sample were separated

by SDS-PAGE, transferred into polyvinyldifluoride membranes (Millipore) and

blotted with anti-phospho[Thr172]AMPKα (2531), anti-ACC (3662), anti-

phospho[Ser79]ACC (3661) from Cell Signaling Technology, Inc, anti-AQP7

(2862), anti-CD36 (9154), anti-TFAM (23588), anti-MFN1 (50330), anti-MFN2

(50331) from Santa Cruz Biotechnology, anti-SREBP1c (3259), anti-OXPHOS

(110413), anti-OPA1 (119685) from Abcam and anti-COX (556433) from BD

Biosciences. The original membrane was stripped and reblotted with β-actin

(1616, Santa Cruz Biotechnology) as a loading protein. Chemiluminescent

detection was performed with horseradish peroxidase-conjugated secondary

antibodies [anti-rabbit (2317), anti-mouse (2317) and anti-goat (2020)] in

ChemiDoc™ XRS+System (Bio-Rad Laboratories, Inc.) and band densities were

quantified using Image Lab™ software 262 5.2.1 (Bio-Rad Laboratories, Inc.).

The mean relative intensities were quantified and normalized to the signal of β-

actin or the signal intensity of total form for phosphorylated proteins.


156

2.7 Gene analysis by real-time PCR

Total RNA of eWAT was extracted by homogenization with an ULTRA-

TURRAX® T 25 basic (IKA® Werke GmbH, Staufen, Germany), purified using

RNeasy® Lipid Tissue Mini Kit (74804, Qiagen) and treated with DNase I (RNase-

free DNase, Qiagen) to remove any trace of genomic DNA. RNA concentration

was determined by reading absorbance at 260 nm. One microgram of purified

RNA worked as template for cDNA production using 200 units of M-MLV reverse

transcriptase (Invitrogen) and random hexamers. Transcript levels for Aqp7

(NM_019157.2) and Cd36 (NM_031561.2) were quantified by real-time PCR

(7300 Real Time PCR System, Applied Biosystems). Primers and probes were

designed using the software Primer Express 2.0 (Applied Biosystems) as follows,

Aqp7: 5’-GGCTTCGTGGATGAGGTATTTG-3’ (sense), 5’-

ACAGTCCAGCACTTCAAGGGAC-3’ (antisense) and FAM-

AGCTGTGTATCTTCGCCATCACG-TAMRA (Taqman® probe); Cd36: 5’-

GACATTTGCAGGTCCATCTATGC-3’ (sense), 5’-

CAGAACCCAGACAACCACTGTTT-3’ (antisense) and FAM-

TTCTTCCAGCCAACGCCTTTGCCT-TAMRA (Taqman® probe) purchased from

Genosys (Sigma). Primers or TaqMan® probes encompassing fragments of the

areas from the extremes of two exons were designed to ensure the detection of

the corresponding transcript avoiding genomic DNA amplification. The cDNA was

amplified at the following conditions: 95 °C for 10 min, followed by 45 cycles of

15 s at 95 °C and 1 min at 59 °C, using the TaqMan® Universal PCR Master Mix

(Applied Biosystems). The primer and probe concentrations were 300 and 200

nmol/L, respectively. All samples were run in triplicate and the average values

were calculated and normalized to 18S rRNA (Applied Biosystems), using the

comparative CT method (2-ΔΔCT) as recommended by Applied Biosystems.

2.8 Statistical analysis

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


157

and differences were considered significant at p≤0.05. Pearson’s correlation was

used to analyse possible associations between variables. Statistical analyses

were performed using SPSS 21.0 for Windows (SPSS Inc.).

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.


158

Ta

ble

1:

An

ima

l cha

racte

ristics a

nd p

lasm

a a

na

lysis

S

S

SV

PA

S

ET

H

S

HV

PA

H

ET

A

NO

VA

Bod

y w

eig

ht (g

) 676.8

±11.5

612.7

6±15.3

559.3

±10.0

a,c

684.3

±14.2

710.0

±10.8

c

592.8

±7.3

b,e

E

Food

-eff

icie

ncy r

atio

6.0

1±0.3

3

5.6

6±0.2

8

5.9

3±

0.3

2

5.8

8±0.3

5

5.9

8±0.2

9

5.9

4±0.3

N

S

Muscle

mass (

%)

0.7

1±0.0

1

0.8

7±0.0

3a

1.0

0±

0.0

3a,c

0.6

6±0.0

2

0.6

5±0.0

1c

0.8

5±0.0

2b,d

,e

DxE

, D

, E

Vis

cera

l ad

iposity (

%)

9.0

1±0.2

1

7.4

9±0.4

6a

4.7

6±

0.4

6a,c

11.7

6±0.3

7a

8.9

2±0.3

7b,c

6.7

7±0.1

45

b,d

,e

D, E

Adip

ocyte

are

a m

ean

(μm

2)

3716

.1±30

1.4

2697

.3±25

3.0

1629

.1±14

6.6

a

5348

.6±47

1.5

a

3636±4

00

b

2429

.4±28

8.2

b

D, E

Pla

sm

a

Insulin

le

vels

(μ

g/L

) 0.2

77±0.0

18

0.2

73±0.0

11

0.1

91±0.0

19

0.3

41±0.0

3

0.3

29±0.0

23

0.2

08±0.0

24

b,e

D, E

Glu

cose levels

(g

/L)

228±18

.68

3

217±15

.59

4

242.5

±15.8

03

258.2

6±3.2

31

235±15

.32

6

190±15

.12

6

NS

HO

MA

-IR

23.1

77±0.3

92

21.9

34±0.4

51

14.4

74±0.3

88

a,c

36.6

31±1.2

33

a

32.6

55±

0.8

88

c

19.8

16±0.9

55

b,e

D

xE

, D

, E

Data

are

ex

pre

sse

d a

s t

he m

ea

n±S

EM

. a v

s.

SS

; b v

s.

HS

; c v

s. S

VP

A;

d v

s. S

ET

; e v

s. H

VP

A.


159

3.2 Effects of diet and exercise on AQP7 and FAT/CD36 expression in eWAT

It is well known that prolonged exercise induces an increase in plasma glycerol

and NEFA levels in response to the elevated release of catecholamines and

decreased production of insulin (Frühbeck et al., 2014). Accordingly, despite no

changes induced by the HFD regimen per se, ET reduced both plasma glycerol

and NEFA levels, while VPA only decreased plasma glycerol levels in animals

SVPA animals compared to their sedentary counterparts (figure 1).

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


160

had no impact on FAT/CD36, while ET increased Cd36 gene expression only in

SET group and FAT/CD36 protein expression in both SET and HET groups

(figure 2). A negative and strong association between FAT/CD36 protein

expression and plasma NEFA (r=-0.70, p<0.001) was observed.

