Adipose tissue (AT) expansion and remodeling that maintains healthy function relies on stromal preadipocytes capable of differentiating into new adipocytes (adipogenesis). During chronic positive energy balance, a relative deficit in adipogenesis, from either a decrease in preadipocyte number or their capacity to differentiate, leads to excessive adipocyte hypertrophy and AT dysfunction. AT contains macrophages whose number and activation state is dynamically regulated with changes in AT mass. This study aims to investigate the effect of macrophage-secreted factors on preadipocyte survival. To assess the effect of macrophage-secreted factors on preadipocytes, murine 3T3-L1 preadipocytes or human primary preadipocytes were incubated with macrophage-conditioned medium (MacCM), prepared from either murine (J774A.1, RAW264.7, bone marrow-derived) or human (THP-1, monocyte-derived) macrophage models, respectively. MacCM inhibited preadipocyte apoptosis and activated pro-survival signaling in both preadipocyte models. Inhibition of PDGFR, Akt, or ERK1/2 reduced the pro-survival effect of MacCM in 3T3-L1 preadipocytes. Inhibition of reactive oxygen species (ROS) generation, or enhancement of ROS clearance, reduced MacCM-dependent 3T3-L1 preadipocyte survival. Whereas anti-inflammatory activated macrophages retained the ability to prevent preadipocyte apoptosis, pro-inflammatory activated macrophages did not. TNF-α immunoneutralization restored the survival activity of pro-inflammatory MacCM on 3T3-L1 preadipocytes. These studies reveal a novel pro-survival effect of MacCM on preadipocytes, and identify signaling molecules (PDGF, Akt, ERK1/2, and ROS) that underlie this action. Macrophage activation was found to regulate the pro-survival activity of MacCM. These in vitro cell culture studies are consistent with a model in which the extent of preadipocyte apoptosis in vivo may determine preadipocyte number and the ability of AT to expand while maintaining healthy function during chronic positive energy balance.

El objetivo principal de esta tesis se ha centrado en el estudio de las diferencias entre ratas macho y hembra en la morfología, la función y la biogénesis mitocondrial del tejido adiposo marrón (TAM) y del hígado, mediante el análisis de distintas subpoblaciones mitocondriales obtenidas a través del fraccionamiento de la población mitocondrial total. Los resultados han puesto de manifiesto que las diferencias entre géneros a nivel mitocondrial tanto en el TAM y como en el hígado podrían ser atribuidas a la existencia de una subpoblación mitocondrial altamente diferenciada en las hembras, hecho que podría ser indicativo de un proceso de biogénesis mitocondrial distinto entre ambos géneros. Los resultados sugieren la existencia de un factor común a ambos tejidos que influiría en la regulación de dicho proceso. En este sentido, las hormonas sexuales podrían ser uno de los factores candidatos responsables de las diferencias observadas en el presente trabajo. / The main goal of this thesis has been focused on the study of gender differences in the mitochondrial morphology, function and biogenesis both in brown adipose tissue (BAT) and in liver, through the analysis of the several mitochondrial subpopulations isolated by means of the fractionation of the whole mitochondrial population. Results have reflected that the gender dimorphism stated in mitochondrial population both in BAT and in liver could be attributed to the existence to more highly differentiated mitochondria in female rats, which could be the result of a different mitochondrial biogenesis process between genders. Since the existence of a common factor which influences this process in both tissues could be hypothesized, sexual hormones could be one of the main factors responsible for the differences described in the present work

