Physical exercise and white adipose tissue plasticity in the context of obesity.

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(1)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.

(2) 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. BEIGING, ADIPOKINES. ii. ADIPOSITY,. MYOKINES,.

(3) 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/DTPDES/7087/2014.. The present work was conducted in the Research Centre in Physical Activity, Health and Leisure (CIAFEL) (FCT, UID/DTP/00617/2013) and in the Metabolic Research Laboratory (Clínica Universidad de Navarra) supported by Fondo de Investigación Sanitaria-FEDER (FIS PI10/01677, PI12/00515 and PI13/01430) from the Spanish Instituto de Salud Carlos III, the Department of Health of the Gobierno de Navarra (61/2014).. iii.

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(5) In memory of my dad, who I love and miss dearly.. v.

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(7) 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.. vii.

(8) 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 chromatrographymass 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.. viii.

(9) To Nuno, thank you for all support, friendship, dedication, affection and love you gave me on this journey. My biggest supporters have always been my family. I am very grateful for the opportunities and encouragement provided by my parents, brothers over the years. Thank you so much for your support, help, and advices through this process. Thank you all for contributing to my success.. ix.

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(11) 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. xi.

(12) General Discussion ................................................................................ 227 CHAPTER V. Conclusions ............................................................................. 243 Conclusions ........................................................................................... 245 References ..................................................................................................... 247 References............................................................................................. 249 FACSIMILE .................................................................................................... ccci. xii.

(13) 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. xiii.

(14) 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. xiv.

(15) 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, Beclin1/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. xv.

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(17) List of Tables. CHAPTER III. Experimental Work 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. xvii.

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(19) 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 adipocytelike (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. xix.

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(21) 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. xxi.

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(23) 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. xxiii.

(24) 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. xxiv.

(25) 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. xxv.

(26) 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. xxvi.

(27) CHAPTER I. General Introduction.

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(29) Chapter I. General Introduction. 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. 3.

(30) Chapter I. General Introduction. mechanism that could be stimulated by distinct pharmacological and nonpharmacological 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. 4.

(31) Chapter I. General Introduction. 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. 5.

(32) Chapter I. General Introduction. proteins also have an important role in the process, strongly reinforcing the link between inflammation and metabolic adipocyte deregulation. Leptin, a product of the ob gene, was discovered as an adipocyte-specific secreted protein that regulates food intake and energy expenditure in an endocrine manner (Zhang et al., 1994). Moreover, several other adipokines are secreted by WAT with important functions involved in the regulation of nutrient metabolism, energy expenditure, insulin sensitivity and inflammatory response (Choe et al., 2016; Flores et al., 2006). However, the production and secretion of adipokines by VAT are dysregulated in obesity (Choe et al., 2016; Gollisch et al., 2009; Lara-Castro et al., 2006). For example, some studies reported that circulating adiponectin (adipQ) levels were reduced in obese individuals (Lara-Castro et al., 2006) and inversely correlated with the degree of adiposity and insulin resistance (Choe et al., 2016), while leptin levels were elevated (Gollisch et al., 2009). On the other hand, hypothalamic leptin resistance aggravates obesity status through appetite control inhibition and lipid oxidation (Choe et al., 2016). Ghrelin was firstly discovered in stomach as a ligand of the growth hormone secretagogue receptor (GHS-R), which is expressed in WAT (Tsubone et al., 2005). Through its receptor, ghrelin acts as a growth hormone (GH) releasing peptide with important roles in appetite stimulation, energy and glucose homeostasis, autophagy and immune function (Dixit et al., 2004; Mao et al., 2015; Tsubone et al., 2005). Therefore, circulating levels of ghrelin may function as an adiposity signal that contributes to weight regain in obese subjects as observed by its increased secretion during weight loss (Aas et al., 2009). Under obese conditions, adiposopathy has been associated with the development of inflammation by increasing secretion of various pro-inflammatory chemokines and cytokines, such as tumor necrosis factor (TNF)-α, interleukin-6 (IL-6) and monocyte chemotactic protein (MCP-1) (Lopategi et al., 2016; Lumeng et al., 2007). Moreover, the macrophage content of VAT is positively correlated with both adipocyte size and body fat mass, and the expression of pro-inflammatory cytokines, such as TNFα, is mostly derived from macrophages rather than adipocytes (Weisberg et al., 2003). Along with the increased number of macrophages in “sick” VAT, obesity has been linked to a phenotypic switch from an anti-inflammatory “M2”. 6.

