A depleção de tecido adiposo é um marcador da caquexia. Neste contexto, o aumento na lipólise decorrente do aumento na expressão da lipase hormônio-sensível (LHS) parece ser o fator-chave. Entretanto, a contribuição das novas proteínas relacionadas à lipólise [adipose triglyceride lipase (ATGL), comparative gene identification 58 (CGI-58) e perilipina] ainda é controversa. Caracterizamos a expressão destas proteínas e de adipocinas na caquexia. Pacientes com câncer caquéticos foram investigados. Um modelo experimental foi também estudado utilizando animais portadores de tumor sacrificados no 7º (TB7) e no 14º dia (TB14) após a inoculação tumoral e controles. Foram analisados no tecido adiposo os aspectos morfológicos, morfométricos e moleculares. Verificamos nos pacientes um aumento na expressão de LHS e ATGL, concomitantemente à redução da perilipina. Nos animais TB7 verificou-se um desequilíbrio na secreção de fatores anti e pró-inflamatórios e no grupo TB14 houve redução na expressão das proteínas analisadas sugerindo comprometimento da função celular. / Loss of fat mass is a hallmark of cachexia. It seems that an increase in lipolysis due to increased expression of hormone-sensitive lipase (HSL) is the key factor behind this effect. However, the contribution of novel proteins related to lipolysis [adipose triglyceride lipase (ATGL), comparative gene identification - 58 (CGI-58) and perilipin 1] is still controversial. We characterized the expression of those proteins and adipokines in cachexia. Subcutaneous adipose tissue from cachetic cancer patients and epidydimal pad from tumour-bearing rats was analysed. Morphological, morphometric and molecular aspects were examined. We found an increased HSL and ATGL expression and reduction in perilipin 1 content in cachectic patients. In rats, at the intermediate stage of the syndrome, there was an imbalance in the secretion of pro and anti-inflammatory factors. In terminal cachexia the expression of almost all proteins analysed was reduced in the animals, suggesting impairment of cellular function.

Orientador: Carlos Alan Candido Dias Junior / Resumo: INTRODUÇÃO: O tecido adiposo perivascular (PVAT) libera sob condições fisiológicas fatores de relaxamento derivados do tecido adiposo (ADRF), que possivelmente desempenham um papel importante na modulação do tônus vascular. Foi demonstrado que a inativação de canais de potássio sensíveis ao ATP (KATP) aboliu o efeito anticontratil do PVAT. Outros estudos mostraram que o sulfeto de hidrogênio (H2S) é produzido pelo PVAT devido a expressão da sua enzima precursora cistationina gamma liase (CSE) e que o H2S atua através ativação dos KATP. Além disso, o PVAT sofre uma disfunção em condições fisiopatológicas como a hipertensão. No entanto, seus efeitos ainda não são bem explorados durante a hipertensão gestacional. Portanto, nosso objetivo foi examinar o envolvimento do H2S derivado do PVAT na modulação do tônus vascular de ratas prenhes hipertensas e normotensas. MÉTODOS: Foram realizadas curvas concentração-resposta induzidas pela fenilefrina na presença e ausência de PVAT e endotélio em ratas prenhes normotensas (Norm- Preg) e hipertensas (HTN-Preg). A pressão arterial materna, os parâmetros fetais e placentários, a angiogenese e os níveis de H2S também foram avaliados. RESULTADOS: A hipertensão gestacional foi associada ao desequilíbrio angiogênico e a restrição do crescimento fetal e placentário e o PVAT não se mostrou disfuncional. Além disso, sob a formação estimulada de H2S pelo PVAT, mas não no endotélio, houve redução nas curvas concentração-resposta à fenilefrina em aor... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: INTRODUCTION: Perivascular adipose tissue (PVAT) releases diffusible adipocyte- derived relaxing factors (ADRFs) under physiological conditions, which possibly play key roles to modulate the vascular tone. It was demonstrated that the inactivation of ATP-sensitive potassium channels (KATP channels) impaired the anticontractile effect of PVAT. Further studies have shown that hydrogen sulfide (H2s) is released by PVAT due to the expression of cystathionine gamma lyase (CSE) and that (H2s) acts through the activation of KATP channels. In addition, PV A T is impaired in pathophysiological situations such as hypertension. However, it is not yet well explorated during hypertension in pregnancy. Therefore, we aimed to examine the involvement of PVAT-derived H2S to modulate the vascular tone in aorta from normotensive and hypertensive pregnant rats. METHODS: Phenylephrine-induced contractions in the presence and absence of PVAT and endothelium in aortas from normotensive pregnant (Norm-Preg) and HTN-Preg rats were investigated. Maternal blood pressure, fetal-placental parameters, angiogenesis and H2S levels were also assessed. RESULTS: Hypertensive pregnancy associated with angiogenic imbalance and fetal- placental growth restrictions revealed that there is no PVAT dysfunction. Moreover, under stimulated H2S formation PVAT, but not endothelium, reduces phenylephrine-induced contractions in aortas from HTN-Preg rats. Also, H2S synthesis inhibitor abolishes anticontractile effects disp... (Complete abstract click electronic access below) / Mestre

