Teses / dissertações sobre o tema "Obesity ; Type 2 diabetes ; Adipose Tissue"

In the past 30 years the prevalence of obesity has almost trebled resulting in an increased incidence of type 2 diabetes mellitus (T2DM) and other co-morbidities. Visceral adipose tissue is believed to play a vital role in these conditions, but underlying mechanisms remain unclear. A close association exists between obesity, diabetes and oxidative stress, resulting in increased reactive oxygen species formation. The experiments in this thesis address this by searching for possible biochemical changes which may be specific for the onset of obesity related T2DM, as well as looking for genetic alterations at molecular and gene expression levels. This thesis also explored various techniques such as polymerase chain reaction (PCR), colorimetric assays and real-time RT-PCR. The aim was to investigate the role of adipose tissue in obesity and T2DM, focusing on markers of oxidative stress and gene expression in human visceral adipose tissue from subjects categorised as lean, obese and obese with T2DM. This cross-sectional study measured two markers of oxidative stress, two markers of DNA damage, gene expression analysis and identification of genes associated with T2DM and obesity. Specific gene sequencing was carried out on the glutathione reductase gene to determine possible gene variants. Results showed a paradoxical decrease in adipose markers of oxidative stress in subjects with obesity and T2DM. There appeared to be a protective mechanism in these subjects, displaying reduced levels of oxidative stress compared to other groups. This could be due to a significant proportion of these subjects being on ACE inhibitor and statin therapy, which may be confounding results and minimising the effects of the oxidative burden. Additionally, the same subjects showed an increased expression of the glutathione reductase gene. It is difficult to conclude if the decreased levels of oxidative stress in these subjects were a result of the increased glutathione reductase expression in the visceral adipose tissue or if there remains an unseen factor influencing the dramatic expression change seen in this group of subjects. No glutathione reductase gene variants were identified in these samples. This analysis highlighted that within this sample set, the impact of oxidative stress is in fact reversible as the antioxidant capacity in these subjects is evident, and in combination with correct drug therapy it may be possible to combat oxidative burden and reduce the subsequent damage inflicted upon the cells.

This thesis sought to investigate how systemic lipids may contribute to the adipocyte derived inflammatory response and highlight how the adipocyte’s function can alter in different metabolic states which could contribute to the pathogenesis of type 2 diabetes mellitus (T2DM) and cardiovascular risk. Specifically, this thesis firstly examined the inflammatory nature of lipoprotein-associated phospholipase A2 (LpPLA2), a member of the phospholipase A2 super family of enzymes which previously has been shown to enhance Ox-LDL production in foam cells during arterial inflammation contributing to coronary artery disease. Therefore initial studies sought to (1) characterise PLA2 isoforms in lean, obese, T2DM abdominal subcutaneous (AbdSc) and omental (Om) in human adipose tissue (AT); (2) evaluate the role of lipids and inflammatory markers on circulating LpPLA2, and (3) determine the in vitro regulation of LpPLA2 in human adipocytes by its influence on LDL and Ox-LDL. AT and sera from lean, overweight, obese and T2DM subjects were taken. PLA2 gene expression was determined by microarray, RT-PCR and Western Blot. Associations between circulating LpPLA2 and metabolic parameters were investigated. The human adipocyte cell line, Chub-S7, was used to assess the effects of oxidized LDL (Ox-LDL) and LDL on PLA2 expression. LpPLA2 mRNA levels were higher in AbdSc AT than Om AT in obesity by 2-fold (P<0.05). The cPLA2 protein expression increased with obesity in AbdSc AT (P<0.01). T2DM showed increased LpPLA2 mRNA levels in AbdSc (P<0.001) and Om AT (P<0.01). Serum LpPLA2 showed positive correlations with cholesterol, TG, LDL, endotoxin and Ox-LDL (P<0.001) in non-diabetic subjects and with Ox-LDL (P<0.001), LDL (P<0.01) and cholesterol (P<0.05) in T2DM. In differentiated pre-adipocytes, activation of LpPLA2 protein expression was noted in response to LDL and Ox-LDL (P<0.001). The adipocyte appeared to be an active source of LpPLA2, altered by fat depot and metabolic state, with LpPLA2 protein expression induced by LDL and Ox-LDL, in vitro. Increased LpPLA2 protein from the adipocyte in obesity and/or T2DM could contribute to raise circulating Ox-LDL, as noted in other studies as well with increasing adiposity, which promotes further inflammation and atherosclerotic risk. Through the development of these current studies it appeared that how the adipocyte managed lipids was important to how the adipocyte may induce an inflammatory response and pathogenic factors. Therefore subsequent studies investigated how the change in metabolic state such as those derived in T2DM patients that undergo bariatric surgery may not necessarily reverse their inflammatory response. Previous studies from the team have shown that lipids may induce an innate immune response via toll like receptor (TLR) activation therefore subsequent investigations sought to consider the potential role of triglycerides (TG) as another mediator of inflammation. As such studies examined the specific impact of TG changes, pre- and post-bariatric surgery, on TLR expression in ex vivo AT and the in vitro effects of triglyceride rich lipoprotein (VLDL), on TLR expression in isolated human differentiated pre-adipocytes. Serum and AT was taken from a cohort of Obese, T2DM, female subjects prior to bariatric surgery and 6 months post-surgery. Human differentiated pre-adipocyte Chub S7 cells were again used to examine transcriptional effects of VLDL on TLR expression. Following surgical intervention, BMI (P<0.001), blood glucose (P<0.001), insulin (P<0.001), HOMA-IR (P<0.001), TG (P<0.05), Cholesterol (P<0.001) and LDL-cholesterol (P<0.05) were significantly improved. There was a significant reduction in TLR-4 mRNA post-surgery (P<0.01) irrespective of surgery type. It was also noted that subjects with the greatest drop (55.5% reduction) in TGs post-surgery (P<0.001) showed a significant correlated reduction in TLR4 mRNA expression (P<0.001). Whilst the in vitro treatment of differentiated Chub S7 cells highlighted VLDL induced TLR 4 mRNA expression (P<0.05) suggesting the inflammatory impact of lipids on adipocytes. These studies further highlighted that the reduction in AT inflammation appears dependent on how successfully subjects reduce their serum triglyceride, which appears supported by the in vitro findings. These studies suggest that bariatric surgery lead to metabolic improvement with weight loss, whilst dietary intervention is still required to ensure TGs reduce to reduce inflammation. Taken together, these studies and thesis highlight the diverse nature of lipids and their interaction with the adipocyte to impact on their inflammatory response. These data also highlight the importance to maintain a good systemic lipid profile low in TG to reduce adipocyte induced inflammation and that AT may represent an important therapeutic target to reduce inflammation, atherosclerotic risk and development of metabolic complications.

The adipose tissue lipid chaperones aP2 and mal1, also known as fatty acid binding proteins (FABPs), are significant molecules contributing to metabolic homeostasis, whereby their absence promotes physiological changes that improve systemic metabolism. Identification of palmitoleate as a lipokine generated in aP2-mal1 deficiency--originating from adipose and directing the lipogenic program in liver, established a role for these chaperones in linking adipocyte and hepatic function. We have recently demonstrated a functional role for secreted aP2 in the activation of gluconeogenesis and hepatic glucose output, further designating this molecule as an adipocyte-derived regulatory factor that influences liver metabolism. Key molecules linking the metabolism of nutrients in energy generating pathways are the nucleotide cofactors NAD and NADH. Together, these molecules function to coordinate the maintenance of redox reactions during normal cellular metabolism and act as required substrates for enzymes such as sirtuins and poly ADP-ribose polymerases. Using global metabolite profiling, we show that combined deficiency of the adipose tissue lipid chaperones aP2 and mal1 leads to a hepatic nucleotide imbalance resulting from metabolic reprogramming in liver. We demonstrate that this reprogramming of metabolite flux is accompanied by significant alterations in liver NAD metabolism and establish a role for aP2 in directing substrate utilization through inhibition of the rate-limiting enzyme for NAD synthesis, nicotinamide phosphoribosyltransferase. Several models for the proposed regulatory pathways that link nutrient metabolism to aging include mechanisms that are NAD dependent. Accordingly, we found that long-term FABP deficiency confers a strong resistance to aging related metabolic deterioration. Together, the findings presented in this thesis support a considerable role for FABPs in the regulation of NAD metabolism and healthspan.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by consistently high blood glucose, resulting from a combination of insulin resistance and reduced capacity of β-cells to secret insulin. While the exact causes of T2DM is yet unknown, obesity is known to be a major risk factor as well as co-morbidity for T2DM. As the global prevalence of obesity continues to increase, the association between obesity and T2DM warrants further study. Traditionally, mathematical models to study T2DM were mostly empirical and thus fail to capture the dynamic relationship between glucose and insulin. More recently, mechanism-based population models to describe glucose-insulin homeostasis with a physiological basis were proposed and offered a substantial improvement over existing empirical models in terms of predictive ability. The primary objectives of this thesis are (i) examining the predictive usefulness of semi-mechanistic models in T2DM by applying an existing population model to clinical data, and (ii) exploring the relationship between obesity and T2DM and describe it mathematically in a novel semi-mechanistic model to explain changes to the glucose-insulin homeostasis and disease progression of T2DM. Through the use of non-linear mixed effects modelling, the primary mechanism of action of an antidiabetic drug has been correctly identified using the integrated glucose-insulin model, reinforcing the predictive potential of semi-mechanistic models in T2DM. A novel semi-mechanistic model has been developed that incorporated a relationship between weight change and insulin sensitivity to describe glucose, insulin and glycated hemoglobin simultaneously in a clinical setting. This model was also successfully adapted in a pre-clinical setting and was able to describe the pathogenesis of T2DM in rats, transitioning from healthy to severely diabetic. This work has shown that a previously unutilized biomarker was found to be significant in affecting glucose homeostasis and disease progression in T2DM, and that pharmacometric models accounting for the effects of obesity in T2DM would offer a more complete physiological understanding of the disease.

Aims and hypothesis: Beyond the insulinotropic effects, glucose-dependent insulinotropic polypeptide (GIP) may regulate post-prandial lipid metabolism by promoting fat deposition and inflammation in adipose tissue after high fat diets. We hypothesised that GIP would have an anabolic action in subcutaneous adipose tissue (SAT) promoting non-esterified fatty acid (NEFA) re-esterification. We speculated these effects may be mediated by changes to the expression of key lipid metabolism enzymes and that GIP may promote inflammation by affecting the expression of key adipokines in SAT. We postulated that these effects may be different according to obesity status or glucose tolerance. Incretins and other gut hormones are affected by medications used in the treatment of T2DM. We hypothesised that metformin, a commonly used drug in T2DM, may influence the secretion of incretin and other gut hormones which may contribute to its pleotropic effects in glucose metabolism. Subjects/Methods: We recruited 31 participants, for 2 different studies. In the first study, 23 men in four categories, normoglycaemic lean (n=6), normoglycaemic obese, (n=6), obese with impaired glucose regulation (IGR) (n=6) and obese, T2DM (n=5) participated in a double-blind, randomised, crossover study involving a hyperglycaemic clamp with a 4-hour infusion of GIP or placebo (normal saline). Serum insulin, plasma NEFA concentrations, SAT triacylglycerol (TAG) content and gene expression of key lipid metabolism enzymes, lipoprotein lipase (LPL), adipose tissue triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) and adipokine gene expression (TNF-α, MCP-1, osteopontin and adiponectin) in SAT were determined before and after the GIP/placebo infusions. In the second study, eight subjects (6 male and 2 female) were studied on two occasions for 6 hours following a standard mixed meal, before and after metformin monotherapy for at least 3 months. Blood samples were taken in the fasted state and at multiple time points after the mixed meal for measuring incretin hormone, glucagon like peptide (GLP-1), ghrelin (appetite regulatory gut hormone) and dipeptidyl peptidase –IV (DPP-IV) activity. Results: Study-1 The insulinotropic effect of GIP vs. placebo was greater in lean, obese and obese IGR groups with no significant effect in obese T2DM. In contrast, GIP lowered NEFA concentrations in obese T2DM concomitantly increasing the SAT-TAG content. Such effects were not observed in other groups. There was no change in gene expression of LPL, ATGL and HSL with GIP or placebo infusions. The gene expression of TNF-α was significantly higher in obese T2DM group and the expression of MCP-1 was higher in lean and obese subjects. Study-2 Metformin monotherapy in obese patients with T2DM was associated with significantly increased postprandial active GLP-1 concentrations. Conclusion: In T2DM, although the insulinotropic effect of GIP is impaired, the ability of GIP to promote fat storage seems intact lowering NEFA concentrations and increasing SAT lipid deposition which may further exacerbate obesity and insulin resistance. Oral hypoglycaemic agent metformin influences the incretin system by increasing GLP-1 concentrations and this may represent another important mechanism of its glucose-lowering effect.