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

The activation of 5’ AMP-activated protein kinase (AMPK) by phosphorylation

triggers the phosphorylation/inhibition of acetyl CoA (ACC) (Long & Zierath,


161

2006). The inhibition of ACC leads to the reduction of malonyl-CoA, an allosteric

inhibitor of carnitine palmitoyltransferase-1, thereby enhancing the mitochondrial

β-oxidation. Long-term HFD had no significant impact on total AMPK and ACC or

their phosphorylated proteins. Similarly, VPA had no impact on AMPK and ACC

expression and phosphorylation, while ET significantly increased basal and

phosphorylated AMPK and decreased total and phosphorylated ACC compared

to sedentary counterparts (figure 3).

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

We also evaluated the protein expression of sterol regulatory element-binding

transcription factor 1 (SREBP1c), a transcription factor involved in the de novo

synthesis of fatty acids. Despite no alterations in full-length SREBP1c protein

expression, animals engaged in the HFD regimen increased truncated/full-length

SREBP1c ratio when compared to standard diet-fed counterparts. The VPA


162

intervention had no impact on SREBP1c levels, while ET decreased full-length

SREBP1c protein expression as well as truncated/full-length SREBP1c ratio

compared to their sedentary counterparts (figure 4).

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;

3.4 Effects of diet and exercise on mitochondrial function, biogenesis and

fusion-related proteins in eWAT

Regarding mitochondrial OXPHOS subunits, HFD only decreased protein

expression of complex IV (C.IV) subunit (MTCO1). VPA increased subunit of C.IV

(MTCO1) and tend to increase C.V (ATP5A) protein expression in standard diet-

fed animals. On the other hand, 8-wks of ET increased protein expression of C.II

(SDH8) in SET group and C.IV (MTCO1) and C.V (ATP5A) in both SET and HET

animals compared to their sedentary counterparts (Fig. 4). Long-term HFD

significantly decreased mitochondrial biogenesis transcript factor A (TFAM)

protein expression, but had effect on either cytochrome c oxidase (COX),

mitofusin 1 (MFN1), mitofusin 2 (MFN2), or optic atrophy 1 (OPA1) protein

expression. The VPA intervention had no effect in the studied proteins related

with mitochondrial bioenergetics, biogenesis or fusion. On the other hand, ET


163

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).

Figure 5: Protein expression of oxidative phosphorylation subunits (A-E) and

representative images of blots (F). Data are expressed as the mean±SEM. DxE,

diet and exercise interaction; D, diet effect; E, exercise effect; NS, not



164

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


165

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;


166

Rodríguez, Catalan, Gomez-Ambrosi, & Frühbeck, 2011). In contrast with our

findings, some studies reported that either circulating glycerol levels or AQP7

expression are elevated in VAT, which results in increased plasma glycerol levels

and hepatic glucose production in obese and insulin-resistant subjects (Catalán

et al., 2008) . The discrepancy on these results may be due to differences in

studied animal species or in the severity of insulin resistance as the regulation of

AQP7 expression in human adipose tissue appears to be more related with

insulin resistance than with obesity (Rodríguez, Catalan, Gomez-Ambrosi, &

Frühbeck, 2011; Rodríguez, Catalan, Gomez-Ambrosi, Garcia-Navarro, et al.,

2011). Although with no alterations in eWAT AQP7 expression, our results are in

line with other studies (Cummins et al., 2014; Sutherland et al., 2008) reporting

that the chronic intake of high-fat-rich diets shifts adipocyte metabolic activity from

energy supply to lipid storage, as seen by increases in lipogenesis markers

(SREBP1c and ACC) expression and hypertrophic adipocyte morphology in HS

animals. Moreover, one of the main findings of the present study is that the

regulation of VAT AQP7 expression by exercise seems to be intensity-dependent

in both diet conditions. In fact, ET but not VPA was able to decrease eWAT AQP7

expression in both standard and HFD conditions, possibly through ET-induced

inhibitory effect of adipocyte lipolytic activity, as described elsewhere (Frühbeck

et al., 2014). Interestingly, only few conflicting results were published concerning

the effects of physical exercise on subcutaneous adipose tissue AQP7

expression (Lebeck et al., 2012; Trachta et al., 2014). Therefore, further studies

are needed to better explore the role of exercise on AQP7 expression in VAT as

the regulation of glycerol transport by aquaglyceroporins contributes to the

control of fat accumulation, glucose homeostasis, among other important

functions.

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;


167

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).

These exercise-induced positive effects observed in the VAT and mitochondrial

lipid metabolism regulators can be explained by AMPK activation (Canto et al.,

2009), which plays a key role in cellular energy homeostasis by increasing fatty

acid oxidation and inhibiting other pathways, such as lipogenesis and lipolysis

(Daval et al., 2005). In the present study, the AMPK activation in endurance-

trained animals was consistent with decreases in circulating NEFA levels and

improvements in mitochondrial metabolism in both diet regimens. Nevertheless,

VPA did not alter AMPK expression, which are in line with findings in other studies

(Canto et al., 2009; Daval et al., 2006), suggesting that exercise intensity may be

a key determinant on its activation, as state above.

Despite the relevance of mitochondria on VAT metabolism, few studies have

been addressed to investigate the hypothetical impact of exercise on

mitochondrial functions and biogenesis, particularly in the context of obesity. The

decrease in WAT mitochondrial function and biogenesis induced by HFD might

be associated with increases in plasma NEFA levels, as reported by others

(Cummins et al., 2014; Sutherland et al., 2008). In fact, an excessive of NEFA

levels might affects mitochondrial function and dynamics as seen by decreased

levels of biogenesis-related factors and mitofusin protein 1 (MFN1), although an

increase of mitofission protein (DRP1) in 3T3-L1 adipocytes has been associated

with smaller and compact mitochondria (Gao et al., 2010). In the present study,

although no alterations in plasma NEFA levels were found, a decrease in the

protein expression of mitochondrial complexes and biogenesis transcript factor

was found in HFD-fed animals, which suggests a tendency toward an impairment

on mitochondrial bioenergetics and possibly contributes to HFD-induced


168

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

In conclusion, this is the first study to demonstrate that an ET program induced

an increase of fusion-related proteins, such as OPA1, MFN1 and 2, suggesting

that exercise might plays an important role in mitochondrial dynamics network in

VAT, under obesity context (Bach et al., 2003). We further observed that

exercise-induced improvements on VAT mitochondrial functions may be related

with reduced lipogenic-related markers, which were observed by decreases in

ACC and SREBP1c expression in endurance-trained animals. Taken together,

data suggest that the alterations induced by exercise reflect a positive remodeling

in mitochondrial function and the overall VAT metabolism even in the context of

obesity.

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


169

ET program with specific characteristics (intensity, duration and frequency)

mitigates several adverse cellular metabolic consequences induced by obesity.

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Metab, 96(4), E586-597.

Rodríguez, A., Ezquerro, S., Mendez-Gimenez, L., Becerril, S., & Frühbeck, G.

(2015). Revisiting the adipocyte: a model for integration of cytokine

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& Saha, A. K. (2003). AMPK as a metabolic switch in rat muscle, liver

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Stallknecht, B., Simonsen, L., Bulow, J., Vinten, J., & Galbo, H. (1995). Effect of

training on epinephrine-stimulated lipolysis determined by microdialysis

in human adipose tissue. Am J Physiol, 269(6 Pt 1), E1059-1066.