(33) Chapter I. General Introduction. 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. 7.

(34) Chapter I. General Introduction. humans (Alkhouri et al., 2010) has been reported. In addition, the autophagic flux seems to be increased in visceral fat of obese individuals (Kovsan et al., 2011) as this process is required for adipocyte differentiation (Sarparanta et al., 2016). Markers of autophagy are correlated with whole body adiposity, visceral fat distribution, and adipocyte hypertrophy (Sarparanta et al., 2016). Given the social, health and economic impact of obesity, clinicians and researchers have focused their attention on a variety of preventive and therapeutic countermeasures to antagonize such phenomenon. These include the development of many pharmacological agents and chemicals and, on the other hand, the study of the powerfulness that life style changes have on the prevention, attenuation and reversion of obesity-associated features. Similarly to its. impact. against. many. other. pathological. disorders,. including. neurodegenerative, cardiovascular and metabolic diseases (Bertram et al., 2016; Safdar et al., 2016), physical exercise is one of the most powerful lifestyle interventions used to prevent and/or mitigate overweight and visceral adiposity accumulation associated with obesity (Bajer et al., 2015; Way et al., 2016). Even when body weight or visceral adiposity is not reduced, physical exercise has significant impact on metabolic health. Accumulating evidence reveal that physical exercise induced favorable metabolic and inflammatory adaptations in VAT, strengthening the metabolic relevance of adipose tissue on whole-body adaptations to physical exercise and being a promising direct target in the treatment of obesity and associated disorders (De Matteis et al., 2013; Giles et al., 2016; Hirshman et al., 1989; Holland et al., 2016; Stanford et al., 2015; Tanaka et al., 2015). Moreover, it has been reported that physical exerciseinduced myokines release mediates some physiological adaptations in VAT, including the modulation of a brown adipocyte-like phenotype (Rodríguez et al., 2016). In the present dissertation, we focused in the cross-talk between skeletal muscle and WAT, and in the potential mediators of this process in an attempt to have a more global view of the benefits of physical exercise on the obesity-related underlying pathways. Although structural and functional differences between white and brown adipose tissues, they have a remarkable plasticity and can acquire features of one another. 8.

(35) Chapter I. General Introduction. under specific physiological conditions and stimuli (Wu et al., 2014), which include physical exercise. Therefore, white adipose cells can initiate adaptive responses to physical exercise and acquire a brown adipocyte-like phenotype (Bostrӧm et al., 2012; Nakhuda et al., 2016; Wu et al., 2014). The browning process relates to an increase in mitochondrial density and function (Laye et al., 2009; Sutherland et al., 2009; Xu et al., 2011), and to an uncoupling of the oxidative phosphorylation (OXPHOS) caused by the increase of uncoupling protein-1/thermogenin (UCP1) expression. Morphologically, brown adipocyte-like phenotype cells are characterized by the presence of small lipid droplets typical of brown adipocytes (Cao et al., 2011). Animals-based studies showed that the expression of browning genes (e.g. Ucp1 and Prdm16) increases in VAT after a chronic ET (Tiano et al., 2015; Wu et al., 2014) or voluntary wheel running (Cao et al., 2011; Tiano et al., 2015), which suggests that physical exercise enhances brown adipocyte progenitor cells in adipose tissue. One potential mechanism underlying the development of brown adipocyte-like phenotype includes the myokines secreted by exercised skeletal muscle (Bostrӧm et al., 2012). In fact, some skeletal muscle-derived myokines, e.g. irisin and IL-6, exert endocrine effects on adipocytes as positive regulators of brown adipocyte-like phenotype (Bostrӧm et al., 2012; Cao et al., 2011; Knudsen et al., 2014), and thus, emerged as new potential candidates to treat obesity and related disorders. However, to our best knowledge, the underlying mechanisms of physical exercise-induced myokines release and its potential signaling inﬂuence on WAT metabolism is still a matter of debate in the context of obesity. An increasing number of studies demonstrate that physical exercise exerts important effects by reducing visceral adiposity and large-sized adipocytes, which consequently improves hypoxiaresponsive markers, e.g. HIF-1α and VEGF, in obesity (Baynard et al., 2012; Yan et al., 2012). Small-sized adipocytes have been associated with a positive impact on the production and secretion of adipose-derived hormones, including leptin and adipQ, which are involved in the regulation of several physiological functions, such as energy balance, appetite, inflammation, and metabolism (Choe et al., 2016). Short-term ET-induced decreases in leptin levels were associated with fat mass loss (Miyatake et al., 2004) and long-term of ET (more than 12 weeks) had. 9.