Le tissu adipeux blanc n'est pas un simple réservoir énergétique, il secrète également de nombreuses molécules appelées adipokines. En condition basale, les adipokines participent à l'homéostasie générale en permettant la régulation de diverses fonctions et voies métaboliques. Au cours du développement de l'obésité, la physiologie du tissu adipeux est fortement perturbée. Cela se traduit par des dysfonctionnements parmi lesquels l'établissement d'un état inflammatoire chronique à bas bruit. Ces perturbations au sein du tissu adipeux concourent à la dysrégulation de l'expression des adipokines, conduisant à un dysfonctionnement de certaines voies métaboliques, au niveau adipocytaire mais aussi au niveau systémique ce qui aboutit à un état d'insulino-résistance pouvant déboucher sur le développement d'un diabète de type 2. De nombreuses études épidémiologiques montrent qu'une carence en vitamine D est associée à diverses pathologies telles que certains cancers, certaines maladies auto-immunes ou maladies cardiovasculaires. Par ailleurs, il existe une corrélation inverse entre les taux plasmatiques de 25(OH)D et la prévalence de l'obésité, de l'hypertension artérielle, et du diabète de type 2. Cependant, le lien de causalité entre carence en vitamine D et obésité n'est à ce jour pas clairement démontré. Le but de cette thèse est donc de mieux comprendre le lien qui existe entre carence en vitamine D, obésité et désordres physiologiques associés
White adipose tissue is not a simple energy reservoir, it also secretes several molecules called adipokines. In standard conditions, adipokines are involved in general homeostasis permitting the regulation of numerous functions and metabolic pathways. During the development of obesity, adipose tissue physiology is severely disrupted. This results in dysfunction such as low-grade inflammatory status. The accumulation of these disturbances within adipose tissue generates a dysregulation of adipokines secretion. This will have for consequences a failure of some metabolic pathways resulting in insulin-resistant state leading to type 2 diabetes. Several epidemiological studies show a link between vitamin D deficiency and numerous pathologies like cancers, immunity deseases or cardiovascular deseases. In addition, there is an inverse correlation between plasma levels of 25(OH)D and the prevalence of obesity, hypertension and type 2 diabetes. However, the link between vitamin D deficiency and obesity is not well established. The aim of this thesis is to better understand the link between vitamin D deficiency, obesity and physiological disorders associated. For this purpose, we have evaluated vitamin D effects on adipose tissue and adipocyte biology (inflammation, glucose uptake) and subsequently effects of vitamin D supplementation on diet-induced obesity. In a first study in vitro, we have showed an anti-inflammatory effect of vitamin D on adipocyte. This effect appears to be VDR-dependant and implies a modulation of NFκB signaling pathway. This study could partly explain le link between vitamine D deficiency and low-grade inflammation associated with obesity

Résumé: Comparées aux classiques insulines humaines régulières (IHR), les insulines analogues ultrarapides (IAUR) ont été conçues pour mieux synchroniser le pic insulinémique avec l’absorption du repas. Le progrès a été démontré chez les patients diabétiques de type 1, mais le contrôle glycémique s’est peu ou pas amélioré chez les patients diabétiques de type 2 (DT2), qu’ils soient sous IAUR ou IHR. Or ces patients constituent 75 % des utilisateurs d’insuline. L’utilité des IAUR est donc toujours débattue. La dose (donc le volume) injectée et le flot sanguin dans le tissu adipeux sous-cutané (FSTA) sont les facteurs majeurs de l’absorption de l’insuline. Les patients DT2, résistants à l’insuline, s’injectent des doses importantes et leur FSTA est de 50 à 70 % plus faible que celui des sujets sains de poids normal (PN). Nous avons montré que l’absorption sous-cutanée des IAUR est diminuée chez les sujets obèses et DT2 (ODT2) par rapport aux sujets PN, que le volume injecté avait un effet délétère additionnel et que le FSTA peut être augmenté de façon pharmacologique avec un agent vasoactif (AV) chez des sujets résistants à l’insuline. Nous suggérons que l’ajout d’un AV à une IAUR va augmenter le FSTA au site d’injection et donc améliorer sa pharmacocinétique (PK) et sa pharmacodynamie (PD). Pour vérifier cette hypothèse, nous avons 1) évalué la réponse du FSTA à 4 AV chez des sujets PN, obèses non-diabétiques et ODT2; 2) évalué la PK/PD et la biodisponibilité de l’IAUR lispro ± AV chez des sujets ODT2; et 3) caractérisé l’expression des cibles des AV dans le tissu adipeux sous-cutané chez les sujets énumérés en 1). Les 4 AV ont augmenté le FSTA des sujets ODT2, mais moins que celui des autres sujets. L’occurrence de la raréfaction et/ou dysfonction microvasculaire chez les sujets ODT2 pourrait expliquer l’hyporéactivité vasculaire aux AV testés. Le plus actif des AV chez les sujets ODT2 a été ajouté à l’IAUR lispro pour améliorer sont absorption sc. Les PK/PD ont été améliorées seulement chez les sujets ODT2 avec une hémoglobine glycosylée A1c ≥ 8 %; c’est-à-dire 4 sujets sur 8. Chez ces derniers, l’absorption de 30 U + AV a été plus rapide de 14 et 71 min à 20 et 80 % de l’aire sous la courbe totale de la lispro plasmatique, respectivement. Chez les 4 autres sujets ODT2, l’absorption de la lispro semble s’être détériorée avec l’AV. Une interaction chimique a peut-être eu lieu entre l’AV et la lispro, ce qui aurait perturbé son absorption. Selon nos résultats, le niveau de contrôle du diabète, le volume d’injection et les caractéristiques chimiques de l’AV seraient des modulateurs de l’efficacité du concept IAUR + AV. Il nous faut maintenant déterminer l’impact de ces facteurs sur la capacité d’un AV à améliorer l’absorption sc de l’IAUR chez les sujets ODT2.
Abstract: Compared to classic regular human insulin (RHI), short-acting insulin analogues were designed to better synchronize plasma insulin increase to food absorption. Although improvements were noted in subjects with type 1 diabetes, slight to no improvement in glycemic control were observed in subjects with type 2 diabetes (T2D) using SAIA instead of RHI. Nevertheless, they represent 75 % of all insulin users. Consequently, the relative useful-ness of SAIA in T2D patients is currently hotly debated. Injected volume and subcutaneous (sc) adipose tissue blood flow (ATBF) are two main factors involved in insulin absorption. In fact, T2D patients use large doses of insulin because of their resistance to insulin and have an ATBF 50 to 70 % lower than lean healthy subjects. We already showed that SAIA absorption is decreased in obese T2D (OT2D) subjects compared to normal weight healthy subjects and that volume has additional detrimental effects. We also showed that ATBF can be increased pharmacologically with vasoactive agents (VA) in healthy and insulin-resistant subjects. Then we suggest that in OT2D subjects, addition of VA to SAIA preparations will locally increase ATBF, improve insulin sc absorption (Pharmacokinetic - PK) and bioavailability, thus insulin hypoglycemic effect (Pharmacodynamic - PD). To test this hypothesis, we 1) assessed ATBF response of 4 selected VA within three experimental groups (normal weight, obese non-diabetic and OT2D subjects); 2) evaluated insulin PK/PD and bioavailability improvement in OT2D subjects after the addition of the best VA to SAIA lispro and 3) characterized expression of selected VA targets in sc adipose tissue biopsies, within equivalent experimental groups, and compared results with ATBF responses. All 4 VA were able to increase ATBF of OT2D subjects but in a less extend than other subjects. The occurrence of microvascular rarefaction and/or dysfunction in OT2D subjects can explain the hyporeactivity to tested VA. Nevertheless, one VA among others was shown more effective to increase ATBF in OT2D subjects and was then tested (mixed) with SAIA lispro. With the AV, PK/PD were improved only in OT2D subjects with A1c glycated hemoglobin ≥ 8 %; 4 subjects on 8. The sc absorption of 30 U + VA was faster by 14 and 71 min for respectively 20 and 80 % of the total area under the lispro plasmatic curve. But the sc absorption with VA appeared blunted with the other subjects. Maybe detrimental chemical interactions occurred between the VA and lispro, which could impede absorption. Our results suggest that diabetes control state, injection volume, and VA chemical characteristics influence the efficacy of our SAIA + VA concept. Further tests are needed to seize the impact of these factors on VA effectiveness in sc absorption improvement of SAIA in OT2D subjects.

Chemerin is a chemoattractant adipokine that regulates adipogenesis and may induce insulin resistance. Chemerin serum concentrations are elevated in obese, insulin-resistant, and inflammatory states in vivo. Here we investigate the role of omental (OM) and subcutaneous (SC) adipose tissue chemerin and CMKLR1 messenger RNA (mRNA) expression in human obesity. In addition, we test the hypothesis that changes in chemerin serum concentrations are primarily associated with reduced body fat mass in the context of 3 weight loss intervention studies. Chemerin serum concentration was measured in 740 individuals in a cross-sectional (n = 629) study including a subgroup (n = 161) for which OM and SC chemerin mRNA expression has been analyzed as well as in 3 interventions including 12 weeks of exercise (n = 60), 6 months of calorie-restricted diet (n = 19) studies, and 12 months after bariatric surgery (n = 32). Chemerin mRNA is significantly higher expressed in adipose tissue of patients with type 2 diabetes mellitus and correlates with circulating chemerin, body mass index (BMI), percentage body fat, C-reactive protein, homeostasis model assessment of insulin resistance, and glucose infusion rate in euglycemic-hyperinsulinemic clamps. CMKLR1 mRNA expression was not significantly different between the 2 fat depots. Obesity surgery–induced weight loss causes a significant reduction on both OM and SC chemerin expression. All interventions led to significantly reduced chemerin serum concentrations. Decreased chemerin serum concentrations significantly correlate with improved glucose infusion rate and reduced C-reactive protein levels independently of changes in BMI. Insulin resistance and inflammation are BMI-independent predictors of elevated chemerin serum concentrations. Reduced chemerin expression and serum concentration may contribute to improved insulin sensitivity and subclinical inflammation beyond significant weight loss.

O tecido adiposo marrom (TAM), caracterizado pela presença da proteína termogênica UCP-1, é conhecido há muitas décadas como um tecido termogênico em mamíferos, porém sua significância clínica em humanos era considerada pequena, com exceção de neonatos, até que o desenvolvimento e uso de métodos de PET-FDG terem demonstrado que humanos adultos também possuem TAM ativo, especialmente após exposição ao frio. Essa descoberta levou a um enorme aumento nas pesquisas sobre o assunto, já que sua ativação, levando a um aumento do gasto energético, poderia, pelo menos na teoria, ser uma possível arma no tratamento da obesidade e diabetes tipo 2 e sua redução ou ausência ser uma causa de ganho de peso. Muitos compostos vêm sendo estudados como possíveis recrutadores e ativdadores desse tecido. A melatonina é um deles, embora nenhum estudo tenha sido feito em humanos. A melatonina, um hormônio pineal sintetizado à noite com um papel crítico na sincronização do ritmo circadiano, é estudado há várias décadas como um regulador chave do metabolismo energético em diversas espécies animais. Ratos pinealectomizados ganham peso e tem distúrbios metabólicos durante sua vida, e a suplementação noturna de melatonina, reverte estas alterações, sem redução da ingesta alimentar. Devido a isso, uma hipótese é que o papel central da melatonina no metabolismo energético inclui sua função no gasto energético, possivelmente relacionado à ativação do TAM. Muitos modelos experimentais, a maioria em animais hibernantes, demonstraram o papel da melatonina no recrutamento do TAM. Nesse estudo, o objetivo é determinar se a suplementação de melatonina para indivíduos e animais de experimentação (ratos Wistar) deficientes de melatonina aumenta sua ativação. Foi encontrado que, em ratos Wistar, animais pinelaectomizados possuem uma capacidade termogênica do TAM reduzida após exposição ao frio comparado com a temperatura ambiente, e a suplementação de melatonina normaliza essa capacidade termogênica. Esse dado sugere um papel da melatonina na resposta máxima de ativação do TAM após um desafio ao frio agudo. Também foi observado um aumento de expressão de UCP-1 (RNA) em animais repostos com melatonina, tanto em controles como em pinealectomizados, e animais pinealectomizados não repostos apresentam uma expressão de UCP-1 menor que um grupo controle. Em humanos, a suplementação de melatonina aumenta o volume e atividade do TAM em quatro indivíduos pinealectomizados (por tumores pineais) com baixo nível de melatonina no basal, analisado por tomografia de emissão de prótons acoplada a ressonância magnética (PET-RM). Embora a análise do TAM em ambos os protocolos tenha sido distinta, seus resultados apontam para a mesma regulação positiva do TAM pela melatonina. A termografia infravermelha (TIV) foi também realizada em humanos, com aumento de atividade de TAM após exposição ao frio, poréma correlação entre as respostas com a TIV e o PET-RM foi moderada e não significativa. Diferenças entre o protocolo frio e limitação da TIV em indivíduos mais obesos podem ter contribuído para esses resultados. Uma relação positiva da suplementação de melatonina nos lípides (principalmente colesterol e triglicérides) também foi encontrada, porém sem impacto na gordura hepática
Brown adipose tissue (BAT), characterized by the presence of the thermogenic protein UCP-1 have long been known as a thermogenic tissue in mammals, however its significance in humans was considered minor, with the exception of newborns, until FDG-PET exams demonstrated that human adults still have active BAT, especially after cold exposure. This prompted to an incredible increase in research on the field, since its activation, leading to increased energy expenditure could, at least theoretically, be a possible tool for the treatment of obesity and type 2 diabetes and its reduction or absence be a cause of weight gain. Many compounds aiming to recruit and activate BAT have been studied. Melatonin has been one of them, although no study has been performed in humans. Melatonin, a pineal hormone synthetized at night with a critical role in the synchronization of circadian rhythms, has long been studied as a key regulator of energy metabolism in many animal species. Pinealectomized rats gain weight and have metabolic disturbances during life, and the circadian supplementation of melatonin, at night, reverts these alterations, without decrease in energy intake. Due to that, it is hypothesized that a main role of melatonin in energy metabolism includes its action on energy expenditure, possibly related to activation of BAT. Many experimental models, mainly in hibernating animals, have shown a role of melatonin on BAT recruitment. In the present study, we ought to determine if the supplementation of melatonin for melatonin deficient subjects and experimental animals (Wistar rats) increases BAT activation. We found, in Wistar rats, that pinealectomized animals have a reduced BAT thermogenic capacity after acute cold exposure compared with ambient temperature, and melatonin supplementation in this animals leads to normalization of BAT thermogenic capacity. This data suggests a role of melatonin in improving the maximal response of BAT after an acute challenge. We also found that melatonin supplementation increases UCP-1 RNA expression both in control and pinealectomized rats, and pinealectomized rats without supplementation have a reduced UCP-1 expression compared with controls. In humans, we found that melatonin supplementation increased BAT volume and activity in four pinealectomized (due to pineal tumors) individuals with low melatonin at baseline, analyzed by Positron Emission Tomography associated with magnetic resonance (PET-MR). Although the analysis of BAT in both studies was different, their results point to the same positive regulation of BAT by melatonin. We also performed infrared termography (IRT) in humans, but the results were not conclusive since although we also found an increase in BAT activity measured in Watts, the correlation between the methods was moderate. The difference may be due to different protocols of cold exposure between methods, probably inadequate in IRT, as well as maybe to a limitation of IRT in more obese individuals. We also found that melatonin supplementation in melatonin deficient humans may have a positive impact on blood lipid concentrations, (mainly total cholesterol and triglycerides) but, at least for the time studied, does not appear to have an impact on liver fat