Obesity is one of the most important public health problems facing the world today. Gene expression studies applied to fat depots from obese subjects have provided important clues about the pathophysiology of adipose tissue. The data collected in this thesis show that the synthesis of fatty acids (lipogenesis) is decreased in the adipose tissue of obese subjects, and describe the behavior of a new lipogenic factor. We also demonstrate that subcutaneous fat (beneath the skin of the buttocks, thighs and abdomen) is characterized by a greater responsiveness to thyroid hormones than the visceral (around the omentum, the intestines and the perirenal areas), and describe the increased activity of enzymes that activate thyroid hormones in adipose tissue of obese patients and the effects on the metabolism. According to these results, the local activation of thyroid hormone and the ability to synthesize fat are altered in adipose tissue from obese patients, and indicate significant differences between visceral and subcutaneous adipose tissue depots. / La obesidad es uno de los problemas de salud pública más importante. Los estudios de expresión aplicados a los depósitos de grasa en sujetos obesos han aportado importantes indicios sobre la fisiopatología del tejido adiposo. Los datos recogidos en esta tesis doctoral demuestran que la síntesis de grasa (lipogénesis) está disminuida en el tejido adiposo del paciente obeso, y describen el comportamiento de un nuevo factor lipogénicos. Se demuestra además que el tejido adiposo subcutáneo (situado bajo la piel de las nalgas , muslos y abdomen) está caracterizado por una mayor capacidad de respuesta a las hormonas tiroideas respecto al adiposo visceral (alrededor del epiplón, los intestinos y las áreas perirrenal) y se describe un incremento en la actividad de las enzimas que activan las hormonas tiroideas en el tejido adiposo del paciente obeso y los posibles efectos de esta eventualidad sobre el metabolismo. Según los resultados recopilados en esta tesis doctoral, la activación local de hormonas tiroideas y la capacidad para sintetizar acidos grasos del tejido adiposo del paciente obeso están alteradas, e indican importantes diferencias entre los depósitos de grasa visceral y subcutáneo. / L’obesitat és un dels problemes de salut pública més important. Els estudis d'expressió aplicats als dipòsits de greix en subjectes obesos han aportat importants indicis sobre la fisiopatologia del teixit adipós. Les dades recollides a aquesta tesi doctoral demostren que la síntesis de greix (lipogènesis) està disminuïda al teixit adipós del pacient obès, i descriuen el comportament d'un nou factor lipogènic. Es demostra, a més a més, que el teixit adipós subcutani (situat sota la pell de les natges, cuixes i abdomen) està caracteritzat per una major capacitat de resposta a les hormones tiroidees respecte a l'adipós visceral (al voltant de l’epipló, els intestins i las àrees perirenals) i es descriu un increment en l’activitat dels enzims que activen les hormones tiroidees al teixit adipós del pacient obès i els possibles efectes d'aquesta eventualitat sobre el metabolisme. Segons els resultats recopilats a aquesta tesi doctoral, l’activació local d'hormones tiroidees i la capacitat per sintetitzar greixos del teixit adipós del pacient obès estan alterades, i indiquen importants diferències entre els dipòsits de greix visceral i subcutani.

Previous investigators demonstrated that epididymal white adipose tissue (EWAT) lipectomy suppressed spermatogenesis and caused atrophy of the seminiferous tubules. EWAT lipectomy, however, may disrupt testicular innervation, which reportedly compromises testicular function. To resolve this confound and better clarify the role of EWAT in spermatogenesis, three experimental groups of hamsters were created in which: i.) the superior and inferior spermatic nerves were transected (SSNx) at the testicular level, ii.) EWAT was extirpated (EWATx), and iii.) testicular nerves and EWAT were left intact (SHAM controls). It was hypothesized that transection of the superior and inferior spermatic nerves would disrupt normal spermatogenesis. The findings indicate a significant reduction in spermatogenic activity and marked seminal tubule atrophy within the EWATx testis, as compared to the SSNx and controls testes, which did not differ significantly from each other. From these data, it is concluded that EWAT, and not testicular innervation, is central to normal spermatogenesis.

Le récepteur CD36 est impliqué dans le transport des acides gras libres non estérifiés (AGNE) au niveau des tissus cardiaque et périphériques. Les dommages tissulaires et la dysfonction cardiaque observés après une ischémie-reperfusion (I/R) du myocarde sont en partie liés à l’internalisation et au métabolisme oxydatif accrus des AGNE dont la concentration sanguine augmente transitoirement après un infarctus du myocarde, contrairement à ce qui est observé chez des souris déficientes en CD36. Nous avons émis l’hypothèse selon laquelle le EP 80317, un ligand synthétique du récepteur CD36, exercerait un effet cardioprotecteur contre les dommages induits par une ischémie transitoire du myocarde. Nos objectifs étaient 1) de vérifier l’effet cardioprotecteur du EP 80317 et 2) de définir son mécanisme, plus précisément de documenter l’effet du traitement sur le métabolisme lipidique. À cette fin, des souris de type sauvage ont été traitées par le EP 80317 (289 nmol/kg) par voie sous-cutanée pendant 14 jours avant d’être soumises à 30 minutes d’ischémie suivant la ligature de l’artère coronaire gauche descendante et de sa reperfusion pendant une période de 6 ou 48 heures. Le cœur et les tissus périphériques (foie, muscle squelettique et dépôts adipeux) ont été prélevés pour déterminer le profil de certains gènes impliqués dans la régulation du métabolisme lipidique. Nos travaux ont montré que l’effet cardioprotecteur d’un traitement préventif par le EP 80317 est associé à une augmentation transitoire du stockage des triglycérides et d’une réduction des AGNE circulants. / The CD36 receptor is involved in the transport of non-esterified fatty acids (NEFA) in cardiac and peripheral tissues. Tissue injury and cardiac dysfunction are in part due to acute internalization and oxidative metabolism of NEFA which concentration rises transiently in the blood following myocardial infarction, in opposition to what is observed in CD36-deficient mice. We hypothesized that EP 80317, a synthetic ligand of the CD36 receptor, provides cardioprotective effect against injuries induced by transient myocardial ischemia. Our objectives were 1) to verify the cardioprotective effect of EP 80317 and 2) to define its mechanism, more precisely to investigate the mechanisms of the treatment on lipid metabolism. For this purpose, wild-type mice were treated with EP 80317 (289 nmol/kg) subcutaneously for 14 days before being submitted to 30 minutes of ischemia following left anterior descending coronary artery ligature and reperfusion for a period of 6 or 48 hours. Heart and peripheral tissues (liver, skeletal muscle and adipose tissue) were harvested to determine the profile of selected genes involved in the regulation of lipid metabolism. Our work has shown that the cardioprotective effect of a pretreatment with EP 80317 is associated with a transient increase of triglycerides storage and reduced circulating NEFA.