Suda, K., Izawa, T., Komabayashi, T., Tsuboi, M., & Era, S. (1993). Effect of

insulin on adipocyte lipolysis in exercise-trained rats. J Appl Physiol

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1083.







Vroegrijk, I. O., van Klinken, J. B., van Diepen, J. A., van den Berg, S. A.,

Febbraio, M., Steinbusch, L. K., Glatz, J. F., Havekes, L. M., Voshol, P.

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Yoshida, Y., Jain, S. S., McFarlan, J. T., Snook, L. A., Chabowski, A., & Bonen,

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and biogenesis. J Physiol, 591(18), 4415-4426.




Chapter III. Experimental Work. Study IV

175

Study IV

Impact of physical exercise on visceral adipose tissue fatty acid profile

and inflammation in response to a high-fat diet regimen

Sílvia Rocha-Rodrigues1, Amaia Rodríguez2, Inês O. Gonçalves1, Elisabete

Maciel3, Ana Moreira3, Sónia Santos4; Maria R. Domingues3, Gema Frühbeck2,5,

António Ascensão1, José Magalhães1





CIBEROBN, Instituto de Salud Carlos III, Pamplona, Spain;

3Mass spectrometry Centre, UI-QOPNA Department of Chemistry, University of

Aveiro, Aveiro

4CICECO Department of Chemistry, University of Aveiro, Campus Santiago,

3810-193 Aveiro, Portugal


Pamplona, Spain

Submitted on: International Journal of Biochemistry and Cell Biology

(under review)


176

Abstract

Purpose: Studies associate specific fatty-acids (FA) with the pathophysiology of

inflammation. We aimed to analyze the impact of exercise on adipose tissue FA

profile in response to a high-fat diet (HFD) and to ascertain whether these

exercise-induced changes in specific FA have repercussions on obesity-related

inflammation.

Methods: Sprague-Dawley rats were assigned into sedentary, voluntary

physical-activity (VPA) and endurance training (ET) groups fed a standard (S,

35kcal% fat) or high-fat (71kcal% fat) diets. VPA-animals had unrestricted access

to wheel-running. After 9-wks, ET-animals engaged a running protocol for 8-wks,

while maintained dietary treatments. The FA content in epididymal white-adipose

tissue (eWAT) triglycerides was analyzed by gas-chromatography and

the expression of inflammatory markers was determined using RT-qPCR,

Western and slot blotting.

Results: Eight-wks of ET reversed obesity-related anatomical features. HFD

increased plasma tumor necrosis factor (TNF)-α content and eWAT monocyte

chemoattractant protein (MCP)-1 protein expression. HFD decreased eWAT

content of saturated FA and monounsaturated FA, while increased linoleic acid

in eWAT triglycerides. VPA decreased visceral adiposity, adipocyte size and

MCP-1 in HFD-fed animals. VPA and ET interventions diminished palmitoleic acid

and increased linoleic acid in HFD-fed groups. ET reduced eWAT inflammatory

markers (TNF-α, IL-6), macrophage recruitment (MCP-1 and F4/80) and

increased IL-10/TNF-α ratio in plasma and in eWAT in both diet types.

Conclusions: Exercise induced FA-specific changes independently of dietary FA

composition, but only ET attenuated the inflammatory response in VAT of HFD-

fed rats. Moreover, the exercise-induced FA changes did not correlate with the

inflammatory response in VAT of rats submitted to HFD.

Keywords: endurance training, visceral adiposity, fatty acids, TNF-α, IL-10


177

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


178

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).

Physical exercise has been well recognized as a strategy to prevent visceral

adiposity accumulation, improve inflammatory process as well as lipid

metabolism, such as fatty acid profile (Gollisch et al., 2009; Jenkins et al., 2012;

Kawanishi et al., 2015; Vieira, Valentine, Wilund, Antao, et al., 2009). In fact,

studies demonstrated that endurance training (ET) induced FA 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).

Moreover, ET decreased MUFA in humans (Danner et al., 1984; Sutherland et

al., 1981) and in rodents (Wirth et al., 1980); however the findings are not

consistent (Rocquelin & Juaneda, 1981; Thorling & Overvad, 1994). The anti-

inflammatory impact of regular physical exercise are 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). In addition, an increased IL-10 expression

was also observed in response to ET (Jenkins et al., 2012; Lira et al., 2010; Lira

et al., 2009), strengthening the anti-inflammatory effects of physical exercise.

However, to our best knowledge, the role of physical exercise as a modulator of

FA profile-specific changes on VAT and concomitantly on the inflammatory

response associated to obesity has never been clarified. Therefore, in the present


179

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.


2.1 Animal care and dietary treatments

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.


180

Ta

ble

1:

Fa

tty a

cid

com

po

sitio

nof

sta

nda

rd a

nd

hig

h-f

at d

iets

S

tan

dard

die

t (g

/L)

HF

D (

g/L

)

Sa

fflo

we

r O

live

oil

Co

rn o

il T

ota

l S

aff

low

er

Oliv

e o

il C

orn

oil

To

tal

C1

8:2

n6

78

7

60

145

78

7

120

205

C1

8:1

n9

12

.2

77

26.5

1

15

.7

12.2

77

53

142

.2

C1

6:0

6

.9

10.5

1

0.8

2

8.2

6

.9

10.5

2

1.6

39

C1

8:0

2

.9

3

2.1

8

2.9

3

4.2

1

0.1

C1

8:3

n3

- 0

.6

0.6

1

.2

- 0

.6

1.2

1

.8

C1

6:1

n7

- 1

.0

- 1

.0

- 1

.0

1

.0

The s

tandard

and h

igh

-fat

die

ts w

ere

purc

hased t

o D

iets

Inc., c

ata

log #

71

002

7 a

nd

71

201

31.

The t

wo d

iets

diffe

red in

th

e a

mount of corn

oil

each o

ther,

40 g

of w

as a

dded to o

bta

in h

igh

-fat d

iet. S

tand

ard

die

t co

nta

in 1

2%

,

39 a

nd 4

9%

of

satu

rate

d f

att

y a

cid

s,

monounsatu

rate

d f

att

y a

cid

s a

nd p

oly

unsatu

rate

d f

att

y a

cid

s,

respectively

.

The

hig

h-f

at

die

t conta

in

12%

, 3

5%

an

d

51%

of

satu

rate

d

fatt

y

acid

s,

monounsatu

rate

d

fatt

y

acid

s

and

poly

unsatu

rate

d f

att

y a

cid

s,

respective

ly


181

2.2 Physical exercise protocols

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.

2.3 Blood sampling and visceral adipose tissue collection

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.

2.4 Determination of adipocyte-size profiling

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


182

mean were determined from 4 sections per rat and 4 rats per group (>1500

adipocytes counted per group).

2.5 Analysis of fatty acid composition in eWAT triglycerides

Lipid extraction: The lipids were extracted with chloroform:methanol:water (4:2:1)

and mechanic disruption, dried under N2 flow, and solubilized in chloroform, as

previously described by (Archer et al., 2013). The total lipid extract was

fractionated into triglycerides using solid phase extraction with 500 mg

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).


183

2.6 Circulating cytokines 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 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.