Overweight and obesity are associated with Type 2 Diabetes, non-alcoholic fatty liver disease, cardiovascular disease and cancer, but all fat is not equal as storing excess lipid in subcutaneous white adipose tissue (SWAT) is more metabolically favorable than in visceral fat. Here, we uncover a critical role for mTORC2 in setting SWAT lipid handling capacity. We find that subcutaneous white preadipocytes differentiating without the essential mTORC2 subunit Rictorexpress mature adipocyte markers but develop a striking lipid storage defect. In vivo,this results in smaller adipocytes, reduced tissue size, lipid re-distribution to visceral and brown fat, and sex-distinct effects on systemic metabolic fitness. Mechanistically, mTORC2 promotes transcriptional upregulation of select lipid metabolism genes controlled by PPARgand ChREBP. These include genes that control lipid uptake, synthesis, and degradation pathways as well as Akt2, the gene encoding its substrate and insulin effector. Finally, we reveal a potential novel mTORC2 target, ACSS2, which might control intracellular acetyl-CoA availability and regulate metabolic gene expression by altering histone modification in white adipocytes. Exploring this pathway may uncover strategies to promote safe lipid storage and improve insulin sensitivity.

L’obésité est une pathologie multifactorielle qui contribue au développement de l’insulinorésistance (l’IR). De nos jours, même si de nombreux travaux ont permis de répondre à plusieurs questions relatives à la pathogenèse de l’IR, les mécanismes cellulaires et moléculaires qui sous-tendent la pathogenèse de l’IR ne sont pas encore complètement élucidés chez l’Homme. Deux hypothèses principales se sont dégagées de ces travaux.D’une part, certaines études suggèrent que le défaut primaire à l’origine de l’IR se trouverait au niveau du tissu adipeux, qui en libérant dans la circulation systémique des adipokines, des cytokines inflammatoires et des acides gras contribue au développement d’une inflammation et d’une hyperlipidémie systémique. Ces deux paramètres exercent des effets délétères sur la sensibilité à l’insuline des autres organes comme le muscle et le foie. D’autre part, le muscle étant l’organe gluco-régulateur par excellence en situation postprandiale, d’autres études le place au cœur de la physiopathologie de l’IR.Malgré ces controverses, aucune étude n’a à ce jour exploré simultanément l’inflammation et l’IR systémique /tissulaire dans le muscle et le tissu adipeux chez un même sujet. Dans ce contexte, l’objectif de ce travail de thèse était de mieux comprendre les mécanismes impliqués dans la mise en place de l’IR. Il s’inscrit dans le cadre d’un projet de recherche translationnel, qui compare dans une cohorte de femmes ménopausées, des sujets minces à des sujets obèses de grade I insulinosensibles (OIS) et insulinorésistants (OIR). Plusieurs paramètres systémiques et tissulaires (lipotoxicité, inflammation et activation du récepteur toll like receptor 4 (TLR4)) impliqués dans la physiopathologie de l’IR ont été analysés chez ces sujets.Les résultats de cette étude mettent en avant l’importance de l’activation des voies de l’immunité innée dans la régulation de la sensibilité à l’insuline. Ainsi, alors qu’aucune inflammation systémique n’est détectée, on observe une activation différentielle des voies de signalisation du récepteur TLR4 de l’immunité innée entre le tissu musculaire et le tissu adipeux des sujets OIR. La voie MyD88-dépendante qui est une voie pro-inflammatoire, est activée dans le muscle squelettique de ces sujets et est associée à une IR au sein de ce tissu. A l’inverse, la voie TRIF-dépendante qui est une voie anti-inflammatoire est activée au sein du tissu adipeux et permet le maintien de la sensibilité à l’insuline. Ce maintien de la réponse à l’insuline se fait grâce l’induction du système interféron et de l’enzyme anti-oxydante manganèse superoxyde dismutase (MnSOD). Dans ce travail, nous montrons d’une part, que le défaut de réponse à l’insuline musculaire se trouve au cœur de la physiopathologie de l’IR chez les sujets obèses de grade I. D’autre part, ces résultats mettent en exergue l’importance de la balance de régulation des voies de l’immunité innée « MyD88 » et « TRIF » dans le maintien de la sensibilité à l’insuline
Obesity is a multifactorial disease promoting the development of insulin resistance (IR). Nowadays, cellular and molecular mechanisms underlying IR pathogenesis in humans are not fully understood although many studies have been attempted to improve our knowledge. Tow hypotheses arose from these researches.On one hand, several studies have led to the idea that a chronic inflammatory state combined with adipose tissue (AT) dysfunction could be the so-called “central mechanism” leading to IR. AT dysfunction is associated with increase in the release of inflammatory adipokines/cytokines and free fatty acid (FFA), leading to systemic inflammation and lipid overload. These latter parameters would have deleterious effects on diverse organs such as muscles and liver by affecting their insulin sensitivityOn the other hand, skeletal muscle (SM) is responsible for 80% of glucose uptake and metabolism in postprandial state and muscle failure in this function is often considered as the first defect causing IR.Interestingly, despite these controversies, in human, insulin sensitivity and the onset of inflammation have so far never been investigated simultaneously at systemic level and locally in SM and AT in the same individual.In this context, the aim of this thesis was to better understand the mechanisms involved in IR development in grade I obese subject. It is based on a translational research project that compares in a cohort of postmenopausal women, lean subjects with grade I obese insulin-sensitive (OIS) and insulin-resistant (OIR) subjects. Several systemic and tissue parameters (lipotoxicity, inflammation and toll like receptor 4 (TLR4) activation), involved in the IR pathophysiology were analyzed in these subjects.Our results highlight the importance of innate immunity pathways activation in the regulation of insulin sensitivity. Thus, while no inflammation was detected at the systemic level, there is a differential activation of innate immune TLR4 signaling pathways between muscle and AT of OIR subjects. The MyD88-dependent pathway, a pro-inflammatory pathway, is activated in their SM and is associated with IR in this tissue. Conversely, the TRIF-dependent pathway which is an anti-inflammatory pathway is activated in the AT thus maintaining insulin sensitivity in this tissue. This is supported by the induction of interferon system and the antioxidant enzyme manganese superoxide dismutase (MnSOD).In this work, we show that SM defect is the central mechanism which determines IR in grade I obese subject. We have also highlighted the importance of regulation of the balance between the two innate immunity pathways "MyD88" and "TRIF" in maintaining insulin sensitivity

Gojaznost se najčešće definiše kao uvećanje udela masne mase u ukupnoj telesnoj masi. Danas gojaznost poprima pandemijske razmere i karakter globalnog zdravstveno-socijalnog problema jer predstavlja faktor rizika za razvoj masovnih nezaraznih bolesti, pre svega tipa 2 šećerne bolesti (T2DM) i kardiovaskularnih bolesti. Adipokini su molekule koje luči masno tkivo i koji imaju značajnu ulogu u regulaciji mnogobrojnih procesa u ljudskom organizmu. Adiponektin (ADN) je adipokin sa antidijabetogenim, antiinflamatornim i antiaterogenim dejstvom. Tokom razvoja, pre svega, centralnog tipa gojaznosti dolazi do poremećaja u sekretornom profilu masnog tkiva, nastaje pad serumske koncentracije ADN i dolazi do razvoja mnogobrojnih kardiometaboličkih poremećaja. Cilj rada je ispitivanje značaja određivanja serumskog ADN u proceni kardiometaboličkog profila i utvrđivanje povezanosti njegovog nivoa sa procenjenim desetogodišnjim rizikom za razvoj T2DM kod gojaznih osoba. Studija preseka sprovedena na Klinici za endokrinologiju, dijabetes i bolesti metabolizma, Kliničkog centra Vojvodine je obuhvatila 65 ispitanika sa hiperalimentacionim tipom gojaznosti. Kod ispitanika je procenjen desetogodišnji rizik za razvoj T2DM i sprovedena su odgovarajuća antropometrijska, laboratorijska i morfološka ispitivanja. Ispitanici sa sniženim serumskim ADN su imali nepovoljniji kardiometabolički profil u odnosu na ispitanike sa normalnom vrednošću serumskog ADN dok nije bilo razlike u nivou desetogodišnjeg rizika za razvoj T2DM između navedenih grupa. Takođe, ispitanici sa metaboličkim sindromom (MS) i ispitanici sa nealkoholnom masnom bolešću jetre (NAFLD) su imali niži serumski ADN u odnosu na osobe bez MS i osobe bez NAFLD. Kod gojaznih osoba postoji značajna povezanost serumskog ADN sa većim brojem pokazatelja kardometaboličkog profila ali ne i sa procenjenim desetogodišnjim rizikom za razvoj T2DM.
Obesity is often defined as the significant increase in proportion of fat mass in total body mass. Nowadays, obesity exhibits pandemic proportions and acquires character of the global health and social problem, as it represents the risk factor for the development of non-communicable diseases, especially type 2 diabetes mellitus (T2DM) and cardiovascular diseases. Adipokines are molecules secreted by adipose tissue which play an important role in the regulation of various processes in the human organism. Adiponectin (ADN) is an adipokine with anti-diabetic, anti-inflammatory and anti-atherogenic effects. During development of, above all, central obesity, disorders in the secretory profile of adipose tissue are arising, decline in serum concentrations of ADN advents, which leads to occurrence of numerous cardiometabolic disorders. The aim of study is to examine the significance of determining serum ADN in assessing the cardiometabolic profile, and determining its association with the estimated ten-year risk of developing T2DM in obese persons. Cross-sectional study conducted at the Clinic for Endocrinology, Diabetes and Metabolic Disorders, Clinical Center of Vojvodina, included 65 persons with hyperalimentation type of obesity. Ten-year risk of developing T2DM was assessed and appropriate anthropometric, laboratory and morphological evaluations were performed. Persons with lower serum ADN had worse cardiometabolic profile compared to those with normal serum ADN value, while there was no difference in the level of ten-year risk of developing T2DM between two groups. Also, subjects with metabolic syndrome (MS) and subjects with nonalcoholic fatty liver disease (NAFLD) had lower serum ADN compared to persons without MS and to those without NAFLD. There is a significant association of serum ADN with a larger number of cardiometabolic profile indicators but not with the estimated ten-year risk of developing T2DM.