Hypertension and salt-sensitivity are independent risk factors for cardiovascular disease. Although both conditions are idiopathic, they develop due to a complex interplay between susceptibility genes and environmental factors. Given that the kidney plays an important role in regulating blood pressure, in particular, by maintaining sodium and water balance via pressure-natriuresis, it is not surprising that disturbances in the proper functioning of this intrarenal mechanism have been linked to these conditions. Although direct coupling of changes in renal arterial pressure (RAP) to renal interstitial hydrostatic pressure (RIHP) and consequent sodium excretion is well established, few studies have characterized the moment-to-moment aspects of this process. Thus, the main focus of the research presented herein was to characterize the moment-to-moment RAP-RIHP relationship, and assess the functioning of this intrarenal mechanism in various animal models of genetic and environmentally-induced hypertension and/or salt-sensitivity. In adult normotensive rats, the response time of RIHP to acute changes in RAP was rapid (<2 seconds), and the moment-to-moment RAP-RIHP relationship was linear over a wide range of pressures. Additionally, the functioning of this relationship was not affected by inhibition of the renin-angiotensin system and autonomic nervous system. Further, the acute RAP-RIHP relationship was impaired in hypertension and/or salt-sensitivity. Specifically, animals with a hypertensive phenotype (i.e. young spontaneously hypertensive rats [SHR] and pro-atrial natriuretic peptide gene-disrupted mice [ANP -/-]) displayed a rightward shift in the moment-to-moment pressure-natriuresis curve towards higher RAP. This rightward shift was associated with increased structurally-based vascular resistance properties in the hindlimb of young SHR versus their normotensive controls. Salt-sensitive phenotypes were associated with a blunting of this acute mechanism. Specifically, this blunting was evident in both the ANP -/-, a transgenic model of salt-sensitive hypertension, and in adult perinatal iron deficient (PID) rats, a developmentally programmed model of salt-sensitivity. It appears that a blunting in the RAP-RIHP relationship is influenced by an imbalance of key blood pressure modulating factors (e.g. ANP). Further, visceral obesity was associated with salt-sensitivity in PID rats; however the mechanism(s) are yet to be elucidated. Novel methodologies (MRI, abdominal girth) were developed for non-invasive assessment of visceral obesity to aid future research. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2010-05-12 10:11:21.197

Adipose tissue (AT) expansion and remodeling that maintains healthy function relies on stromal preadipocytes capable of differentiating into new adipocytes (adipogenesis). During chronic positive energy balance, a relative deficit in adipogenesis, from either a decrease in preadipocyte number or their capacity to differentiate, leads to excessive adipocyte hypertrophy and AT dysfunction. AT contains macrophages whose number and activation state is dynamically regulated with changes in AT mass. This study aims to investigate the effect of macrophage-secreted factors on preadipocyte survival. To assess the effect of macrophage-secreted factors on preadipocytes, murine 3T3-L1 preadipocytes or human primary preadipocytes were incubated with macrophage-conditioned medium (MacCM), prepared from either murine (J774A.1, RAW264.7, bone marrow-derived) or human (THP-1, monocyte-derived) macrophage models, respectively. MacCM inhibited preadipocyte apoptosis and activated pro-survival signaling in both preadipocyte models. Inhibition of PDGFR, Akt, or ERK1/2 reduced the pro-survival effect of MacCM in 3T3-L1 preadipocytes. Inhibition of reactive oxygen species (ROS) generation, or enhancement of ROS clearance, reduced MacCM-dependent 3T3-L1 preadipocyte survival. Whereas anti-inflammatory activated macrophages retained the ability to prevent preadipocyte apoptosis, pro-inflammatory activated macrophages did not. TNF-α immunoneutralization restored the survival activity of pro-inflammatory MacCM on 3T3-L1 preadipocytes. These studies reveal a novel pro-survival effect of MacCM on preadipocytes, and identify signaling molecules (PDGF, Akt, ERK1/2, and ROS) that underlie this action. Macrophage activation was found to regulate the pro-survival activity of MacCM. These in vitro cell culture studies are consistent with a model in which the extent of preadipocyte apoptosis in vivo may determine preadipocyte number and the ability of AT to expand while maintaining healthy function during chronic positive energy balance.