Thermogenic brown and beige adipocytes convert chemical energy to heat by metabolizing glucose and lipids via uncoupling protein 1 (Ucp1), a process known as non-shivering thermogenesis. Serotonin (5-HT) neurons in the ventral medulla are known to regulate sympathetic efferent neurons in the intermediolateral nucleus (IML) necessary to maintain brown adipose tissue (BAT) activity. Previous studies show that mice lacking central 5-HT neurons are incapable of maintaining body temperature in cold, ambient conditions. Due to this direct linkage between 5-HT and thermoregulation, we hypothesized that central 5-HT neurons may be vital to the regulation of brown and beige adipocyte activity. Given that BAT consumes large amounts of substrate when active, we also hypothesized that inactivation of BAT due to deletion of the regulatory neural circuitry (5-HT neurons) would cause metabolic dysregulation. To test this, we generated mice in which the human diphtheria toxin (DT) receptor was selectively expressed in central 5-HT neurons under control of a Pet-1 promoter. Pet-1 is a transcription factor selectively located in mature, central 5-HT neurons. Coincidentally, some cells within pancreatic islets also express Pet-1, and contain adequate machinery to produce, release, and uptake 5-HT. Systemic treatment with DT eliminated 5-HT neurons and caused loss of thermoregulation, BAT steatosis, and a >50% decrease in Ucp1 expression in BAT and beige fat, indicative of reduced thermal production. In parallel, blood glucose increased 3.5-fold, free fatty acids 13.4-fold and triglycerides 6.5-fold. Intracerebroventricular (ICV) treatment with 1/30th the systemic dose of DT induced an even greater thermoregulatory impairment. The metabolic deficits following systemic DT treatment indicate that central 5-HT neurons are essential for proper metabolic regulation. However, such high levels of glucose and lipids also indicate failure of the pancreatic endocrine program following systemic treatment, likely due to moderate destruction of β-cells expressing Pet-1 and the DT receptor. Because ICV treatment caused even greater thermoregulatory and metabolic deficits, where little, if any, of the toxin would spread systemically, central 5-HT neurons are clearly essential for normal central regulation of metabolism. Interestingly, similar BAT and beige fat defects occurred in Lmx1bf/f/p mice, in which 5-HT neurons fail to develop in utero. Assessment of systemically treated animals using a euglycemic/hyperinsulinemic clamp showed extensive fasting hyperglycemia and systemic insulin resistance, coinciding with reduced glucose uptake in skeletal muscle and BAT. The hyperinsulinemic clamp failed to suppress hepatic glucose and fatty acid production, leading to the conclusion that loss of central 5-HT neurons disrupts central hepatic regulation. In attempts to induce BAT thermogenesis and metabolism, we optogenetically stimulated 5-HT neurons in the rostral raphe pallidus and measured BAT and body temperature along with blood glucose. Unfortunately, these stimulations were incapable of increasing BAT temperature and lowering blood glucose, perhaps limiting therapeutic potential of these 5-HT neurons. We conclude that 5-HT neurons are major players in central regulation of metabolic homeostasis, in part through recruitment and activation of brown and beige adipocytes and hepatic substrate production. Data also suggest that 5-HT neurons regulate glucose homeostasis via undefined neural mechanisms independently of BAT activity and pancreatic insulin secretion. Cumulative data on central 5-HT neurons indicate they are master regulators of whole-body metabolism.

Type 2 diabetes is caused by defect pancreatic islet β-cells together with peripheral insulin resistance. The disease is often accompanied by obesity with associated low-grade visceral adipose tissue inflammation, which contributes to insulin resistance. As a consequence of, and a possible compensation for the increased insulin demand, blood flow to the pancreatic islets is increased in animal models of diabetes. This increased blood perfusion might with time affect the vascular network as well as β-cells within the islets. This thesis investigates the role of changes of blood perfusion in pancreatic islets and adipose tissues, as well as the recruitment to and composition of leukocyte subpopulations in insulin-sensitive tissues in experimental models of diabetes. Blood flow measurements in islets and adipose tissues of rats and mice were performed using the microsphere technique, while leukocyte recruitment was studied in the mouse cremaster muscle using intravital microscopy. Increased islet blood flow was observed in the GK rat model of type 2 diabetes, which was decreased by acute as well as continuous 2-week inhibition of β3-adrenoceptors without affecting plasma insulin concentrations. Increased inflammatory leukocyte recruitment was observed in both alloxan-induced and high-fat diet-induced diabetes. However, an impaired bacterial clearance was observed in diabetic mice, which was due to impaired phagocytosis. A gender difference was detected in mice fed a high-fat diet, since obese female mice did not show increased levels of pro-inflammatory circulatory markers or inflammatory leukocytes in the adipose tissue. The main effector cell in the adipose tissue inflammation in high-fat-fed male mice seemed to be the pro-inflammatory macrophage. The Treg population in adipose tissue was increased in female mice, but remained unchanged in male mice on high-fat diet. In conclusion, increased islet blood flow in type 2 diabetes could be reversed by β3-adrenoceptor inhibition, which may maintain islet function. The diabetes-associated hyperglycemia activated leukocytes but impaired their phagocytic ability. High-fat-fed female mice showed less peripheral inflammation due to a smaller number of recruited inflammatory macrophages and a high-fat diet-induced Treg population in intra-abdominal adipose tissues.