Le diabète de type 2 constitue un enjeu majeur de santé publique et la mise au point de stratégies insulino-sensibilisantes est un défi permanent pour les scientifiques. Cette étude montre qu’un traitement chronique au myo-inositol améliore la sensibilité à l’insuline, réduit l’accrétion adipeuse et augmente la capacité de survie des souris au paraquat. L’effet insulino-sensibilisant semble passer, au moins en partie, par un effet direct sur la voie de signalisation insuline (éventuelle implication de médiateurs de type inositol glycanes). La diminution de l’accrétion adipeuse semble, quant à elle, liée à une réduction de l’activité de lipogenèse de novo et doit probablement aussi contribuer à l’effet insulino-sensibilisant sur le long terme. Une supplémentation en myo-inositol a également amélioré la sensibilité à l’insuline et réduit l’accrétion adipeuse chez la souris sous régime riche en graisses, mais n’a pu prévenir le dévelopement d’une obésité et d’une insulino-résistance associée à une lipotoxicité. Par ailleurs, chez des souris âgées obèses et au contrôle glycémique altéré, la supplémentation en myo-inositol fut inefficace. Cette réduction ou perte d’effet insulino-sensibilisant dans ces deux modèles murins pourrait être liée à la perte d’efficacité du myo-inositol sur la réduction de la masse adipeuse dans un contexte d’obésité déjà installée (souris âgées) et d’activité de lipogenèse de novo réduite (régime gras). De plus, la génération de messagers secondaires putatifs de l’insuline de type inositol glycanes est probablement réduite en cas d’insulino-résistance et pourrait aussi expliquer la perte d’efficacité du myo-inositol dans ces deux cas. Finalement, le myo-inositol seul et/ou utilisé dans le contexte d’une suralimentation chronique n’est pas une stratégie viable de prévention ou de traitement de la résistance à l’insuline. Par contre, son association avec d’autres stratégies insulino-sensibilisantes pourrait potentialiser son/leurs action(s) et éventuellement aider à réduire l’utilisation de stratégies médicamenteuses
Insulin resistance is the first step in the development of type 2 diabetes so finding insulin-sensitizing strategies is challenging for scientists. Some inositol isomers or derivatives have been reported to exert insulin-mimetic activity. myo-Inositol being the most abundant stereoisomeric form of inositol in foodstuffs, we tested its insulin-mimetic potential in the long term and as a nutritional strategy for insulin resistance prevention and/or treatment. This study demonstrates that chronic myo-inositol treatment improves insulin sensitivity, reduces white adipose tissue accretion and improves mice survival mice to paraquat challenge. The insulin-sensitizing effect seems to be related to a direct effect on insulin signaling pathway. Reduction in adipose tissue mass also probably contribute to the long term effect of myo-inositol on insulin sensitivity. Myo-Inositol supplementation also improved insulin sensitivity and reduced white adipose tissue deposition in mice fed a high fat diet, but did not prevent insulin-resistance or obesity development. On one year-old mice with established obesity and altered glycemic control, myo-inositol supplementation showed no beneficial effect. myo-Inositol apparently acts on adipose tissue through reduction of de novo lipogenesis rather than stimulation of lipolysis. This may explain the lack or loss of myo-inositol efficiency in reducing adipose tissue mass in contexts of already well-established obesity (old mice) or reduced de novo lipogenesis (high fat diet feeding). Generation of inositol glycan putative insulin second messengers is probably reduced in context of insulin resistance which may explain the reduced effect of myo-inositol in both obese mice models. Moreover, myo-Inositol did not prevent lipotoxicity and so the associated insulin-resistance in high fat diet fed mice. In conclusion, myo-inositol alone and/or in a context of overnutrition is not a suitable strategy for the prevention or treatment of insulin resistance. Combining it with other insulin sentitizing strategies may however potentiate their action and help reducing insulin-sensitizing drugs use

Le tissu adipeux (TA) contribue à l’inflammation chronique associée aux complications de l’obésité (diabète, ). Au début de l’obésité, l’inflammation est de bas grade suggérant des mécanismes immunosuppresseurs, dont l’activité décroit avec l’obésité, accélérant la survenue des complications. Les cellules souches adipocytaires (CSA) participent au développement puis à l’homéostasie des dépôts de TA sous-cutanés (SAT) et viscéraux (VAT). Sous l’effet de stimuli inflammatoires, elles exercent in vitro des fonctions immunosuppressives, ces propriétés sont cependant perdues quand elles sont isolées de TA inflammatoires dans l’obésité avancée. Le rôle des CSA dans l’homéostasie immunitaire du TA est peu connu. Nous avons émis l’hypothèse que les CSA ont une fonction immunosuppressive active dès le début de l’obésité et que son altération participe à la survenue des complications de l’obésité. Pour aborder cette question, j’ai mis en œuvre deux protocoles d’obésité induite par la diète chez la souris C57Bl/6 et exploré les fonctions des CSA isolées des dépôts sous-cutanés (CSA-S) et viscéraux (CSA-V). Mes travaux confirment d’importantes différences à l’état sain, dans le potentiel de prolifération et de différenciation des CSA-S et -V. Nous montrons que ces différences correspondent à des métabolomes distincts avec une plus forte activité glycolytique des CSA-V, et mitochondriale des CSA-S. Grâce à ce modèle, nous avons pu étudier les propriétés immunosuppressives des CSA dans les phases précoces de l’obésité et dans les deux types de dépôts de TA. Une cinétique de 6, 10 et 14 semaines (s) de régime riche en gras (HF) nous a permis de couvrir plusieurs étapes de l’obésité, depuis l’intolérance au glucose dès 6 s, sans inflammation des TA, jusqu’à la résistance à l’insuline à 14 s de régime et l’inflammation du VAT mais pas du SAT. Mes résultats montrent que dès 6 s, les CSA-V en réponse au HF attirent les macrophages tout en inhibant leur polarisation M1 pro-inflammatoire. En parallèle, les CSA-S induites par le régime HF suppriment la prolifération des lymphocytes T activés et inhibent fortement leur migration. Cette étude montre (i) que les CSA des 2 dépôts sont activées par le régime HF indépendamment de l’inflammation et de la durée du régime, (ii) que les propriétés immunosuppressives induites concernent des cibles différentes selon les dépôts, (iii) qu’elles sont encore présentes au début de la résistance à l’insuline. L’analyse des TA fraichement isolés montre que la progression de l’obésité diminue la prolifération et la proportion des CSA. A ce stade de l’obésité précédant l’inflammation du SAT, le caractère souche des CSA in vitro n’est pas touché mais l’exposition à l’environnement obésogène empêche leur maintien dans les dépôts. Dans un second protocole, j’ai exploré l’impact de la surnutrition maternelle pendant l’allaitement sur les propriétés des CSA de la descendance adulte. Ce contexte prédispose la descendance au développement de maladies métaboliques. Nous voulions savoir si les CSA sont des cibles du régime maternel et conservent des altérations à long terme. J’ai isolé les CSA-S et -V de la descendance 6 s après le sevrage et exploré l’impact d’un régime HF post-sevrage. L’analyse par RNAseq montre que le régime maternel altère le transcriptome des CSA à l’âge adulte et induit une intolérance au glucose, même avec un régime standard post-sevrage. Trois catégories de gènes sont particulièrement affectées par les régimes HF : mortalité, stress métabolique, synthèse des protéines et ribosomes. D'autres ne sont affectées que par le régime de la mère. Pour les gènes associés à la mortalité, une accentuation du différentiel est observée lors du cumul des régimes HF maternel et post-sevrage. Nous montrons que les CSA sont altérées avant les complications de la descendance adulte. Les CSA peuvent donc être programmées par la surnutrition des mères et pourraient être des vecteurs des dysfonctionnements futurs du TA
Obesity induces a chronic inflammation responsible for complications (diabetes, cardiovascular diseases) where adipose tissue plays a central role. At the onset of obesity, inflammation is at low grade suggesting the control by immunosuppressive mechanisms that decrease with obesity, promoting complications. Adipose stem cells (ASC) support the development and the homeostasis of adipose tissue in subcutaneous and visceral adipose depots. Inflammatory stimuli induce immunosuppressive functions in ASC in vitro, but published data report that ASC isolated from inflammatory adipose tissues in established obesity have lost these properties. The role of ASC in the immune homeostasis of adipose tissue is poorly known. We made the hypothesis that immunosuppressive properties of ASC are induced since the onset of obesity and that their alterations contribute to complications. To address this question, I performed two protocols of diet induced obesity in mice and I explored the functions of ASC isolated from subcutaneous (S-ASC) and visceral (V-ASC) adipose depots. My results show major differences in the proliferation and the differentiation potential of ASC from distinct adipose depots, according to published data. We reveal that these differences correlate with distinct metabolomes, V-ASC having lower mitochondrial and higher glycolytic activity than S-ASC. Using this model, we studied the immunosuppressive functions of ASC in early obesity in both adipose depots. To this end we performed a kinetic of 6, 10 and 14 weeks of high fat diet (HF) in C57Bl/6 mice. This timing covered low grade inflammation progress from glucose intolerance with 6 weeks of diet in the absence of adipose tissue inflammation, to insulin resistance at 14 weeks of diet associated with visceral, but not subcutaneous, adipose tissue inflammation. My results show that at 6 weeks of HF diet, V-ASC attracted macrophages and inhibited the pro-inflammatory M1 polarization of these cells. At the same time, S-ASC completely suppressed the proliferation of activated T lymphocytes and strongly inhibited their migration. This study shows (i) that CSA from both adipose depots are activated by HF diet, independently of inflammation and diet time, (ii) that the induced immunosuppressive properties target distinct immune cells, (iii) that they are maintained with resistance to insulin. The analysis of adipose tissue composition showed that the ASC population decreased and had lower proliferation rate with HF diet. This indicate that at the onset of obesity, intrinsic properties of ASC were maintained in vitro but their environment altered their maintenance in both adipose depots. In a second protocol, I explored the consequences of maternal overnutrition during lactation on the properties of ASC in the adulthood. It has been reported that these conditions favorize the development of metabolic diseases in the offspring. We wondered whether ASC are targeted by maternal diet and develop long term alterations. I isolated S- and V-ASC from the offspring 6 weeks after weaning and I explored the influence of a HF post-weaning diet. RNA seq analysis showed that maternal diet altered the transcriptome of ASC in the adulthood and induced glucose intolerance, even in animal fed with a standard diet after weaning. Gene expression altered in ASC support cell death, metabolic stress, transcription, protein synthesis. Some genes are only affected by the mother’s diet. For genes associated with cell death, differential expressions induced by HF diet are increased when cumulated with mother’s HF diet. These results show that alterations of ASC precede the complications in the adult offspring. ASC can thus be programmed by maternal overnutrition and are the probable vectors of later adipose tissue dysfunctions

Despite the importance of muscle in the development of type 2 diabetes, few studies have investigated the effect of polychlorinated biphenyls (PCBs) on muscle energy metabolism. Previous results from our lab suggested that PCB126 exposure induced an indirect negative effect on muscle mitochondrial function. Since PCBs are stored in adipose tissue, we hypothesized that PCB126 alters adipokine secretion which in turn affects muscle metabolism. Objectives. Study the adipose-to-muscle communication in PCB126-induced metabolic defects. Methods. Communication between adipocytes and myotubes was reproduced by exposing C2C12 or mouse primary myotubes to the conditioned medium (CM) of 3T3L1 adipocytes exposed to environmentally relevant PCB126 levels. Results. PCB126 significantly increased adipokine secretion and decreased mitochondrial function, glucose uptake and glycolysis in insulin resistant (IR) but not in insulin sensitive 3T3L1. However, exposure of myotubes to CM of IR 3T3L1 only decreased glucose uptake and insulin sensitivity, without altering myotubes glycolysis or mitochondrial function. Conclusion. Our results suggest that the increased adipokine secretion by adipocytes could explain the decreased muscle glucose uptake and insulin sensitivity when exposed to PCB126.

Le tissu adipeux brun (TABr) est devenu ces dix dernières années le centre d’intérêt de nombreux laboratoires en raison de sa capacité à dissiper l’énergie apportée par les substrats sous forme de chaleur. Chez les mammifères, le développement du TABr intervient à la fin de la gestation et devient fonctionnel à la naissance. Sa capacité thermogénique permet aux nouveau-nés de s’adapter face à l'environnement extra-utérin, puis son activité régresse avec l’âge. Ceci suggère que l’environnement intra-utérin joue un rôle important dans la programmation de la physiologie et du métabolisme du TABr. Dans un modèle bien décrit de retard de croissance intra-utérin, qu’est la carence protéique maternelle (CP), la jeune progéniture CP est normoglycémique malgré un défaut de sécrétion de l'insuline mais développe avec l'âge une résistance à l'insuline et une hyperglycémie. Lors de mon arrivée au laboratoire, des résultats suggéraient un rôle du TABr dans les changements dynamiques du profil métabolique de la progéniture CP en fonction de l'âge. En effet, le TABr des rats CP est hyperactif à 3 mois par rapport aux animaux contrôles alors qu’il revient au niveau des contrôles chez la progéniture CP âgée de 18 mois, ce qui corrèle avec l’apparition des troubles métaboliques caractéristiques du diabète de type 2. Durant ma thèse, mon premier objectif a été de démontrer le rôle causal du TABr dans le maintien de l’homéostasie glucidique chez les jeunes animaux CP. Pour ce faire, nous avons exposé au froid de jeunes rats CP pour solliciter leur TABr et nous avons procédé à l’ablation chirurgicale de ce tissu. Nos résultats montrent que la jeune progéniture CP est mieux protégée que les contrôles à une exposition au froid grâce à l’activité thermogénique accrue de leur TABr
Brown adipose tissue (BAT) has grown over the last ten years into the center of interest for many laboratories due to its capacity to burn energy derived from metabolic substrates into heat. Indeed, in mammals, the development of BAT occurs at the end of gestation to become fully functional at birth. Its thermogenic capacity allows newborns to face extrauterine environment, and thereafter its activity declines with age. This suggests that the intrauterine environment plays an important role in the programming of BAT physiology and metabolism. In a well-known model of intrauterine growth retardation (IUGR), the maternal protein restriction model (called LP for low protein), the young LP progeny is normoglycemic despite an insulin secretion defect but develops insulin resistance and hyperglycemia with age. When I started my thesis work, available results in the laboratory suggested a role of BAT in the dynamic changes of the LP progeny metabolic profile according to the age. Indeed, BAT of young LP rats is hyperactive at 3 months compared to controls while this activity drops back to control levels in old 18-months LP progeny, consistent with the appearance of a type 2 diabetic phenotype. During my thesis, the first objective was to search for the causal role of BAT in the maintenance of glucose homeostasis in young LP progeny. Using a first strategy, we exposed young LP progeny to a cold challenge to activate their BAT. In a second approach, we performed surgical ablation of their BAT. Our results show that young LP progeny is more protected against a cold challenge than controls, due to the high thermogenic capacity of their BAT. However, BAT ablation induces hyperglycemia in young LP animals showing that this tissue is required to maintain their normoglycemia. This work, published in Diabetes in March 2017, suggests that a deleterious fetal environment could reprogram BAT metabolism. The second objective of my thesis was to identify the molecular mechanisms allowing the maintenance of active BAT in young LP progeny. To do so, we compared two models of BAT activation, ie our LP model and a well-known model of BAT activation with an agonist of β-3 adrenergic receptors. In both cases, when BAT is active, we observed a global increase in microRNA (miRNA) expression associated to augmented miRNA machinery expression, and in particular AGO2 expression. Interestingly, when BAT is inactive in old LP animals, miRNA expression and miRNA machinery expression return to control levels. While activation of mature brown adipocytes in vitro leads to an increase in AGO2 protein expression, partial deletion of this protein is sufficient to decrease the thermogenic activity of these cells. Collectively our data suggest that AGO2 and increased miRNA expression contribute to BAT activation. The manuscript concerning this research is in the review process at Molecular Metabolism. In the third part of my PhD research efforts, I have found that in the BAT of young LP progeny several miRNAs are robustly downregulated. We have focused on let-7cp and miR-22-3p, which have the most severe decrease in expression. Our key finding is that these two miRNAs act synergistically to hinder mature brown adipocyte thermogenic activity. This work is in the process of being finalized for publication. In conclusion, during my PhD training I have revealed several novel findings, which lead to a better understanding of BAT physiology and its dysregulation in situations eventuating in perturbed glucose homeostasis. While additional efforts are certainly needed, these contributions advance our vision to leverage BAT as a promising target for the prevention and/or treatment of metabolic perturbations associated to obesity and type 2 diabetes