The global epidemic of obesity is rapidly becoming a major public health problem in many parts of the world. Unhealthy diets and physical inactivity are two modifiable risk factors for prevention of obesity and its associated chronic diseases. Their influence on muscle energy metabolism and fat mass is not completely elucidated. A decreased capacity for fatty acid oxidation (FAO) may be a metabolic risk factor for weight gain and is found to be depressed in obese individuals; and exercise training may promote an increased capacity for FAO. In addition to the interest in whole-body FAO, the role of site specific lipid accumulation including visceral adipose tissue (VAT), intrahepatic lipids (IHL) and intramyocellular lipids (IMCL) has become a focus of interest because of their reported association with insulin resistance (IR), a key metabolic defect associated with obesity and type 2 diabetes mellitus (T2DM). However, ambiguity persists regarding the importance of IMCL as a metabolic substrate for energy production in obesity. A better understanding of the factors regulating FAO, body fat distribution and IMCL mobilisation is important for the development of interventions allowing effective treatment of conditions in which these are disturbed. The study of individuals with metabolic myopathies can give more information about the energy metabolism of muscle. McArdle disease (MD) affects glucose availability to muscle for energy production. Investigations into IMCL storage and mobilisation in MD have not been reported. The aims of this thesis are to investigate 1) the effects of weight-loss via dietary restriction plus modest but clinically-relevant exercise training on FAO, body fat distribution and mobilisation of IMCL during exercise in obese non-diabetic adults; 2) the effect of an exercise training intervention on IMCL storage and mobilisation in a subject with MD. All obese subjects underwent a 4 month lifestyle intervention with weekly meetings with a dietitian and an exercise physiologist. Of the 92 subjects, 73 completed the intervention. They showed significant decreases in body weight (8%), fat mass (14%) and total cholesterol (5%). The exercise prescription of 1500 kcal.week-1 resulted in variable compliance with the prescription (1224 ± 1085 kcal.week-1) measured by heart rate monitor. Those who did most exercise and also those who had less weekly variability in their exercise, had greater reductions in body weight and fat mass. The total activity energy expenditure measured by accelerometry did not change post-intervention but there was a reduction in low intensity activity and an increase in moderate and high intensity activity. A submaximal treadmill test and resting metabolic rate (RMR) using indirect calorimetry was measured before and after the intervention to investigate factors regulating FAO and energy expenditure. Subjects showed increases in FAO without change in energy expenditure for the same walking speed post intervention, but the volume of exercise completed during the intervention was not associated with these changes. To investigate body fat distribution in obesity, VAT, IHL and soleus muscle IMCL was measured in a sub-group of 18 males by magnetic resonance imaging (MRI) and spectroscopy (MRS) along with measurement of maximal aerobic capacity. Fitness increased significantly with significant decreases in VAT (29%) and IHL (54%), without significant change in IMCL. Subjects who had the greatest decrease in VAT were those who exercised for longer durations during the intervention. IHL was the only measure of excess lipid that correlated with IR. The measurement of IMCL before and after 1-hour of cycle ergometer exercise showed no significant mobilisation of IMCL either at baseline or after the lifestyle intervention. The intensity of the acute exercise was adjusted to correspond to each individual’s maximal fatty acid oxidation (MFAO) which increased by over 60% post intervention. In the subject with MD, an 8 week exercise training intervention without dietary intervention increased IMCL stores by 27%, but there was no marked change in IMCL with acute exercise at both time points. The findings of this thesis demonstrate that a clinically relevant and achievable lifestyle intervention incorporating weight loss through diet and objectively measured exercise can achieve improvements in blood lipid profile, body composition and FAO. The differential effects of the intervention on the various fat depots and their associations to metabolic markers suggest that individualised strategies may be required dependent upon body fat distribution. The non detection of mobilisation of IMCL by MRS suggests that these lipids may not be present as a substrate source in this population but rather an ectopic lipid depot related to increased energy consumption in diet. The relatively low capacity for FAO in both the obese and MD subjects may have affected the results. This thesis discusses implications for clinical practice, discusses novel findings related to the energy metabolism in obesity and MD and informs clinical and basic science about important future directions.