2.7 Epididymal WAT protein analysis by Western Blot

Protein samples (50 μg) were separated by SDS-PAGE, transferred into

polyvinyldifluoride membranes (Millipore), as described by (Goncalves et al.,

2016), and blotted with anti-IL-6 (ab6672), anti-TNF-α (ab80039), anti-IL-10

(ab9969), anti-MCP-1 (ab25124), anti-F4/80 (ab6640) purchased from Abcam

(Cambridge, UK). The original membrane was stripped and reblotted with β-actin

(sc1616, Santa Cruz Biotechnology, Dallas, TX, USA) for loading protein.

Chemiluminescent detection was performed with horseradish peroxidase-

conjugated secondary antibodies [anti-rabbit (2317), anti-mouse (2317) and anti-

goat (2020)] in ChemiDoc™ XRS+System and band densities were quantified

using Image Lab™ software 262 5.2.1 (Bio-Rad Laboratories, Inc.).

2.8 Epididymal WAR gene analysis by qRT-PCR

RNA isolation and purification was performed, as described earlier (Catalán et

al., 2008). Transcript levels for Il6 (NM_012589), tnfa (NM_012675), Ccl2 (Mcp1)

(NM_031530), were quantified by real-time PCR (7300 Real Time PCR System,

Applied Biosystems (Foster City, CA, USA). Primers and probes were designed

using the software Primer Express 2.0 (Applied Biosystems) as follows, il6: 5’-

AAAGAGTTGTGCAATGGCAATTC-3’ (sense), 5’-

CTTTCTTGTTATCTTGTAAGTTGTTCTTCAC-3’ (antisense) and FAM-


184

TCTGCTCTGGTCTTCTGGAGTTCCGTTTCTA-TAMRA (Taqman® probe); tnfa:

5’- CCCAGACCCTCACACTCAGATC-3’ (sense), 5’-

AGGTACAGCCCATCTGCTGGTA-3’ (antisense), FAM-

TAGCCCACGTCGTAGCAAACCACCAA-TAMRA (Taqman® probe); ccl2 5’-

TCACAGTTGCTGCCTGTAGCAT-3’ (sense), 5’-

GGATCATCTTGCCAGTGAATGAG-3’ (antisense), FAM-

CTCAGCCAGATGCAGTTAATGCCCCAC-TAMRA (Taqman® probe) and were

purchased from Genosys (Sigma). Primers or TaqMan® probes encompassing

fragments of the areas from the extremes of two exons were designed to ensure

the detection of the corresponding transcript avoiding genomic DNA

amplification. The cDNA was amplified at the following conditions: 95 °C for 10

min, followed by 45 cycles of 15 s at 95 °C and 1 min at 59 °C, using the TaqMan®

Universal PCR Master Mix (Applied Biosystems). The primer and probe

concentrations were 300 and 200 nmol/L, respectively (Catalán et al., 2008). All

samples were run in triplicate and the average values were calculated and

normalized to 18S rRNA (4310893E, Applied Biosystems), using the comparative

CT method (2-ΔΔCT) as recommended by Applied Biosystems.

2.10 Calculations and statistics

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.).


185

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 5000μm2. 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).


186

Ta

ble

2:

Bo

dy w

eig

ht,

to

tal e

ne

rgy in

take

, vis

cera

l a

dip

osity,

an

d a

dip

ocyte

siz

e d

ete

rmin

atio

ns

S

S

SV

PA

S

ET

H

S

HV

PA

H

ET

A

NO

VA

Initia

l bo

dy

weig

ht (g

)

286±3.6

9

284±4.8

3

288.4

±4.6

9

279.1

7±3.8

8

286.5

±3.8

4

292.4

3±5.4

5

NS

Bod

y w

eig

ht

at

week 9

(g

)

401.1

8±30.7

8

386.3

5±28.9

6

402.9

6±30.8

9

394.6

4±30.2

7

394.2

2±28.6

413.5

±31.8

6

NS

Fin

al bo

dy

weig

ht (g

)

654.5

±10.8

9

654±53

.74

611.0

±48.0

8a

664.7

5±24.2

9

706.2

±22.4

1

579.7

±14.1

1b,d

E

Energ

y inta

ke

(kcal)

1437

.19±1

8.5

1475

.11±4

8.4

2

1440

.26±1

3.6

1465

.44±3

6.3

1496

.14±2

9.2

5

1433

.83±2

9.4

8

NS

Vis

cera

l

adip

osity (

%)

9.0

1±0.2

1

7.4

9±0.4

6a

4.7

6±0.4

6a,c

11.7

6±0.3

7a

8.9

2±0.3

7b

6.7

7±0.1

45

b,d

D

, E

Ad

ipo

cyte

are

a

Adip

ocyte

are

a

mean (

μm

2)

3716

.1±30

1.4

2697

.3±25

3.0

1629

.1±14

6.6

a

5348

.6±47

1.5

a

3636±4

00

b

2429

.4±28

8.2

b

D, E

Adip

ocyte

are

a

<50

00μ

m2 (%

)

71.1

3±4.3

8

88.8

6±3.1

8

92.3

0±0.7

8a

56.3

6±4.1

9

76.4

9±4.5

4b

99.1

7±5.6

1b,d

E

Adip

ocyte

are

a

≥5000μ

m2 (%

)

18.0

1±4.3

0*

5.3

4±2.5

7*

1.2

5±0.5

9a, *

43.9

8±4.1

2a

23.4

4±4.5

6b, *

7.8

8±3.3

2b,d*

D, E

Data

are

ex

pre

ss

ed

as

th

e m

ea

n±S

EM

. D

xE

, d

iet

an

d e

xe

rcis

e i

nte

rac

tio

n;

D,

die

t e

ffe

ct;

E,

exe

rcis

e e

ffe

ct;

NS

, n

ot

sig

nif

ica

nt;

a v

s.

SS

; b v

s. H

S;

c v

s.

SV

PA

; d

vs

. S

ET

; e v

s. H

VP

A


187

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


189

3.3 Effects of diet and physical exercise on systemic inflammation

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


190

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).

3.4 Effects of diet and physical exercise on eWAT cytokines

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).


191

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



192

3.5 Effects of diet and physical exercise on eWAT macrophage infiltration-

specific markers

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).

Figure 4: Monocyte/macrophages infiltration and migration markers in eWAT. The gene

and protein expression of Ccl2/MCP1 (A and B), F4/80 (C) and representative imagens

of blots (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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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.

The FA esterified to triglycerides have been suggested to play critical roles in

WAT (Chan et al., 2015; Finucane et al., 2015; Lumeng et al., 2007). Using GC-

MS analysis, we determined the FA profile of triglycerides, as they represent 90%

of adipose tissue lipids (Rodríguez, Ezquerro, et al., 2015), in order to understand

the FA-specific changes in response to a HFD regimen and two physical exercise

models (VPA and ET). Considering that the dietary FA composition influences, at

least in part, the FA profile within WAT (Thorling & Overvad, 1994), the sedentary


194

HFD-fed animals showed an increased linoleic acid (C18:2n6) relative content as

this HFD contain higher levels of this FA than the standard diet (120g/L vs. 60g/L).