Evidências na literatura demonstraram que o aumento da adiposidade confere maior suscetibilidade ao desenvolvimento de diabetes tipo 2, pois o tecido adiposo branco (TAB) atua na regulação da homeostasia energética e da sensibilidade à insulina através da sua atividade endócrina e de interações com reguladores neuroendócrinos. O treinamento físico aeróbio tem sido fortemente recomendado para a prevenção e tratamento do diabetes tipo 2, pois promove adaptações no metabolismo energético que contribuem diretamente para a melhora da resposta glicêmica e para o controle de peso corporal. Embora esteja claro na literatura o papel do treinamento físico contra o desenvolvimento de distúrbios no metabolismo da glicose e obesidade, uma lacuna de conhecimento ainda existe quando buscamos informações a respeito da participação metabólica do TAB na prevenção do diabetes tipo 2 através do treinamento físico aeróbio. Dessa forma, o presente estudo teve como objetivo testar a hipótese de que o efeito protetor do treinamento físico contra o desenvolvimento de diabetes tipo 2 é mediado por adaptações funcionais do TAB. Para isso, foram utilizados camundongos alimentados com dieta normocalórica e de cafeteria submetidos ou não ao treinamento físico aeróbio. O treinamento físico aeróbio foi eficaz para a prevenção do diabetes tipo 2, e essa resposta foi associada à menor adiposidade corporal resultante do aumento da lipólise e da capacidade oxidativa do TAB induzido pela maior ativação via da AMPK/ACC
Evidence in the literature have shown that increased adiposity confers greater susceptibility to developing type 2 diabetes and white adipose tissue (WAT) acts in the regulation of energy homeostasis and insulin sensibility through its endocrine activity and interaction with neuroendocrine regulators. Aerobic physical training has been strongly recommended for the prevention and treatment of type 2 diabetes because it promotes adaptations in the energy metabolism that contribute directly to the improvement of glycemic metabolism and body weight control. Although it is clear in the literature the role of physical training against the development of disturbances in the glucose metabolism and obesity, the role of WAT to prevent type 2 diabetes through physical training was poorly investigated. Thus, the present study aimed to test the hypothesis that the protective effect of physical training against the development of type 2 diabetes is mediated by functional adaptations of WAT. For this, we used mice fed with control or cafeteria diet and submitted or not to aerobic physical training. The physical training was effective for the prevention of type 2 diabetes, and this response was associated with lower body fat due to increased lipolysis and oxidative capacity of WAT induced by the activation of AMPK/ACC

Le diabète de type 2 résulte d’un déséquilibre entre les capacités de sécrétion de l’insuline par les cellules bêta-pancréatiques et son action au niveau de ses tissus cibles. Dans le prédiabète, l’hypersécrétion d’insuline compense l’insulino-résistance et cet état est généralement associé à l’obésité et à l’accumulation de tissu adipeux.L’objectif de ma thèse a été d’étudier la biocommunication entre le tissu adipeux viscéral et la cellule bêta-pancréatique lors du prédiabète et du diabète de type 2, en me focalisant sur deux médiateurs originaux, les isoprostanes et les miARNs. Nous avons observé une diminution de la sécrétion d’isoprostanes par le tissu adipeux péripancréatique au cours de l’obésité chez le rat Zucker fa/fa. Spécifiquement observé dans ce tissu adipeux ectopique, ce résultat s’explique par une induction des principales enzymes antioxydantes et une réduction de l’expression de la sPLA2 IIA chez les animaux obèses. Remarquablement, une des isoprostanes, la 15-F2t-Isoprostane ainsi que son épimère aux concentrations de 10 nM et 10 μM inhibent la sécrétion d’insuline gluco-stimulée dans les îlots pancréatiques isolés de rat Wistar. Cet effet pourrait s’expliquer par la liaison de cette isoprostane avec le récepteur au thromboxane A2, dont l’expression génétique et protéique a été mise en évidence pour la première fois dans les îlots de Langerhans et les cellules bêta. La réduction de l’inhibition de la sécrétion d’insuline chez le rat Zucker fa/fa, par une biocommunication paracrine, pourrait favoriser les mécanismes de compensation bêta-cellulaire. Par ailleurs, la production de miARNs, contenus dans des vésicules extracellulaires, par le tissu adipeux omental a été analysée chez l’homme par small RNAseq. Chez des patients obèses, la production de miARNs est modifiée lors de l’insulino-résistance et du diabète de type 2 avec des conséquences possibles sur la fonctionnalité des cellules bêta. Des miARNs différentiellement exprimés lors du diabète de type 2 pourraient ainsi participer à son apparition et représenter de nouveaux biomarqueurs et cibles thérapeutiques. Pour conclure, ces travaux de thèse ont permis de mettre en évidence de nouveaux mécanismes de biocommunication entre le tissu adipeux et les cellules bêta-pancréatiques
Type 2 diabetes occurs as a result of an unability of pancreatic beta-cells to meet the insulin demand in its target tissues. During prediabetes insulin hypersecretion compensate for insulin resistance and this state is usually associated with obesity and excess body fat.The aim of my thesis was to study the biocommunication between visceral adipose tissue and pancreatic beta-cells during prediabetes and type 2 diabetes, with a focus on two original mediators, isoprostanes and miRNAs. We observed a decrease in isoprostane secretion by peripancreatic adipose tissue during obesity in Zucker fa/fa rats. In this ectopic adipose tissue, this observation may be related to an induction of some antioxidant enzymes and a reduction of the expression of sPLA2 IIA in obese animals. Remarkably, 15-F2t-Isoprostane as well as its epimer used at concentrations of 10 nM and 10 μM inhibited glucose-stimulated insulin secretion in isolated pancreatic islets. This effect could be explained by the binding of isoprostanes to the thromboxane A2 receptor, whose gene and protein expression has been demonstrated for the first time in islets and beta-cells. In Zucker fa/fa rats, less inhibition of insulin secretion through a paracrine biocommunication, could favor beta-cell compensatory mechanisms. Furthermore, the production of miRNAs, contained in extracellular vesicles released by omental adipose tissue, was analyzed in humans by small RNAseq. In obese patients, miRNAs production is altered during insulin resistance and type 2 diabetes with possible consequences for beta-cell function. Differentially expressed miRNAs in type 2 diabetes may participate in its development and represent novel biomarkers and therapeutic targets. In conclusion, this thesis highlighted new biocommunication mechanisms between adipose tissue and beta-pancreatic cells

Le tissu adipeux sous-cutané (TAsc) est le site préférentiel du stockage postprandial des triglycérides (TG). Quand les capacités d’accrétion sont dépassées, le stockage des TG se fait dans des sites ectopiques du TA et dans des tissus non adipocytaires, par exemple foie et muscles, ce qui entraine de multiples dysfonctionnements dans ces organes et tissus, et permet le développement du syndrome d’insulinorésistance. Chez les sujets obèses, la période postprandiale est caractérisée par des anomalies métaboliques, immunitaires, hormonales, et également par une diminution importante du flot sanguin dans le tissu adipeux (FSTA) sous-cutané. Ce blocage de la perfusion postprandiale du TA a aussi été montrée chez des individus minces qui avaient de très lourds antécédents familiaux de maladies cardiométabolique (obésité, diabète de type 2, maladies cardiovasculaires). Dans cette thèse, on classifiera ces individus comme « non-répondeurs ». À ce jour, peu d’attention a été accordée à ce phénomène. L’hypothèse qui sous-tend cette thèse est que les anomalies du FSTA sont innées ou primaires et sont impliquées très tôt dans le développement de la résistance à l’insuline (RI), du diabète de type 2 et du syndrome métabolique. Le but de notre recherche était donc de vérifier si les altérations du FSTA sont présentes chez les personnes saines et minces, mais à très haut risque de développer une RI ou une maladie cardiométabolique. Nous avons aussi cherché à déterminer les facteurs liés à la non-réponse. Pour cela il nous a fallu explorer certains facteurs hormonaux impliqués dans la régulation du FSTA. Nos résultats montrent que le FSTA est très diminué, à jeun et en postprandial, chez les sujets à haut risque de maladies cardiométaboliques mais encore minces et métaboliquement sains, sans RI. Nous avons aussi montré, pour la première fois, l’effet vasodilatateur du peptide intestinal vasoactif (VIP) dans le TAsc, tout comme le rôle stimulant du système cholinergique dans la régulation postprandiale du FSTA. Cependant, aucun de ces facteurs ne participe au dysfonctionnement du FSTA postprandial chez les non-répondeurs. Des taux répétés de TG plus élevés chez les non-répondeurs et l’association du FSTA avec certains indices de la RI décrits dans la littérature suggèrent que l’altération du métabolisme lipidique suite à la diminution du FSTA puisse servir de médiateur à la détérioration de la sensibilité à l’insuline.
Abstract : Subcutaneous adipose tissue (SCAT) is the preferential site of triacylglycerols (TAG) postprandial disposal. When the buffering capacity of SCAT for lipids is exceeded, TAG are disposed in ectopic adipose tissue depots and in non-adipose tissues, such as liver and muscles. Consequently, multiple dysfunctions of these organs and tissues develop including insulin resistance (IR). In obese people, the postprandial period is characterized by metabolic, immune and hormonal alterations, but also by severely altered adipose tissue blood flow (ATBF). Nevertheless, significant alteration of postprandial ATBF was also found in lean individuals with highly positive familiar history of cardiometabolic diseases (obesity, type 2 diabetes, cardiovascular diseases). In the thesis, we term them as "non-responders". Up to date, little attention has been payed to this phenomenon. The underlying hypothesis of this thesis is that alterations in ATBF are inborne or very early and that they participate on the development of IR, type 2 diabetes and metabolic syndrome. Consequently, the aim of our research was to verify if the alterations in ATBF are present in healthy, normal-weight subjects, but at very high risk for development of IR or cardiometabolic diseases. Simultaneously, we searched for factors linked with nonresponsiveness phenomenon. To do this, we examined some hormonal factors in ATBF regulation. Our results confirm the presence of altered fasting and postprandial ATBF in at highrisk subjects for cardiometabolic diseases, but still lean and metabolically healthy, without IR. For the first time, we have also demonstrated the role of cholinergic system in postprandial ATBF regulation, and vasodilatory effect of vasoactive intestinal peptide (VIP) in SCAT. However, none of these factors takes part in postprandial ATBF dysfunction in non-responders. Higher TAG levels repeatedly found in non-responders and the association of ATBF with some indices of insulin sensitivity described in the literature suggest that alteration of lipid metabolism as a result of low ATBF may mediate deterioration of insulin sensitivity.

Diabetes mellitus tipo 2 (DM2) está associado a um estado de inflamação crônica e ativação do estresse do retículo endoplasmático (ERE). Nesse contexto, são necessários estudos para encontrar alternativas que melhorem o quadro inflamatório, como os ácidos graxos poli-insaturados ômega 3 (n-3 PUFA), um conhecido agente anti-inflamatório. Esse estudo teve por objetivo avaliar o efeito da suplementação do ácido alfa-linolênico (ALA, um n-3 PUFA) no estresse do retículo endoplasmático e no estado inflamatório no tecido adiposo subcutâneo abdominal (TASC) em pacientes com DM2. Foi conduzido um estudo duplo-cego, prospectivo, placebo-controlado. Vinte pacientes com DM2 foram randomizados para suplementação com 3g/dia de ALA ou placebo durante 60 dias. O tecido adiposo foi coletado através de punção aspirativa por agulha fina do abdome antes e após a suplementação e os genes e proteínas foram avaliados através de PCR em tempo real e western blot. Foi encontrada, após suplementação, uma redução da expressão gênica do XBP1 (20%), sXBP1 (70%) e aumento da expressão gênica do GRP78 (150%), confirmado na expressão proteica. Além disso, foi encontrado aumento da expressão gênica da adiponectina (90%) e redução da expressão gênica do IL-6 (80%) e IRS-1 (60%), sem correlação com a expressão proteica, no tempo pós-suplementação com ALA. Portanto, foi demonstrado que o ALA pode modular o ERE através da via da IRE1/XBP, levando ao aumento das chaperonas (BIP/GRP78), além de um efeito adicional na expressão gênica da adiponectina, IL-6 e IRS-1, o que pode demonstrar um potencial terapêutico do ALA em pacientes com DM2.
Type 2 diabetes mellitus (T2DM) is a state of chronic inflammation and activation of endoplasmic reticulum stress (ERS). In this context, studies are necessaries to find new possibilities to improve this inflammation such as the n-3 polyunsaturated fatty acid (n-3 PUFA) acting as an anti-inflammatory agent. In this study, we aimed to evaluate the effect of n-3 PUFA alpha-linolenic acid (ALA, a n-3 PUFA) supplementation in T2DM patients on the molecular expression of ERS genes in abdominal subcutaneous adipose tissue (SAT). We performed a placebo-controlled study, in a double-blind design with 20 T2DM patients, receiving, randomly, 3g/day of ALA or placebo for 60 days. The adipose tissue was collected by fine-needle aspiration in abdomen before and after the supplementation and the genes and proteins were evaluated by real-time PCR and western blot. It was seen, after the supplementation, a reduction in XBP1 (20%), sXBP1 (70%) and an increase in Grp78 (150%) gene expression, likewise same results in protein concentration. Furthermore, it was observed an increase in adiponectina (90%) gene expression and reduction in IL-6 (80%) and IRS-1 (60%) gene expression, with no correlation to protein expression after supplementation of ALA. Therefore, we have provided evidence that ALA may modulate ERS by the IRE1/XBP pathway leading to an increase in chaperones (BIP/GRP78), additionally its effect on adiponectina, IL-6 and IRS-1 gene expression can demonstrate a therapeutic potential in T2DM.