Background Excess adipose tissue and low physical activity are two major determinants for chronic diseases such as type 2 diabetes and cardiovascular disease.  Understanding these relationships requires accurate and precise measures of body composition and physical activity, and most existing observational studies lack such measures. Paper I to III in this thesis addresses the validity of measures of physical activity and abdominal adipose mass. In paper IV and V, we explore the relationships between obesity and physical activity on metabolic health in non-pregnant and pregnant women and their offspring. Methods and Results Two hundred men and women representative of the Northern Sweden EPIC cohort were recruited for Paper I. A questionnaire on physical activity (PAQ) was validated against objectively measured physical activity energy expenditure (PAEE). A categorical physical activity index (Cambridge index) calculated from PAQ showed strongest correlation with PAEE (r=0.33 p<0.05). In Paper II, abdominal adiposity were assessed in 29 adult men and women using anthropometric measurements, dual energy x-ray absorptiometry (DXA) and ultrasound and were compared to computed tomography (CT). Waist circumference showed the highest correlation with CT-assessed visceral (r=0.85, p<0.0001) and subcutaneous adipose tissue (r=0.86, p<0.0001). Adipose thickness was best assessed with ultrasound. In Paper III, the validity of a wrist-worn triaxial accelerometer was assessed in 32 pregnant and 74 non-pregnant women using double-labeled water method (DLW) as the criterion measure. The output from the accelerometer explained 24% (p <0.001) of the variation in PAEE in non-pregnant and 11% (p<0.05) in the pregnant women. In Paper IV, 35 pregnant and 73 non-pregnant women underwent a 75g oral glucose tolerance test and habitual energy expenditure and physical activity was assessed objectively. Total physical activity was inversely associated with early insulin response in both pregnant (r=-0.47, p=0.007) and non-pregnant (r=-0.36, p=0.004) women. In, Paper V, 32 women and their offspring (n=33) were studied 4 months post-partum. Body composition was quantified using DXA in the women and air-displacement plethysmography  in the infants. Mid-pregnancy weight gain was significantly associated with infant fat mass (r=0.41, p=0.022), whereas late-pregnancy weight gain associated to infant fat-free mass (r=0.37, p=0.04). Conclusion This work describes new methods as well as conventional anthropometric estimates and a questionnaire, that provide relatively strong estimates of body composition and physical activity which could be used in larger studies. Pregnant women were shown to have more sedentary behavior than non-pregnant but physical activity appeared to have equal effect on glucose homeostasis in both groups, which may help guide lifestyle interventions in pregnancy. The impact of weight gain during the different trimesters seems to differentially affect the offspring’s body composition in early infancy, which might give us clues to when different aspects of fetal development and growth occur and how modifiable lifestyle behaviors might be intervened upon to improve long-term health. / Embargo

Lipoprotein lipase (LPL) is an enzyme to hydrolyze triglycerides in lipoproteins and thereby make the fatty acids available for cellular metabolic reactions. Short-term fasting down-regulates LPL activity in adipose tissue. This regulation is through post-translational mechanism. The objective of this work was to investigate (1) The molecular mechansim for regulation of LPL activity in adipose tissue; (2) The basis for the tissue-specific regulation of LPL in adipose tissue, heart and skeletal muscle. LPL in adipose tissue can be found both inside (intracellular) and outside adipocytes (extracellular). Within adipocytes, neither LPL mass nor the distribution of LPL between active and inactive forms changed on fasting. Extracellular LPL mass also did not change significantly, but shifted from predominantly active to predominantly inactive. Activie, extracellular LPL was distributed in a similar way in the two nutritional states. The down-regulation during fasting is due to a decline of extracellular LPL activity. The up-regulation of LPL activity induced by re-feeding did not need new mRNA. The down-regulation of LPL activity induced by fasting did not occur when mRNA synthesis was inhibited. LPL activity in adipose tissue from fasted rats was fully restored by actinomycin. So fasting switches on a gene, whose product suppresses LPL activity. Similar results were also obtained in experiments on mice. When food was removed from young rats in the early morning, adipose tissue TNF-α activity increased and LPL activity decreased within six hours. There was a negative correlation between TNF-α and LPL activities. Pentoxifylline, that inhibits biosynthesis of TNF-α, almost abolished the rise of TNF-α and the decrease of LPL activity. Actinomycin D virtually abolished the response of LPL activity to fasting or exogenous TNF-α. This study suggests that fasting signals via TNF-α to a gene whose product causes a rapid shift of newly-synthesized LPL molecules towards an inactive form.