In addition, both VPA and ET interventions increased linoleic acid relative

content independently of diet composition. In accordance, other (Dvorakova &

Bass, 1970; Petridou et al., 2005; Wirth et al., 1980) reported that physical

exercise regimens induced FA profile changes in WAT triglycerides by increasing

the percentage of longer chain and PUFA, possibly due to stimulated chain

elongation, polydesaturation and/or depressed monodesaturation (Petridou et al.,

2005; Thorling & Overvad, 1994; Wirth et al., 1980). Surprisingly, the HFD

decreased palmitic acid (C16:0) and stearic acid (C18:0) content in eWAT

triglycerides, which was unexpected as both diets used in the current study

contain the same percentage of SFA (12%). These findings may be justified by

the increased PUFA/SFA ratio in eWAT triglycerides (data not shown) observed

in HFD-fed animals as SFA substrates, mainly palmitic acid, is repressed by

PUFA dietary intake (Coelho et al., 2011). Moreover, the ET intervention reduced

C16:0 content in eWAT triglycerides in standard and high-fat diets-fed animals,

which is also corroborated by other studies (Dvorakova & Bass, 1970; Petridou

et al., 2005; Rocquelin & Juaneda, 1981). Evidence showed that a decreased

SFA content has been associated with improvement in obesity-related features

(Coelho et al., 2011), such as reduced visceral adiposity, which are in line with

observations in SET, HVPA and HET groups. Another surprising result was the

decreased content of palmitoleic acid in all HFD-fed groups as the HFD contains

the same amount of palmitoleic acid as the standard diet. Moreover, the observed

decrease in HVPA and HET groups, as also reported by others (Petridou et al.,

2005; Rocquelin & Juaneda, 1981), was greater than that observed in HS

animals, which was consistent with the decreased desaturase activity

(C16:1n7/C16:0 ratio) (data not shown).

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


195

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).

Physical exercise is also known for enhancing the expression of anti-

inflammatory markers, such as IL-10 (Jenkins et al., 2012). This cytokine has the

ability to selectively inhibit the expression of pro-inflammatory cytokines (e.g.

TNF-α) and simultaneously stimulate the anti-inflammatory gene expression

program (Tedgui & Mallat, 2006). In line, we observed in the current study that

ET, but not VPA, shifted the inflammatory response toward an anti-inflammatory

phenotype by increasing IL-10 expression and IL-10/TNF-α ratio, an indicator of

the inflammatory state and associated diseases (Kaur et al., 2006; Rosa Neto et

al., 2009), which are in line with other findings (Jenkins et al., 2012; Speretta et

al., 2012).

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


196

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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Invest, 40(1), 55-62.

Chapter III. Experimental Work. Study V

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Study V

Effects of endurance training on autophagy and apoptotic signaling in

visceral adipose tissue of long-standing high fat diet-fed rats

Sílvia Rocha-Rodrigues1, Inês O. Gonçalves1, António Ascensão1, José

Magalhães1

1 CIAFEL - Research Centre in Physical Activity, Health and Leisure, Faculty

of Sport, University of Porto, Porto, Portugal

Submitted for publication on: European Journal of Nutrition (under review)


204

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.

Keywords: Exercise; visceral adiposity; Beclin-1; p62; Bcl-2


205

1. Introduction

Western lifestyle, characterized by energy-rich diets and lack of exercise, has

led to a global obesity epidemic (Fruhbeck & Yumuk, 2014). In fact, in

response to this positive energy balance, visceral adipose tissue (VAT) mass

and adipocyte size increase (McLaughlin et al., 2016). Both represents the

typical features of the emerged concept, adiposopathy or “sick fat” (Bays et al.,

2008), which most likely result in obesity-related cellular metabolic

disturbances (Jacobs et al., 2016; Lopes et al., 2016).

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


206

an increase of anti-apoptotic gene expression in mature epididymal adipocytes

(Sakurai et al., 2005) after acute high-intensity endurance exercise and a

reduced rate of apoptosis (Sertie et al., 2013) after 12 wks of endurance

training in lean rats. To our best knowledge, the effects of physical exercise on

autophagy and apoptotic signaling in VAT of obese models are poorly

investigated. In fact, understanding how exercise modulates autophagy and

apoptotic signaling in VAT may contribute to identify potential therapeutic

molecular targets to improve adipose tissue function in obesity conditions.

Therefore, with the present study we aimed to analyze the effects of ET on

autophagy and apoptotic signaling in VAT of long-standing HFD-fed rats.

2. Material and Methods

2.1 Animals and diets:

Six-week old male Sprague-Dawley rats were purchased (Charles River

Laboratories) and housed in cages (2 rats per cage) with an enriched,

temperature- and humidity-controlled environment and maintained on a 12:12h

light-dark cycle. Animals (initial body weight 229.9±2.6 g) were divided into the

following groups (n=8/group): standard diet and sedentary (STD+SED); HFD

sedentary (HFD+SED), standard diet plus endurance training (STD+ET) and

HFD plus endurance training (HFD+ET). Afterwards, animals were fed a

nutritionally adequate isoenergetic and isoproteic standard (35Kcal% fat,

47Kcal% carbohydrates, and 18Kcal% protein) or HFD (71Kcal% fat, 11Kcal%

carbohydrate, 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 (40 g) in each other, which was added and

mixed into the diet to obtain high-fat diet (HFD) (Lieber et al., 2004). As

previously reported by our group (Goncalves, Passos, Rocha-Rodrigues,

Torrella, et al., 2014) and others (Lieber et al., 2004), this isoenergetic pair-

feeding HFD is a suitable model to induce an accumulation of visceral adipose

tissue and obesity-related abnormalities.


207

2.2 Endurance Training:

Animals of the endurance-trained groups were gradually submitted to a

continuous running protocol on a motor-driven rodent treadmill (Le8700,

Panlab Harvard Apparatus), 5 days per week for 8 weeks, as previously

described (Rocha-Rodrigues, Rodriguez, et al., 2016b). Briefly, exercise

intensity was progressively increased from 15 min per day at 15 m min-1 up to

60 m at 25 m min-1 in the last 4 weeks of the program. The exercise-trained

animals were sacrificed 48h after the last training session to avoid exercise-

related acute effects. The non-exercised groups (SS and HS) were placed on

a non-moving treadmill to be exposed to the same potential environment and

handling stress.

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.

2.3 Body weight, energy efficiency and visceral adiposity assessments:

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.

2.4 Plasma analysis:

Using a standardized method for an automated clinical chemistry analyzer

(Olympus AU54001) plasma glucose levels were quantified. Plasma insulin


208

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).

2.5 Histological analysis:

The adipose tissue samples were fixed in 10% formalin, 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. The images of histological sections were cap using

a light microscope (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, as suggested by

[14]. The distribution of adipocyte areas were determined from 4 sections per

rat and 4 rats per group (>1500 adipocytes counted per group).