Le diabète est aujourd’hui la 7ème cause de mortalité dans le monde. Dans cette population, la cardiomyopathie diabétique est la principale cause de morbi-mortalité. Le développement de cette complication débutant dès l’apparition du diabète, un dépistage et une prise en charge précoces de cette pathologie sont donc de première importance et sont les deux objectifs visés par ces travaux. La première étude que nous avons réalisée a permis de démontrer l’utilité d’une évaluation compréhensive de la fonction régionale myocardique gauche par 2D speckle-tracking imaging, en conditions de stress à la dobutamine, dans le dépistage précoce de la cardiomyopathie diabétique chez des patients diabétiques de type 2 asymptomatiques. La deuxième partie de ces travaux a, quant à elle, donné lieu à deux études. L’étude clinique a permis de mettre en évidence une amélioration de la réponse au stress de la fonction régionale myocardique après 3 mois de supplémentation en vitamine D3 chez des patients carencés. L’étude expérimentale a, pour sa part, souligné les effets bénéfiques sur le remodelage et la fonction cardiaques de cette supplémentation, en prévention secondaire, dans un modèle murin de diabète de type 2 induit par un régime gras et sucré. Par ailleurs, cette étude a mis en lumière l’implication potentielle d’une modulation des taux myocardiques en espèces lipotoxiques par la vitamine D3 dans ces effets. L’ensemble de ces travaux de thèse a ainsi permis, d’une part, de proposer une technique de dépistage des signes précoces d’altération de la fonction cardiaque chez le patient diabétique de type 2 et, d’autre part, de montrer les effets bénéfiques d’une supplémentation en vitamine D3 dans ce contexte
Diabetes has reached the 7th place in the world’s top ten mortality causes. In this population, morbi-mortality mainly originates from diabetic cardiomyopathy. This complication evolving from the onset of diabetes, early diagnosis and care are of paramount importance and are the two purposes of this work. In our first study, we demonstrated the relevance of a comprehensive 2D speckle-tracking imaging analysis, under dobutamine stress, in unmasking early left ventricular regional myocardial dysfunction in a population of asymptomatic type 2 diabetic patients. In the second part of this work, we performed two studies. In the first one, we brought to light an improvement of regional myocardial function response to dobutamine stress after a three-month vitamin D3 supplementation protocol, in deficient patients. The second study was performed in a mouse model of diet-induced type 2 diabetes. In this last work, we put forward the beneficial effects of vitamin D3 supplementation, in secondary prevention, on cardiac remodeling and function. These cardioprotective effects may be, at least in part, on account of modulatory effects of vitamin D3 on myocardial lipotoxic species levels. This whole work allow us to propose a tool enabling recognition of early cardiac function impairments in type 2 diabetic patients and to demonstrate the beneficial effects of vitamin D3 supplementation in this context

Dissertação de Mestrado em Química Medicinal apresentada à Faculdade de Ciências e Tecnologia
Diabetes mellitus (DM) is a metabolic pathology with increasing prevalence in the western society. DM is characterized by hyperglycemia resulting from defects in insulin secretion or action, or both. Therefore, chronic hyperglycemia is, in long-term, associated with damage, dysfunction and failure of various organs. Diabetes is closely linked to endothelial dysfunction, atherosclerosis, dyslipidemia and obesity. Insulin resistance, which accompanies obesity and type 2 diabetes, is a condition in which cells are resistant to insulin action and are unable to absorb glucose from the bloodstream and, thus, lead to hyperglycemia.Over the last years, perivascular adipose tissue (PVAT) has emerged as an active component of the blood vessel wall, with endocrine and paracrine functions, that regulates vascular homeostasis and that plays a role on the pathogenesis of cardiovascular diseases. In physiological conditions, this tissue synthetizes a variety of adipokines that contribute to regulation of vascular tone and local inflammation, due to the balance between nitric oxide and endothelin-1. On the other hand, in vascular pathologies, PVAT increases in volume, becomes dysfunctional and leads to a series of physiological complications.This work utilizes two different models with expectations to bring new insights on the field. These two models were used to study the impact of western hypercaloric diets (high-fat diet and fat diet with methylglyoxal) on PVAT and endothelial function. The first animal model evaluates the impact of type 2 diabetes on PVAT and its vascular implications. The second model allows an assessment of a diet rich in fat and methylglyoxal in PVAT to study its vascular outcome. In fact, our results suggest that the fat diet alone induces not only insulin resistance, but also a considerable degree of PVAT dysfunction. Surprisingly, we showed some similarity between the models in terms of the loss of the anticontractile effect of perivascular adipose tissue.
A diabetes mellitus (DM) é uma patologia metabólica com uma prevalência crescente na sociedade ocidental. A DM é caracterizada por hiperglicémia resultante de defeitos na secreção ou ação de insulina, ou ambos. Por conseguinte, a hiperglicémia crónica está associada, a longo prazo, a danos, disfunção e falha de vários órgãos. A diabetes encontra-se intimamente associada a disfunção endotelial, aterosclerose, dislipidemia e obesidade. A resistência à insulina, que acompanha a obesidade e a diabetes tipo 2, é uma condição na qual as células apresentam resistência à ação da insulina e não são capazes de absorver glicose da corrente sanguínea que, desta forma, conduz a hiperglicémia.Nos últimos anos, o tecido adiposo perivascular (do inglês, PVAT) tem captado a atenção da comunidade científica devido às suas funções endócrinas e parácrinas como constituinte da parede dos vasos sanguíneos. Desempenha, ainda, um papel fundamental no desenvolvimento de doenças cardiovasculares. Em condições fisiológicas, este tecido sintetiza uma série de moléculas que contribuem para a regulação do tónus vascular e da inflamação local, devido ao equilíbrio entre substâncias vasodilatadoras, como o óxido nítrico, e vasoconstritoras, como a endotelina-1. Por outro lado, em situações patológicas, o PVAT aumenta de volume, torna-se disfuncional e leva a várias complicações micro e macrovasculares.Este trabalho utiliza dois modelos animais diferentes, com o propósito de estudar o impacto de dietas ocidentais hipercalóricas (dieta rica em gordura e dieta gorda com metilglioxal) no PVAT e avaliar a disfunção endotelial associada. O primeiro modelo animal estuda o impacto da diabetes tipo 2 no PVAT e suas implicações vasculares. O segundo modelo permite fazer uma avaliação de uma dieta rica em gordura com metilglioxal no PVAT e estudar o seu impacto vascular. De facto, os nossos resultados sugerem que a dieta gorda, por si só, induz não só insulinorresistência, como também um grau de disfunção do PVAT bastante considerável. Surpreendentemente, mostrámos alguma similaridade entre os modelos em termos da perda do efeito anticontrátil do tecido adiposo perivascular.

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
A modulação da angiogénese do tecido adiposo (TA) tem sido sugerida como alvo terapêutico na prevenção da disfunção do TA e no aumento da sensibilidade à insulina. O Liraglutido, um análogo do peptídeo semelhante a glucagon-1 (GLP-1), aprovado no tratamento da diabetes tipo 2 pela sua ação na secreção e na sensibilidade à insulina e na homeostase lipídica, tem sido igualmente referido como modulador da angiogénese. Assim, o nosso objetivo consistiu na avaliação do papel do Liraglutido na melhoria da angiogénese do TA e do perfil metabólico. Estudámos, por isso, 4 grupos de ratos Wistar (W) e ratos Goto-Kakizaki (GK), um modelo de diabetes tipo 2 não obeso, ambos com 14 semanas de idade: dois grupos, W Lira e GK Lira, constituídos por ratos W e GK tratados com injeções subcutâneas de Liraglutido (200μg/Kg, duas vezes por dia) durante 14 dias e dois grupos controlo, W e GK, formados por ratos W e GK não submetidos a tratamento. Avaliámos o perfil metabólico sistémico e marcadores da angiogénese no tecido adiposo epididimal (TAE). Os ratos GK, relativamente aos ratos W, apresentaram níveis diminuídos dos fatores pro-angiogénicos, uma marcada intolerância à insulina e uma menor ativação do recetor da insulina (RI) no TAE. O Liraglutido induziu um aumento parcial da tolerância à insulina e um aumento significativo da ativação do RI nos ratos GK, assim como uma melhoria no perfil lipídico e uma perda significativa de peso. No TAE dos ratos GK não se verificaram alterações significativas relativas aos níveis do marcador de célula endotelial CD31, da angiopoietina-2 (Ang-2) e do seu recetor Tie-2, mas observou-se um aumento significativo do fator de crescimento endotelial vascular (VEGF), do seu recetor VEGFR2 e do fator induzido por hipóxia-2a (HIF-2a). Por conseguinte, conclui-se que o Liraglutido promove a angiogénese do TA que está associada a um aumento da sinalização da insulina no TA e a um melhor perfil metabólico, na diabetes tipo 2.
Modulation of angiogenesis in adipose tissue (AT) has been suggested as a potential target in preventing AT dysfunction and improving insulin sensitivity. Liraglutide is a glucagon-like peptide-1 (GLP-1) analogue, approved for type 2 diabetes treatment, known for its effects in insulin secretion and sensitivity and lipid homeostasis. However, besides its glucose-lowering properties, Liraglutide has also been suggested to be a modulator of angiogenesis. Thus, our aim was to assess the role of Liraglutide in improving AT angiogenesis and the metabolic outcome. Therefore, we divided 14-week-old Wistar (W) and non-obese type 2 diabetic Goto-Kakizaki (GK) rats into four groups: two groups of W and GK rats injected subcutaneously with Liraglutide (200μg/Kg, twice a day) during 14 days (W Lira; GK Lira) and two control groups comprising W and GK rats with no treatment (W; GK). The systemic metabolic profile and markers of angiogenesis in epididymal adipose tissue (EAT) were evaluated. GK rats showed decreased levels of proangiogenic factors as well as a marked insulin intolerance and a lower activation of the insulin receptor (IR) in EAT when compared to W group. Liraglutide partially improved insulin tolerance and completely restored IR activation in GK rats. Liraglutide treatment also led to a marked weight loss and an improved lipid profile in GK rats. In the EAT of GK rats no significant alterations were observed in the levels of the endothelial cell marker CD31, angiopoietin-2 (Ang-2) and its receptor Tie-2, but a significant increase of vascular endothelial growth factor (VEGF), its receptor VEGFR2 and the hypoxia inducible factor-2a (HIF-2a) was observed. Thus, this study suggests that Liraglutide improves AT angiogenesis which is associated with an improved AT insulin signalling and a better metabolic outcome in type 2 diabetes.