The global epidemic of obesity is rapidly becoming a major public health problem in many parts of the world. Unhealthy diets and physical inactivity are two modifiable risk factors for prevention of obesity and its associated chronic diseases. Their influence on muscle energy metabolism and fat mass is not completely elucidated. A decreased capacity for fatty acid oxidation (FAO) may be a metabolic risk factor for weight gain and is found to be depressed in obese individuals; and exercise training may promote an increased capacity for FAO. In addition to the interest in whole-body FAO, the role of site specific lipid accumulation including visceral adipose tissue (VAT), intrahepatic lipids (IHL) and intramyocellular lipids (IMCL) has become a focus of interest because of their reported association with insulin resistance (IR), a key metabolic defect associated with obesity and type 2 diabetes mellitus (T2DM). However, ambiguity persists regarding the importance of IMCL as a metabolic substrate for energy production in obesity. A better understanding of the factors regulating FAO, body fat distribution and IMCL mobilisation is important for the development of interventions allowing effective treatment of conditions in which these are disturbed. The study of individuals with metabolic myopathies can give more information about the energy metabolism of muscle. McArdle disease (MD) affects glucose availability to muscle for energy production. Investigations into IMCL storage and mobilisation in MD have not been reported. The aims of this thesis are to investigate 1) the effects of weight-loss via dietary restriction plus modest but clinically-relevant exercise training on FAO, body fat distribution and mobilisation of IMCL during exercise in obese non-diabetic adults; 2) the effect of an exercise training intervention on IMCL storage and mobilisation in a subject with MD. All obese subjects underwent a 4 month lifestyle intervention with weekly meetings with a dietitian and an exercise physiologist. Of the 92 subjects, 73 completed the intervention. They showed significant decreases in body weight (8%), fat mass (14%) and total cholesterol (5%). The exercise prescription of 1500 kcal.week-1 resulted in variable compliance with the prescription (1224 ± 1085 kcal.week-1) measured by heart rate monitor. Those who did most exercise and also those who had less weekly variability in their exercise, had greater reductions in body weight and fat mass. The total activity energy expenditure measured by accelerometry did not change post-intervention but there was a reduction in low intensity activity and an increase in moderate and high intensity activity. A submaximal treadmill test and resting metabolic rate (RMR) using indirect calorimetry was measured before and after the intervention to investigate factors regulating FAO and energy expenditure. Subjects showed increases in FAO without change in energy expenditure for the same walking speed post intervention, but the volume of exercise completed during the intervention was not associated with these changes. To investigate body fat distribution in obesity, VAT, IHL and soleus muscle IMCL was measured in a sub-group of 18 males by magnetic resonance imaging (MRI) and spectroscopy (MRS) along with measurement of maximal aerobic capacity. Fitness increased significantly with significant decreases in VAT (29%) and IHL (54%), without significant change in IMCL. Subjects who had the greatest decrease in VAT were those who exercised for longer durations during the intervention. IHL was the only measure of excess lipid that correlated with IR. The measurement of IMCL before and after 1-hour of cycle ergometer exercise showed no significant mobilisation of IMCL either at baseline or after the lifestyle intervention. The intensity of the acute exercise was adjusted to correspond to each individual’s maximal fatty acid oxidation (MFAO) which increased by over 60% post intervention. In the subject with MD, an 8 week exercise training intervention without dietary intervention increased IMCL stores by 27%, but there was no marked change in IMCL with acute exercise at both time points. The findings of this thesis demonstrate that a clinically relevant and achievable lifestyle intervention incorporating weight loss through diet and objectively measured exercise can achieve improvements in blood lipid profile, body composition and FAO. The differential effects of the intervention on the various fat depots and their associations to metabolic markers suggest that individualised strategies may be required dependent upon body fat distribution. The non detection of mobilisation of IMCL by MRS suggests that these lipids may not be present as a substrate source in this population but rather an ectopic lipid depot related to increased energy consumption in diet. The relatively low capacity for FAO in both the obese and MD subjects may have affected the results. This thesis discusses implications for clinical practice, discusses novel findings related to the energy metabolism in obesity and MD and informs clinical and basic science about important future directions.

Diss. (sammanfattning) Umeå : Umeå universitet, 2006. / Härtill 4 uppsatser.