2.6 Western Blot determinations:

The eWAT was homogenized in ice-cold RIPA buffer supplemented with

protease inhibitors cocktail using a Polytron homogenizer for 30 sec. The

homogenates were centrifuged at 13000 g for 10 min at 4 ºC to remove

insoluble material. The infranatant was harvested and used for protein

quantification by Bradford method (Bradford, 1976), then proteins were boiled

for 5 min in 2×Laemmli buffer containing 710 mmol/L β-mercaptoethanol.

Afterwards, 50 μg of protein extracts obtained from each sample were

separated by SDS-PAGE, transferred into polyvinyldifluoride membranes and

blotted with anti-Bax (2772), anti-Bcl-2 (2870) and Beclin-1 (3495) from Cell

Signaling Technology, anti-p62 (56416) from Abcam, anti-DLK/PREF1

(2103823) from Sigma Aldrich, anti-Lc3 (152-3) from MBL International

Corporation. All primary antibodies were at 1:1,000 dilution. Control for protein

loading was confirmed by Ponceau S staining. The original membrane was

stripped and reblotted with β-ACTIN (1616, Santa Cruz Biotechnology) for

normalization of quantitative protein. Chemiluminescent detection was


209

performed with horseradish peroxidase-conjugated secondary antibodies

[anti-rabbit (SC2317), anti-mouse (SC2317) and anti-goat (SC2020)] in

ChemiDoc™ XRS+System and band densities were quantified using Image

Lab™ software 262 5.2.1 (Bio-Rad Laboratories, Inc.).

2.7 RT-qPCR analysis:

Total RNA of eWAT was extracted by homogenization with an ULTRA-

TURRAX® T 25 basic (IKA® Werke GmbH, Staufen, Germany), purified using

RNeasy® Lipid Tissue Mini Kit (74804, Qiagen) and treated with DNase I

(RNase-free DNase, Qiagen) to remove any trace of genomic DNA. RNA

concentration was determined by reading absorbance at 260 nm. One

microgram of purified RNA worked as template for cDNA production using 200

units of M-MLV reverse transcriptase (Invitrogen) and random hexamers.

Transcript levels for Dlk1 (NM_053744) were quantified by qPCR (7300 Real

Time PCR System, Applied Biosystems). Primers and probes were designed

using the software Primer Express 2.0 (Applied Biosystems) as follows, Dlk1:

5’-AACCTCCCCTGGCTGTGTTAA-3’-AGAGGTGCAAGCCCGAATATC and

FAM-AGAACCATGGCAGTGTGTCTGCAAGGA-TAMRA (Taqman® probe)

purchased from Genosys (Sigma). Primers or TaqMan® probes

encompassing fragments of the areas from the extremes of two exons were

designed to ensure the detection of the corresponding transcript avoiding

genomic DNA amplification. The cDNA was amplified at the following

conditions: 95 °C for 10 min, followed by 45 cycles of 15 s at 95 °C and 1 min

at 59 °C, using the TaqMan® Universal PCR Master Mix (Applied Biosystems).

The primer and probe concentrations were 300 and 200 nmol/L, respectively.

All samples were run in triplicate and the average values were calculated and

normalized to 18S rRNA (Applied Biosystems), using the comparative CT

method (2-ΔΔCT).

2.8 Caspase-like activity determination:

To measure caspase 3-, 8- and 9-like activities, aliquots of eWAT homogenate

were incubated in a reaction buffer containing 25mM Hepes (pH 7.5), 10%


210

(w/v) sucrose; 10 mM dithiothreitol, 0.1% CHAPS and 100 μM caspase

substrate Ac (N-acetyl)- LEHD-pNA (Calbiochem, UK) for 2 h at 37°C.

Caspase 3-, 8- and 9-like activities were determined by following the detection

of the chromophore p-nitroanilide after cleavage from the labeled substrate Ac-

LEHD-p-nitroanilide at 405 nm. The method was calibrated with known

concentrations of p-nitroanilide (Calbiochem, UK). Caspase-like activity was

calculated by the p-Na released for equal protein loaded.

2.9 Statistical analysis:

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.


211

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

3.2 Adipocyte cellularity:

Although without statistical meaning, the HFD tended to decrease adipocytes

area range 1000-2999 μm2 and significantly increased adipocytes greater than

9000 μm2 compared to STD-fed group. On the other hand, the ET increased

adipocytes area range between 350 and 2999 μm2 and decreased adipocytes

area greater than 6000 μm2. The protein expression of pre-adipocyte factor

(DLK1/PREF1) decreased in sedentary HFD-fed animals, and its gene and

protein expression increased in response to 8-wks of ET (figure 2).


212

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

3.3 Autophagy markers in eWAT:

As seen in figure 3, the HFD regimen had no significant impact in Beclin-1,

microtubule-associated protein-light chain 3 (Lc3II) or in p62 protein

expression compared to standard diet-fed animals. However, 8-wks of ET

increased Beclin-1 protein expression in both diet types (vs. both sedentary

groups) and tended to increase Lc3II in STD-fed animals although without

reaching statistical significance. The p62 protein expression significantly

increased in both ET groups (vs. both sedentary groups).


213

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

3.4 Apoptosis markers in eWAT:

The HFD significantly increased the pro-apoptotic Bax protein expression,

Bax/Bcl-2 ratio and caspase 3-like activity, when compared to standard diet-

fed groups, while no alterations were observed in Bcl-2 expression or

caspases 8 and 9-like activities. On the other hand, eight-wks of ET increased

protein expression of anti-apoptotic Bcl-2 in both diet types (vs. sedentary

groups) and decreased Bax expression, caspases 8 and 9-like activities (only

in HFD+ET group), Bax/Bcl-2 ratio and caspase 3-like activity in both STD+ET

and HFD+ET groups (figure 4).


214

Figure 4: Effect of diet and endurance training on the expression of apoptosis

markers. Bcl-2 (A), Bax (B), Beclin-1/Bcl-2 ratio (C), Bcl-2/Bax ratio (D) and the

representative images blots of each protein (E). Activity of initiator caspases,

caspase-8 and caspase-9 (E and F), and effector caspase-3 (G). Data are expressed

as the mean±SEM. DxE, diet and exercise interaction; D, diet effect; E, exercise

effect; NS, not significant **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

4. Discussion

The ET has been demonstrated to promote chronic adaptations on adipose

tissue, highlighting the relevance of this tissue on whole-body adaptions to

physical exercise and a promising target to treat obesity and its comorbidities

(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). In contrast, obesity

represents a challenging condition at tissue and cellular levels. In fact, chronic

HFD feeding induces an increased rate of synthesis and degradation of cellular

components in metabolically relevant tissues, such as VAT (Sarparanta et al.,


215

2016). In this context, several studies suggest that autophagy and

apoptosis are two critical processes that play important roles on the

maintenance of cellular homeostasis and metabolism. Therefore, with this

study we intended to analyze how ET modulates autophagy and apoptotic-

related signaling in VAT of long-standing HFD-fed rats. The overall findings

demonstrate that 8 wks of ET reverted the apoptotic signaling induced by HFD;

however, neither HFD nor ET had any impact in VAT autophagy markers.