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2018
In the last decades, there has been a huge increase in the prevalence of metabolic disorders, like obesity and Type 2 diabetes (T2D). Brown adipose tissue (BAT) has been described as a potential target to control and treat these disorders, since is involved in body thermogenesis, which is regulated by UCP1, and in energy expenditure. Moreover, BAT activity is inversed correlated with obesity and insulin resistance. However, there is not enough knowledge about its function and how this tissue is affected in these disorders. It is consensual that higher BAT activity is associated with a better metabolic profile, meaning that higher BAT activity is associated with body weight reduction and improved glucose homeostasis. Brown adipocytes phenotype is quite different from the white adipocytes exhibiting higher mitochondrial content. BAT is activated by the sympathetic nervous system (SNS), which is involved in thermogenesis regulation and when active, BAT is capable of producing several bioactive substances, like adenosine. Adenosine is a purine nucleoside that has been shown to be involved in glucose homeostasis. It is involved in the regulation of lipolysis and inflammation, being expressed in adipose tissue. Recently adenosine has been shown to be involved in BAT activation through SNS via A2A adenosine receptor. However, the role of adenosine in BAT regulation is not yet well defined and clear, with a lot of controversial data in literature. Herein, we hypothesize that the adenosine metabolic pathway is essential to maintain BAT function and that adenosinergic system is impaired in metabolic pathologies as obesity and T2D, being a likely therapeutic target for metabolic diseases. Therefore, the general aim of the present study was to investigate if alterations in adenosine metabolism and function in BAT can contribute to metabolic dysfunction in obesity and T2D. The experiments were performed in male Wistar rats of 8 weeks old. Two groups of animals were used: 1) the high-fat high-sucrose animal model (HFHSu), obtained by submitting the animals to a 60% lipid-rich diet (5.1Kcal/g) and 35% sucrose in drinking water for 25 weeks, and 2) an age-matched control group, fed with standard chow (2.56Kcal/g). Fasting glucose and insulinemia were monitored during diet-induced T2D. Glucose tolerance was evaluated by an oral glucose tolerance test and insulin sensitivity by an insulin tolerance test. At a terminal experiment, animals were anesthetized with pentobarbital (60 mg/kg, i.p) and BAT and white adipose tissue (WAT) around BAT was collected and weighted. Glucose uptake was evaluated in BAT through the uptake of 2-deoxy-D-1,2-3H-glucose (2-DG) in the presence and/or absence of adenosine receptors agonists in animal fed with standard diet. The effect of the HFHSu diet in BAT morphology was evaluated by H&E staining. Also, the effect of the diet on BAT dysfunction and inflammation was evaluated through the quantification of mitochondrial markers, hypoxic signaling molecules and inflammatory markers. To evaluate alterations in adenosine metabolism and function in BAT, the expression of adenosine receptors and proteins involved in adenosine metabolism were also assessed, as well as the adenosine content and release in BAT. In this study it was observed an increase in weight gain and caloric intake as consequence of the HFHSu diet, these animals also exhibited decreased insulin sensitivity and glucose tolerance. Moreover, the hypercaloric diet promoted an increase in BAT and WAT around BAT, and a morphologic dysfunction in the tissue demonstrated by a change in BAT color and an increase in adipocytes size in the H&E staining. Beside was also observed a decrease in the expression of mitochondrial markers as PGC-1α and UCP1, alterations in the expression of hypoxia markers as VEGF, HIF-1α and HIF-2α and an increase in inflammatory markers expression as IL1R and IL6R. Moreover, it was shown that adenosine controls glucose uptake in BAT mainly via A1 receptors, since we observed an increase in glucose uptake under incubation with an A1 receptor agonist and decreased glucose uptake with A2A and A2B adenosine receptors agonists. Furthermore, it was demonstrated that HFHSu diet lead to dysregulation of adenosine receptors expression, with overexpression of A2A and downregulation of A2B. Regarding alterations in adenosine metabolism it was observed that adenosine content and release is decreased in the disease model and incubation with EHNA reestablish adenosine levels to values similar to control values, Furthermore it was observed an overexpression of CD73 and ENT1 in the HFHSu diet in BAT, proteins involved on adenosine metabolism. To conclude, this study demonstrate that the targeting of adenosine might contribute to improve metabolic function in obesity and T2D and the modulation of adenosine receptor expression or adenosine levels in BAT could be a therapeutic tool to treat metabolic dysfunction related disorders.
Nas últimas décadas temos assistido a um aumento na prevalência de doenças crónicas e metabólicas como a obesidade e a diabetes tipo 2 (DT2). O tecido adiposo castanho (BAT) tem sido descrito como um potencial alvo no controlo e tratamento destas doenças metabólicas, uma vez que se encontra envolvido na termogénese corporal, regulada pela proteína UCP1, e no gasto de energia. Além disso, sabe-se que a atividade do BAT está inversamente relacionada com a obesidade e a resistência à insulina. No entanto, ainda não há conhecimento suficiente acerca da sua função e sobre como o BAT está a ser afetado nestas doenças. Contudo, é consensual que uma elevada atividade do BAT está associada a uma melhoria do perfil metabólico, o que significa que uma elevada atividade deste tecido está associada a uma diminuição do peso corporal e a uma melhoria na homeostasia da glucose. O fenótipo dos adipócitos castanhos é diferente do apresentado pelos adipócitos brancos, uma vez que os adipócitos castanhos apresentam um elevado conteúdo mitocondrial. Está descrito que o BAT é ativado pelo sistema nervoso simpático (SNS), que se encontra envolvido na regulação da termogénese, e quando ativo produz diversas substâncias bioativas, entre elas a adenosina. A adenosina é um nucleósido purinérgico, que exerce a sua ação através dos recetores A1, A2A, A2B e A3, A adenosina tem-se demostrado estar envolvida na homeostasia da glucose e na regulação da lipólise e da inflamação, sendo expressa no tecido adiposo. Recentemente, demostrou-se que a adenosina está também envolvida na ativação do BAT através do SNS, pelo recetor de adenosina A2A. No entanto, a sua importância na regulação do BAT continua por esclarecer, havendo estudos muito controversos. Assim, a hipótese deste trabalho é que a via metabólica da adenosina é essencial para manter a função do BAT, e que o sistema adenosinérgico se encontra alterado em patologias como a obesidade e a DT2, sendo um possível alvo terapêutico para o tratamento de doenças relacionadas com a disfunção metabólica. Assim, o objetivo geral deste estudo consiste em investigar se alterações no metabolismo da adenosina e da sua função no BAT poderão contribuir para a disfunção metabólica na obesidade e na DT2. As experiências foram realizadas em ratos Wistar machos com 8 semanas de idade. Os animais foram distribuídos aleatoriamente por 2 grupos: 1) um grupo submetido a uma dieta hipercalórica combinada com 60% gordura na dieta (5.1Kcal/g) e 35% de sacarose em água, denominado HFHSu, durante 25 semanas e, 2) o grupo controlo de idade correspondente, alimentado com uma dieta padrão (2.56Kcal/g). A glicemia em jejum e a sensibilidade à insulina foram controladas durante o tempo e que os animais foram submetidos à dieta. A tolerância à glucose oral foi avaliada por um teste de tolerância oral à glucose e a sensibilidade à insulina através de um teste de tolerância à insulina. Após as 25 semanas os animais foram anestesiados com pentobarbital (60 mg/kg, i.p) e o BAT e o tecido adiposo branco em volta do BAT foram recolhidos e pesados. A captação de glucose no BAT foi avaliada através da captação de 2-deoxyglucose na presença e/ou ausência de agonistas dos recetores de adenosina em animais alimentados com a dieta standard. O efeito da dieta hipercalórica na morfologia do BAT foi avaliado em cortes histológicos através de uma coloração com eosina e hematoxilina, tendo sido também estudado o efeito desta dieta na disfunção e inflamação do BAT através da quantificação de marcadores mitocondriais, moléculas sinalizadoras de hipóxia e marcadores inflamatórios. Para avaliar alterações no metabolismo e função da adenosina no BAT foi também avaliado a expressão dos recetores de adenosina e de proteínas envolvidas no metabolismo da adenosina, bem como o conteúdo e a libertação de adenosina no BAT através da incubação do tecido com e sem inibidores farmacológicos tais como o EHNA (inibidor da adenosina desaminase e o NBTI (inibidor dos transportadores de adenosina). Neste estudo, observou-se que os animais submetidos à dieta hipercalórica apresentavam um aumento no ganho de peso, bem como no consumo calórico. Além disso estes animais exibiam um aumento na glicémia basal em jejum, uma diminuição da sensibilidade à insulina bem como da tolerância oral à glucose. A dieta hipercalórica originou um aumento da quantidade de BAT nestes animais, e também na quantidade de WAT em volta do BAT, bem como alterações na morfologia do BAT. Estas alterações foram observadas através de visualização macroscópica onde foi bem visível uma mudança de cor no tecido, passando de uma cor castanha-avermelhado para um castanho-esbranquiçado, além disso a coloração dos cortes histológicos com H&E mostrou ainda um aumento no tamanho dos adipócitos, sugerindo uma alteração nas caraterísticas do tecido de castanho para branco através de um possível processo de “whitening”. Este modelo patológico desenvolveu também uma disfunção no BAT, tendo sido observada uma diminuição da expressão de marcadores mitocondriais como o PGC-1α e a UCP1, alterações na expressão de moléculas sinalizadoras de hipóxia com diminuição da expressão de VEGF e aumento na expressão de HIF-1α e HIF-2α e ainda um aumento na expressão dos marcadores inflamatórios IL1R e IL6R. Observou-se também que a adenosina está envolvida na captação de glucose no BAT em animais controlo, sobretudo através da ação dos recetores A1, uma vez que a incubação do tecido na presença de um agonista dos recetores A1 levou a um aumento da captação de glucose neste tecido, sendo que a presença de agonistas dos recetores A2A e A2B demostraram uma diminuição da captação de glucose no BAT. Além disso observou-se que a dieta HFHSu leva a uma desregulação nos recetores de adenosina, tendo sido observado um aumento na expressão dos recetores de adenosina A2A e uma diminuição da expressão do recetor A2B no BAT, sem qualquer alteração ao nível da expressão do recetor A1 neste tecido. Este modelo patológico induziu também alterações ao nível do metabolismo da adenosina no BAT, observando-se uma diminuição quer no conteúdo quer na libertação de adenosina no BAT no modelo HFHSu em comparação com o grupo controlo. Na quantificação de adenosina no BAT este efeito foi avaliado apenas no tecido imediatamente após dissecção, onde é evidente uma diminuição no conteúdo de adenosina no BAT no nosso modelo patológico, bem com no tecido incubado durante 10, 30 e 60 minutos na presença e ausência de diferentes inibidores farmacológicos tais com o EHNA, um inibidor da adenosina desaminase e o NBTI, um inibidor dos transportadores de adenosina. Aqui observou-se que o uso de EHNA levava a um aumento do conteúdo e libertação de adenosina no tecido no BAT de animais controlo e submetidos à dieta hipercalórica sendo que no grupo HFHSu o uso de EHNA reestabelece as concentrações de adenosina para níveis similares ao grupo controlo. Além disto foi ainda quantificado a expressão de proteínas envolvidas no metabolismo adenosinérgico, sendo observado um aumento da expressão de CD73, uma proteína envolvida na conversão de AMP em adenosina, bem com um aumento na expressão de ENT1, um transportador equilibrativo de adenosina, no BAT do grupo submetido à dieta HFHSu. Assim pode concluir-se com este estudo que o controlo do metabolismo da adenosina pode contribuir para melhorar a função metabólica do BAT na obesidade e na diabetes DT2, e que a modulação dos recetores de adenosina e dos níveis de adenosina no BAT poderão ser um potencial alvo terapêutica no tratamento de doenças relacionadas com a disfunção metabólica, aumentando a atividade do BAT e evitando o processo de “whitening” no tecido.

Dissertação de Mestrado em Patologia Experimental apresentada à Faculdade de Medicina
Introdução e Objetivos: A obesidade está associada ao desenvolvimento de insulino-resistência e disfunção do tecido adiposo, conduzindo a alterações do metabolismo lipídico e glicémico, e em última instância, diabetes tipo 2 (DMT2). A bromocriptina, um agonista dopaminérgico D2, mostrou-se promissora no tratamento da DMT2 contribuindo para a diminuição dos níveis da glicemia. No entanto, os mecanismos de ação da bromocriptina bem como a sua ação nos tecidos sensíveis à insulina não são conhecidos. Neste estudo tivemos como objetivo avaliar o potencial terapêutico da bromocriptina na modulação da sensibilidade à insulina em ratos diabéticos tipo 2 com obesidade induzida pela dieta, bem como a sua ação no tecido adiposo periepididimal (TAE).Métodos: Foram utilizados ratos Wistar (W) alimentados com dieta normal (grupo 1) e ratos Goto-Kakizaki (GK) diabéticos tipo 2 não obesos divididos em 4 grupos: GK com dieta normal (grupo 2), GK com obesidade induzida por uma dieta rica em gordura e sacarose (grupo 3 - GKHFD), GKHFD tratados com bromocriptina (10mg/Kg/dia) durante 30 dias (grupo 4 - GKHFDBr) e GKHFD tratados com veículo (grupo 5 - GKHFDVh) (n=8/grupo). Além da avaliação do perfil glicémico e lipídico, foi a avaliada a tolerância à insulina (prova de tolerância à insulina), bem como os mecanismos associados à captação e utilização da glicose e dos ácidos gordos no TAE.Resultados: Os ratos GK obesos apresentaram um agravamento da tolerância à insulina e dos níveis em jejum da glicemia, colesterol e dos triglicerídeos, em relação aos seus controlos não obesos. O tratamento com bromocriptina reduziu a intolerância à insulina, e a glicemia em jejum. No TAE, aumentou os níveis do transportador da glicose GLUT4, a expressão dos recetores dopaminérgicos D1 e D2, e de marcadores relacionados com a oxidação lipídica.Conclusões: Embora os mecanismos subjacentes sejam ainda desconhecidos, estes resultados sugerem que a bromocriptina melhora o perfil glicémico e a resistência à insulina na DMT2,aumentando a sinalização dopaminérgica no tecido adiposo e alterando o metabolismo dos ácidos gordos. Estudos futuros permitirão desvendar os mecanismos envolvidos.
Introduction and objectives: Obesity is associated with the development of insulin resistance and dysfunction of adipose tissue, leading to changes in lipid and glycemic metabolism, and ultimately type 2 diabetes (DMT2). Bromocriptine, a D2 dopaminergic agonist, has been shown to be promising in the treatment of DMTII contributing to the reduction of glycemic levels. However, the mechanisms involved as well as its action on insulin sensitive tissues are not known. Thus, our goal was to evaluate the therapeutic potential of bromocriptine in the modulation of peripheral sensitivity to insulin in an obese type 2 diabetic animal model, as well as insulin signaling in epididymal adipose tissue (TAE).Methods: Wistar (W) rats fed with normal diet (group 1) and non-obese type 2 diabetes, Goto-Kakizaki (GK) were divided into 4 groups: GK with normal diet (group 2), GK with diet-induced obesity (group 3 - GKHFD), GKHFD treated with bromocriptine (10mg / kg / day) for 30 days (group 4 - GKHFDBr) and GKHFD treated with vehicle (group 5 - GKHFDVh) (n = 8 / group). In addition to the evaluation of the glycemic and lipid profile and insulin tolerance, the mechanisms of glucose and fatty acids uptake and oxidation were evaluated in the TAE.Results: Obese GK rats had lower insulin tolerance and increased fasting levels of glucose, cholesterol and triglycerides in relation to their non-obese controls. The treatment with bromocriptine reduced insulin intolerance and fasting glycemia. In adipose tissue, the expression of GLUT4, D1 and D2 receptors, as well as markers related to lipid oxidation were also improved.Conclusions: Although the underlying mechanisms are still unknown, these results suggest that bromocriptine improves the glycemic profile and insulin resistance in DMT2 by increasing dopaminergic signaling in adipose tissue. Future studies will disclose the mechanisms involved.
FCT