Brown adipose tissue (BAT) is a critical organ for non-shivering thermogenesis, which is under control of both sympathetic and sensory neural innervation. We utilized both a retrograde sympathetic nerve tract tracer pseudorabies virus and an anterograde sensory tract tracer the H129 strain of herpes simplex virus-1 to locate individual neurons across the neuroaxis that are part of the SNS outflow from brain to interscapular BAT and are part of the sensory input to the brain. We found specific neuronal phenotype of the double-infected neurons distributed from the hindbrain to the forebrain with highest densities in several discrete brain regions: the paraventricular hypothalamus (PVH), lateral hypothalamus (LHA), parabrachial nucleus (PB) and raphe pallidus (RPa). The neuroanatomical reality of the SNS-sensory feedback loops suggests coordinated control of BAT thermogenesis at several sites and indicates plasticity of SNS-sensory crosstalk.

Androgens (such as testosterone) have been shown to increase lean body mass and reduce fat body mass in men through activation of androgen receptors (AR). While this suggests a potential clinical use for androgens, attempts at utilization of this class of hormones as a therapeutic are limited by side effects due to indiscriminate AR activation in various tissues. Thus, a greater understanding of the tissues and cells involved in promoting these changes would be beneficial. Here we show that selective overexpression of AR in muscle cells of transgenic (HSA-AR) rodents both increases lean muscle mass and significantly reduces fat mass in males. Similar effects can be induced in HSA-AR females treated with testosterone. Metabolic analyses of HSA-AR males show that these animals demonstrate increased O2 consumption and hypermetabolism. Thus, targeted activation of AR in muscle regulates body composition and metabolism, suggesting a novel target for drug development.

Androgens (such as testosterone) have been shown to increase lean body mass and reduce fat body mass in men through activation of androgen receptors (AR). While this suggests a potential clinical use for androgens, attempts at utilization of this class of hormones as a therapeutic are limited by side effects due to indiscriminate AR activation in various tissues. Thus, a greater understanding of the tissues and cells involved in promoting these changes would be beneficial. Here we show that selective overexpression of AR in muscle cells of transgenic (HSA-AR) rodents both increases lean muscle mass and significantly reduces fat mass in males. Similar effects can be induced in HSA-AR females treated with testosterone. Metabolic analyses of HSA-AR males show that these animals demonstrate increased O2 consumption and hypermetabolism. Thus, targeted activation of AR in muscle regulates body composition and metabolism, suggesting a novel target for drug development.

Obesity is a metabolic disorder that results in elevated levels of free fatty acids and triglycerides in the blood circulation, which further leads to accumulation of lipids within various tissues. Like in other similar metabolic disorders, obesity is thought to be originated from structural and regulatory changes in macromolecular assemblies. This current study aims to investigate the effects of obesity on macromolecular alterations in order to characterize Berlin fat mouse inbred (BFMI) lines which arenew models for the obesity research that may contribute to understanding of spontaneous obesity without induction of a high fat diet. Attenuated Total Reflectance - Fourier Transform Infrared (ATR-FTIR) spectroscopy was used to characterize content and structure of macromolecules in male and female control (DBA/2J) and BFMI lines / namely BFMI856, BFMI860 and BFMI861, in two different adipose tissues / inguinal fat (IF) which is subcutaneous adipose tissue (SAT), gonadal fat (GF) which is visceral adipose tissue (VAT). Structural and compositional alterations in proteins, lipids, saturated and unsaturated lipid contents, nucleic acid, collagen and glycogen contents and variations in the lipid chain length were determined. BFMI860 and BFMI861 lines were found to be the most affected lines since they showed the indications of lipid peroxidation and insulin resistance more severely as they had lower glycogen content in all tissues and lower unsaturated lipid content especially in IF adipose tissues. Moreover, structural changes in lipids were observed especially in male GF adipose tissues of BFMI856 and BFMI861 lines. Protein content decreased significantly specifically in female IF adipose tissues but no change was observed in the structure. Furthermore, BFMI852 line was found to be affected different than other lines and had an effect on especially female IF. To conclude, obesity induced changes differ in BFMI lines according to the gender, adipose tissue type and distinctness in the strains.