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


216

(Thorburn, 2008). In the present study, despite no statistical significant

alterations on autophagy-related proteins were found (discussed below), an

upregulation of the apoptotic effector caspase 3-like activity and Bax protein

expression in VAT of sedentary obese animals argue in favor of a pro-apoptotic

phenotype induced by the HFD. These data are supported by others (2010;

Feng et al., 2011) and reinforce the potential contribution of apoptosis in the

pathogenesis of obesity and associated diseases (Boutens & Stienstra, 2016).

On the other hand, regular physical exercise has been widely recognized as a

relevant strategy against several cellular abnormities induced by HFD feeding,

such as apoptosis (Goncalves, Maciel, et al., 2014; Sakurai et al., 2005). In

this context, we observed that 8 wks of ET induced an anti-apoptotic impact

on VAT and also increased DLK1/PREF1 expression in animals fed with both

diet types, which are in agreement with other observations (Sakurai et al.,

2010; Sakurai et al., 2005; Sertie et al., 2013). These findings suggest that ET

was able to induce remodeling on VAT mass even in the context obesity, likely

through the inhibition of adipogenesis of adipocyte precursor cells.

When autophagy is activated, the autophagosome engulfs cytoplasmic

constituents and fuses with lysosomes to complete degradation. Although the

number of autophagosomes correlates with the level of LC3II, other markers

acting upstream (e.g. Beclin-1) or downstream (e.g. p62) are better to

understand changes in autophagy flux (Sarparanta et al., 2016). The Beclin-1

is required for the initiation of the autophagosome formation and for the

recruitment of other autophagy-related proteins (Salminen et al., 2013). On the

other hand, p62 is localized on the autophagosome for degradation, being their

levels strictly regulated by continuous degradation through basal autophagy

(Sahani et al., 2014). Thus, a decreased p62 expression indicate an

autophagic activity whereas its accumulation indicate defective autophagy

(Sahani et al., 2014). Given the role of autophagy-driving adipocyte

differentiation, autophagy activity increases during adipose tissue expansion

(Sarparanta et al., 2016), as observed in visceral fat of obese patients (Kovsan

et al., 2011). Surprisingly, our data showed that HFD feeding had no significant

impact on any autophagy markers despite a tendency to decrease p62


217

expression. In addition, we observed an increase of hypertrophied adipocytes

and decreased DLK1/PREF1 expression, an important gatekeeper of

adipogenesis (Hudak & Sul, 2013) in sedentary HFD-fed animals. These

findings are in line with others (Kovsan et al., 2011) and suggest that the

degree of autophagy activity in VAT correlated with the degree of obesity,

visceral fat and adipocyte hypertrophy.

Several others studies have reported that an increased basal autophagy is

essential for physical exercise-induced skeletal muscle adaptations and to

potentially protect against obesity (Goncalves et al., 2016; Greene et al., 2015;

Lira et al., 2013). However, the role of physical exercise on VAT-related

autophagy mechanisms is scarcely known. In the present study, 8 wks of ET

increased p62 expression in VAT of endurance-trained animals submitted to a

HFD, which suggest that ET suppressed autophagic activity in eWAT;

however, an increased Beclin-1 was also found, which is scientifically

challenging and deserves for better understanding. These data are consistent

with newly findings of Tanaka and coworkers (Tanaka et al., 2015) reporting

that ET suppressed autophagy activity in eWAT. Curiously, also found

increased autophagy activity in inguinal WAT and stromal vascular fraction

collected from eWAT, which suggest that autophagic adaptations are depot-

specific and may, at least in part, explain some of our conflicting results. In

fact, in the present study we used the whole adipose tissue, composed by

distinct cells types, which may had represented a confounding factor to

unraveling the role of ET on VAT autophagy activity in the context of obesity.

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


218

needed to unravel the overall potential therapeutic role of physical exercise on

obesity-related diseases.

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CHAPTER IV. General Discussion

Chapter IV. General Discussion

227

General Discussion

The findings comprised in this dissertation suggest that physical exercise

modulated lipid accumulation, and induced adaptations on adiposopathy

markers. Inflammatory mediators, cytokines and adipokines as well as apoptotic

signaling regulators that positively influence VAT plasticity and remodeling in

obesity were also altered by physical exercise. Moreover, data clearly

demonstrate that physical exercise stimulated skeletal muscle myokines that

were associated with a “brown”-like phenotype in VAT, strengthening the

remarkable effects of physical exercise in VAT plasticity in the context of obesity.

Altogether, data from the studies encompassed in this thesis highlight the role of

exercise, particularly endurance exercise training, as a powerful tool able to

dynamically modulate VAT phenotype in obese conditions. Moreover, this

dissertation contributes to the better understanding of the mechanisms

underlying the morphological, metabolic, inflammatory, apoptotic adaptations and

the “brown”-like phenotype behind VAT “functional boosting”.

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,


228

apoptotic signaling markers (increases on Bax expression and caspase 3-like

activity), and MCP-1 were also observed in VAT of HFD-fed rats from the present

work. The MCP-1 plays a critical role in regulating monocytes/macrophages

migration and infiltration, and subsequent chronic inflammation (Gogebakan et

al., 2015; Kawanishi et al., 2015; Kawanishi et al., 2010; Lumeng et al., 2007), a

pathophysiological mechanism that links obesity to disease risk. Moreover,

increased circulating and tissue levels of pro-inflammatory markers (TNF-α and

leptin) and decreased anti-inflammatory adipQ levels in the HFD-fed animals

suggest that HFD feeding induced changes towards a (mild) pro-inflammatory

state.

It is known that physical exercise is an important strategy to counteract obesity-

induced cellular disturbances (Goncalves, Passos, Rocha-Rodrigues, Torrella, et

al., 2014; Kawanishi et al., 2015; Vieira, Valentine, Wilund, Antao, et al., 2009).

Therefore, with the present dissertation we aimed to analyze the cross-tolerance

effects of physical exercise against the dynamic modulation of the VAT

phenotype induced by obesity. In accordance with others (Gollisch et al., 2009;

Linden et al., 2014; Miyatake et al., 2004; Reseland et al., 2001), data from our

studies (I-V) demonstrated that physical exercise regimens, particularly ET, were

able to counteract HFD-induced adiposopathy, tissue hypoxia, apoptotic

signaling, macrophage infiltration and increased production of inflammatory-

related markers. Moreover, particularly ET, increased the expression of anti-

inflammatory markers (adipQ and IL-10) in both SET and HET animals. These

findings are in accordance with the well-described anti-inflammatory effect of

physical exercise (Gollisch et al., 2009; Kawanishi et al., 2013; Kawanishi et al.,

2015; Kawanishi et al., 2010). This improved inflammatory state has been

associated with the reduction of adipocyte hypertrophy and visceral adiposity

(Gollisch et al., 2009; Miyazaki et al., 2010; Vieira, Valentine, Wilund, Antao, et

al., 2009), which are in line with our findings (study I and IV) in trained animals.