(36) Chapter I. General Introduction. 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.. 10.

(37) Chapter I. General Introduction. Existing evidence showed that physical exercise increased the expression of VAT mitochondrial content and biogenesis markers (Laye et al., 2009; Sutherland et al., 2009; Xu et al., 2011), such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and mitochondrial transcript factor A (TFAM) (Sutherland et al., 2009; Xu et al., 2011). In addition, the protein and gene expression of OXPHOS subunits increased after voluntary free-wheel and ET in DIO mice (Xu et al., 2011), as well as in hyperphagic obese rats (Laye et al., 2009). These physical exercise-induced benefits in the metabolism of VAT mitochondria seem to be explained by increased 5' AMP-activated protein kinase (AMPK) activation (Canto et al., 2009). The AMPK activation has been associated with increases of PGC-1α expression, which stimulates mitochondrial biogenesis (Chen et al., 2015), and also potentially shifts adipocyte metabolism toward fat utilization instead of storage (Chen et al., 2015). These metabolic effects of physical exercise are regularly accompanied by a reduction of the inflammatory status (Goto-Inoue et al., 2013; Oliveira et al., 2011). The anti-inflammatory impact of regular physical exercise is well documented (Gollisch et al., 2009; Jenkins et al., 2012; Kawanishi et al., 2015), and rely on several modulatory effects, such as decreased expression of pro-inflammatory cytokines (TNF-α and IL-6) and macrophage recruitment and infiltration (Gollisch et al., 2009; Kawanishi et al., 2010), independently of body weight reduction (Vieira, Valentine, Wilund, Antao, et al., 2009). Furthermore, physical exercise increases “M2” macrophages activation, which was proposed as a potential mechanism by which exercise reduces inflammation in adipose tissue (Kawanishi et al., 2010). The “M2” macrophages release anti-inflammatory cytokines and are associated with increased FFA oxidation and decreased availability of potentially toxic lipid species. Interestingly, Kawanishi et al (Kawanishi et al., 2010) demonstrated that mice feed with DIO and submitted to ET exhibited reduced pro-inflammatory cytokines in epididymal WAT even without reductions on fat mass. Those changes were associated both with a suppression of macrophages infiltration and with macrophages phenotype switch from M1 to M2 (Kawanishi et al., 2010). The specific changes on fatty acid profile in adipose tissue triglycerides may also be an important adaptation induced by physical exercise. In fact, this may. 11.

(38) Chapter I. General Introduction. contribute to the attenuation of the inflammatory state as some fatty acids have been described to be involved in the inflammatory process (Oliveira et al., 2015; Vaughan et al., 2015). In fact, ET programs seem to induce fatty acids profilespecific 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 antiinflammatory 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 antiapoptotic markers in VAT (Sertie et al., 2013). Both autophagy and apoptoticrelated 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. 12.

(39) Chapter I. General Introduction. 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 nonpharmacological 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.. 13.

(40) Chapter I. General Introduction. 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, TNFα and IL-10);. 14.

(41) Chapter I. General Introduction. 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 HFDfed 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.. References Aas, A. M., Hanssen, K. F., Berg, J. P., Thorsby, P. M., & Birkeland, K. I. (2009). Insulin-stimulated increase in serum leptin levels precedes and correlates with weight gain during insulin therapy in type 2 diabetes. J Clin Endocrinol Metab, 94(8), 2900-2906. Alkhouri, N., Gornicka, A., Berk, M. P., Thapaliya, S., Dixon, L. J., Kashyap, S., Schauer, P. R., & Feldstein, A. E. (2010). Adipocyte apoptosis, a link between obesity, insulin resistance, and hepatic steatosis. J Biol Chem, 285(5), 3428-3438. Bajer, B., Vlcek, M., Galusova, A., Imrich, R., & Penesova, A. (2015). Exercise associated hormonal signals as powerful determinants of an effective fat mass loss. Endocr Regul, 49(3), 151-163. Balagopal, P., George, D., Yarandi, H., Funanage, V., & Bayne, E. (2005). Reversal of obesity-related hypoadiponectinemia by lifestyle intervention: a controlled, randomized study in obese adolescents. J Clin Endocrinol Metab, 90(11), 6192-6197.. 15.

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