Several studies reported that the reduction of hypertrophic adipocyte contributes

to the increases of adipQ and decreases in leptin production and secretion

(Miyazaki et al., 2010), which are involved in a broad range of system-wide

actions including energy balance, inflammation, insulin sensitivity/resistance and


229

metabolism (Choe et al., 2016; Flores et al., 2006). AdipQ, an adipocyte-secreted

cytokine, has been described to have anti-obesity and anti-diabetic effects by

stimulating lipid oxidation and an (anti)-inflammatory response (Kusminski &

Scherer, 2012; Vernochet et al., 2009). Data from studies I and IV, demonstrating

the ET-mediated increases in the expression of adipQ (plasma and eWAT), were

consistent with improvement of the insulin sensitivity (assessed by QUICKI) and

of the anti-inflammatory phenotype (increases in IL-10) observed in HET animals

(Choe et al., 2016; Numao et al., 2011). Leptin is predominantly secreted by

adipocytes into circulation to regulate energy homeostasis, but also stimulate pro-

inflammatory cytokines production (Choe et al., 2016). Leptin levels are elevated

in obesity (Gollisch et al., 2009; Linden et al., 2014), but hypothalamic leptin

resistance aggravates obesity status and are positively correlated with body fat

and adipocyte size (Choe et al., 2016), which are in line with our observations in

HS animals (study I). Both used models of physical exercise (VPA and ET)

reversed eWAT leptin expression, being this decrease associated with reduced

adipocyte hypertrophy and visceral adiposity, which is consistent with others

(Bradley et al., 2008; Chapados et al., 2008; Gollisch et al., 2009; Huang et al.,

2010; Jenkins et al., 2012; Linden et al., 2014; Zachwieja et al., 1997).

Accumulated evidence attribute several important roles to ghrelin on energy

production, adiposity signal, and inflammation (Leidy et al., 2004; Mao et al.,

2015; Purnell et al., 2003; Zhou & Xue, 2009). Data from study I confirmed

previous findings reporting an inverse correlation between ghrelin levels and

body weight, visceral adiposity and adipocyte size (Purnell et al., 2003). On the

other hand, physical exercise, particularly ET, ameliorated plasma ghrelin levels

with a concomitant decrease in body weight and adiposopathy-related features,

which are in line with findings of Tiryaki-Sonmez et al. (Tiryaki-Sonmez et al.,

2013). Moreover, as ghrelin actions are mediated through its receptor, the growth

hormone secretagogue receptor (GHS-R) (Mihalache et al., 2016), reduced

GHS-R expression in HVPA and HET groups were observed (study I). This

finding is in line with previous reports (Soares & Leite-Moreira, 2008) suggesting

that GSH-R have intriguingly subtle but opposite physiological actions to ghrelin.

Ghrelin has also been described as a negative regulator of autophagy


230

(Rodríguez, Ezquerro, et al., 2015). Autophagy is an important self-degradation

process involved in cellular homeostasis and metabolism (Benbrook & Long,

2012; Salminen et al., 2013). In fact, several studies demonstrated that

autophagy is increased in VAT of obese individuals (Haim et al., 2015; Kovsan et

al., 2011; Sarparanta et al., 2016) in an attempt to limit excessive adipocyte

hypertrophy, inflammation, and consequently prevent VAT dysfunction

(Rodríguez, Ezquerro, et al., 2015). In line, and considering that ET reduced

obesity-related features, a decreased autophagy, as suggested by increased p62

expression, was expected in HET animals (study V). Considering ghrelin as a

negative regulator of autophagy (Rodríguez, Ezquerro, et al., 2015), this finding

might be related to physical exercise-mediated increases in ghrelin levels;

however further studies are needed to better understand this relevant topic.

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


231

and IL-6 overexpression, two important factors modulated by intensity of exercise

(Horowitz, 2003), were observed in ET groups, but not in VPA groups (studies II

and III). A prolonged AMPK activation in WAT has been shown to decrease body

fat content, increase energy expenditure, improve lipolysis and oxidative

metabolism versus lipid storage pathways (Chen et al., 2015; Gaidhu et al., 2009;

Hashimoto et al., 2013). Accordingly, data from studies II and III showed that ET

positively modulated the expression of AQP7 (decreased) and FAT/CD36

(increased), two important intracellular lipid accumulation regulators, along with

lower levels of NEFA and glycerol in plasma of HFD-fed rats. These reduced

levels of NEFA and glycerol, two lipolytic products, were possibly related to the

effect of physical exercise at counteracting the increased lipolytic activity

described in obesity conditions (Jacobs et al., 2016; Lopes et al., 2016). These

data are supported by other studies (Chapados et al., 2008; Pistor et al., 2015)

reporting an inhibitory effect of physical exercise on the adipocyte lipolytic activity.

The AMPK activation suppresses the expression of sterol regulatory element-

binding protein (SREBP)-1c, the major transcription factor involved in the fatty

acid biosynthesis and lipid uptake (Daval et al., 2005). Accordingly, our data

(study III) demonstrated that ET reverted HFD-induced increases in SREBP1c

and positively modulated its downstream target acetyl-CoA carboxylase (ACC),

a lipogenic enzyme, in HFD-fed animals. This possible AMPK-dependent

suppression SREBP1c pathway may underlie, at least in part, the cross-tolerance

effects of physical exercise on VAT plasticity and remodeling in the context of

obesity.

An increased AMPK activity has also been associated with improvements in

mitochondrial function and biogenesis (Gaidhu et al., 2011), and consequently

with a brown-like phenotype in WAT (Wang et al., 2015; Zhang et al., 2015; Zhu

et al., 2016). During the browning process, increased mitochondrial function and

density, and uncoupling of oxidative phosphorylation (OXPHOS) induced via

increased UCP1 expression have been reported (Laye et al., 2009; Sutherland

et al., 2009; Xu et al., 2011). Moreover, other regulators of this process have been

described (Lo & Sun, 2013). A critical role for BMP7 in brown adipogenesis is

known [28] by activating a full program of brown adipogenesis, including the


232

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.


233

Fig

ure

1:

Su

mm

ary

of

the

syste

mic

and

ad

ipose

tis

sue a

da

pta

tio

ns ind

uce

d b

y H

FD

and

th

e p

reve

ntive

– V

PA

and t

hera

pe

utic –

ET

eff

ect of

exe

rcis

e.


234

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CHAPTER V. Conclusions

Chapter V. Conclusions

245

Conclusions

Based on the results that emerged from the studies comprised in this dissertation,

the main conclusions are summarized below:

i) both VPA and ET conferred protection against adiposopathy-related

features, VAT lipid accumulation and macrophage infiltration induced by

obesity;

ii) the ET-induced myokine production was associated with a brown

adipocyte-like phenotype, increased pre-adipocytes and mitochondrial

content, biogenesis and fusion-related proteins in VAT of HFD-fed rats;

iii) the ET reduced lipogenic-related markers in HFD-fed rats;

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;

v) the ET shifted toward an anti-inflammatory phenotype 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;

vii) The ET was effective in attenuating apoptotic-related signaling in long-

standing HFD-fed rats.

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

Chapter V. Conclusions

246

findings contributed to better understand the involvement of systemic and

adipose tissue-driven mechanisms in exercise-induced protection against

obesity. Moreover, the data obtained in this thesis reinforce that exercise

characteristics, namely intensity and duration, might limit intervention program

beneficial effects.

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