Subclinical inflammation plays a pivotal role in the development of obesity- related complications including type 2 diabetes mellitus, hypertension and cardiovascular diseases. Macrophages are considered important players participating in the initiation and progression of subclinical inflammation within as well as outside of adipose tissue. The first part of this dissertation thesis was focused on macrophage characterization and their quantitative and qualitative changes accompanying metabolic improvements after bariatric surgery. We have demonstrated that the number of macrophages in subcutaneous adipose tissue is reduced regardless of their M1 or M2 polarization manifested as positivity of both the M1- associated CD40 antigen and the M2-associated CD163 and CD206 antigens 6 months after bariatric surgery. Thus, as suggested by previously published data, subcutaneous adipose tissue macrophages seem to have a mixed phenotype. We further confirmed a higher number of non- classical monocytes, which play a role in the control of vascular integrity, in obese subjects as well as a classical monocyte-derived origin of CD163 positive monocytes. Our data also support the previous suggestion of the soluble form of CD163 antigen being a suitable marker of metabolic complications of obesity. The second part...

Tese no âmbito do Programa de Doutoramento em Ciências da Saúde, ramo de Ciências Biomédicas, apresentada à Faculdade de Medicina da universidade de Coimbra.
Background and aims: Adipose tissue has a crucial role in metabolism and is nowadays recognized as an endocrine organ that produces and potentially secrets more than 600 factors involved in several physiological processes. Adipose tissue dysfunction is associated with increased insulin resistance and systemic dysmetabolism, which contributes to the development of metabolically unhealthy obesity and type 2 diabetes. Methylglyoxal-induced glycation is commonly associated with microvascular dysfunction, namely during chronic hyperglycemia, being a hypothetic factor underlying the impairment of adipose tissue microvasculature and ultimately, tissue dysfunction. Glucagon-like peptide-1 (GLP-1) based therapeutic approaches, specifically metabolic surgery and GLP-1 analogue treatment, have pleiotropic effects, including improved adipose tissue homeostasis, insulin signalling and glucose tolerance. Thus, we hypothesised that methylglyoxal-induced glycation has adverse effects on periepididymal adipose tissue vasculature, expandability and function, which could be targeted by GLP-1-based strategies through glyoxalase-1-dependent mechanisms. Accordingly, our main goal was to evaluate the role of glycation-induced microvascular dysfunction in adipose tissue function, exploring the mechanisms involved and the role of GLP-1-based therapies. Methods: Wistar rats supplemented with methylglyoxal and/or maintained with high-fat diet, were studied regarding periepididymal adipose tissue vasculature and function, namely addressing tissue hypoxia and local and systemic insulin resistance. For the first time, dynamic contrast-enhanced magnetic resonance imaging and adipose tissue angiogenesis assay have been used to quantify rat periepididymal adipose tissue blood flow and angiogenesis, respectively. Additionally, the role of glyoxalase-1 activity in human visceral adipose tissue and systemic metabolism was assessed in a cohort of obese patients (diabetic and non-diabetic). The role of GLP-1-based therapies on adipose tissue glycation was studied in type 2 diabetic Goto-Kakizaki rats (GK) maintained with high-calorie diet and submitted to sleeve gastrectomy (surgical model) and liraglutide-treated GK rats (pharmacological model). The glyoxalase-1-dependent mechanisms in adipose tissue and systemic metabolic profile were studied, as well as the role of liraglutide in preventing methylglyoxal-induced impairment of angiogenesis and modulating glyoxalase-dependent angiogenesis in adipose tissue by matrigel plug and adipose tissue angiogenesis assays, respectively. Kruskal-Wallis test (all pairwise multiple comparisons) was applied, being p<0.05 considered as the criterion for significance. Results: For the first time, our group demonstrated that methylglyoxal supplementation to high-fat diet-induced obese rats impairs periepididymal adipose tissue capillarization and blood flow, causing increased periepididymal adipose tissue hypoxia and insulin resistance as well as systemic and muscle insulin resistance and glucose intolerance. In turn, the adipose tissue angiogenic assay showed decreased capillarization after dose-dependent methylglyoxal exposure and glyoxalase-1 inhibition. Insulin resistant patients have shown a progressive decline in pancreatic -cell and adipose tissue function, which were exacerbated in diabetic obese patients, including decreased serum HDL cholesterol and adiponectin and visceral adipose tissue glyoxalase-1 activity. In visceral adipose tissue from obese patients the activity of this enzyme was negatively correlated with HbA1c. Animal models of vertical sleeve gastrectomy and liraglutide treatment have shown restored periepididymal adipose tissue levels and activity of glyoxalase-1. Liraglutide-treated rats also showed increased levels of periepididymal adipose tissue angiogenic and vasoactive factors, and insulin signalling. Moreover, liraglutide prevented negative methylglyoxal-induced effects on matrigel plug assay, being also effective in increasing periepididymal adipose tissue angiogenesis in a glyoxalase-1-dependent manner. Conclusions: Methylglyoxal-induced glycation impairs periepididymal adipose tissue expandability and function, contributing to insulin resistance and to the onset of metabolically unhealthy obesity. Lower visceral adipose tissue glyoxalase-1 activity follows the progressive decline of adipose tissue function, and is negatively associated with metabolic dysregulation in obese patients. Furthermore, glyoxalase-1 is a target for GLP-1-based therapeutic approaches, improving periepididymal adipose tissue function and insulin sensitivity, preventing early metabolic dysregulation, metabolically unhealthy obesity and type 2 diabetes development.

L’obésité est une pathologie multifactorielle qui contribue au développement de l’insulinorésistance (l’IR). De nos jours, même si de nombreux travaux ont permis de répondre à plusieurs questions relatives à la pathogenèse de l’IR, les mécanismes cellulaires et moléculaires qui sous-tendent la pathogenèse de l’IR ne sont pas encore complètement élucidés chez l’Homme. Deux hypothèses principales se sont dégagées de ces travaux.D’une part, certaines études suggèrent que le défaut primaire à l’origine de l’IR se trouverait au niveau du tissu adipeux, qui en libérant dans la circulation systémique des adipokines, des cytokines inflammatoires et des acides gras contribue au développement d’une inflammation et d’une hyperlipidémie systémique. Ces deux paramètres exercent des effets délétères sur la sensibilité à l’insuline des autres organes comme le muscle et le foie.D’autre part, le muscle étant l’organe gluco-régulateur par excellence en situation postprandiale, d’autres études le place au cœur de la physiopathologie de l’IR.Malgré ces controverses, aucune étude n’a à ce jour exploré simultanément l’inflammation et l’IR systémique /tissulaire dans le muscle et le tissu adipeux chez un même sujet.Dans ce contexte, l’objectif de ce travail de thèse était de mieux comprendre les mécanismes impliqués dans la mise en place de l’IR. Il s’inscrit dans le cadre d’un projet de recherche translationnel, qui compare dans une cohorte de femmes ménopausées, des sujets minces à des sujets obèses de grade I insulinosensibles (OIS) et insulinorésistants (OIR). Plusieurs paramètres systémiques et tissulaires (lipotoxicité, inflammation et activation du récepteur toll like receptor 4 (TLR4)) impliqués dans la physiopathologie de l’IR ont été analysés chez ces sujets.Les résultats de cette étude mettent en avant l’importance de l’activation des voies de l’immunité innée dans la régulation de la sensibilité à l’insuline. Ainsi, alors qu’aucune inflammation systémique n’est détectée, on observe une activation différentielle des voies de signalisation du récepteur TLR4 de l’immunité innée entre le tissu musculaire et le tissu adipeux des sujets OIR. La voie MyD88-dépendante qui est une voie pro-inflammatoire, est activée dans le muscle squelettique de ces sujets et est associée à une IR au sein de ce tissu. A l’inverse, la voie TRIF-dépendante qui est une voie anti-inflammatoire est activée au sein du tissu adipeux et permet le maintien de la sensibilité à l’insuline. Ce maintien de la réponse à l’insuline se fait grâce l’induction du système interféron et de l’enzyme anti-oxydante manganèse superoxyde dismutase (MnSOD).Dans ce travail, nous montrons d’une part, que le défaut de réponse à l’insuline musculaire se trouve au cœur de la physiopathologie de l’IR chez les sujets obèses de grade I. D’autre part, ces résultats mettent en exergue l’importance de la balance de régulation des voies de l’immunité innée « MyD88 » et « TRIF » dans le maintien de la sensibilité à l’insuline
Obesity is a multifactorial disease promoting the development of insulin resistance (IR). Nowadays, cellular and molecular mechanisms underlying IR pathogenesis in humans are not fully understood although many studies have been attempted to improve our knowledge. Tow hypotheses arose from these researches.On one hand, several studies have led to the idea that a chronic inflammatory state combined with adipose tissue (AT) dysfunction could be the so-called “central mechanism” leading to IR. AT dysfunction is associated with increase in the release of inflammatory adipokines/cytokines and free fatty acid (FFA), leading to systemic inflammation and lipid overload. These latter parameters would have deleterious effects on diverse organs such as muscles and liver by affecting their insulin sensitivityOn the other hand, skeletal muscle (SM) is responsible for 80% of glucose uptake and metabolism in postprandial state and muscle failure in this function is often considered as the first defect causing IR.Interestingly, despite these controversies, in human, insulin sensitivity and the onset of inflammation have so far never been investigated simultaneously at systemic level and locally in SM and AT in the same individual.In this context, the aim of this thesis was to better understand the mechanisms involved in IR development in grade I obese subject. It is based on a translational research project that compares in a cohort of postmenopausal women, lean subjects with grade I obese insulin-sensitive (OIS) and insulin-resistant (OIR) subjects. Several systemic and tissue parameters (lipotoxicity, inflammation and toll like receptor 4 (TLR4) activation), involved in the IR pathophysiology were analyzed in these subjects.Our results highlight the importance of innate immunity pathways activation in the regulation of insulin sensitivity. Thus, while no inflammation was detected at the systemic level, there is a differential activation of innate immune TLR4 signaling pathways between muscle and AT of OIR subjects. The MyD88-dependent pathway, a pro-inflammatory pathway, is activated in their SM and is associated with IR in this tissue. Conversely, the TRIF-dependent pathway which is an anti-inflammatory pathway is activated in the AT thus maintaining insulin sensitivity in this tissue. This is supported by the induction of interferon system and the antioxidant enzyme manganese superoxide dismutase (MnSOD).In this work, we show that SM defect is the central mechanism which determines IR in grade I obese subject. We have also highlighted the importance of regulation of the balance between the two innate immunity pathways "MyD88" and "TRIF" in maintaining insulin sensitivity

Adipose tissue as an active endocrine organ is explored in a number of processes associated with metabolic disorders. This work is aimed on studying the effect of endoscopically implantable weight-reducing device - duodeno-jejunal bypass liner - on subclinical inflammation of adipose tissue in obese patients, which contributes to the development of type 2 diabetes mellitus. Specifically, for patients implanted with duodeno- jejunal bypass liner we determined anthropometric, biochemical and hormonal characteristics, and mRNA expression of subcutaneous adipose tissue proinflammatory (TNF-α, leptin, CCL- 2, CX3CL1, CD40, CD80, CD86, CD206, CD163 and IL-6) and anti-inflammatory genes (Adiponektin, IL-10) before, one month and ten months after the intervention. The implantation of duodeno-jejunal bypass liner significantly decreased body weight, waist circumference, and improved metabolic and glycemic control. In addition, C-reactive protein a highly sensitive indicator of inflammatory processes in the body was reduced ten months after implantation. In the case of mRNA expression of CD86 (a marker of activated B lymphocytes and macrophages) there was temporarily increase in adipose tissue one month after the surgery and the subsequent significant decrease after ten months. mRNA expression of other...

Chemerin is a chemoattractant adipokine that regulates adipogenesis and may induce insulin resistance. Chemerin serum concentrations are elevated in obese, insulin-resistant, and inflammatory states in vivo. Here we investigate the role of omental (OM) and subcutaneous (SC) adipose tissue chemerin and CMKLR1 messenger RNA (mRNA) expression in human obesity. In addition, we test the hypothesis that changes in chemerin serum concentrations are primarily associated with reduced body fat mass in the context of 3 weight loss intervention studies. Chemerin serum concentration was measured in 740 individuals in a cross-sectional (n = 629) study including a subgroup (n = 161) for which OM and SC chemerin mRNA expression has been analyzed as well as in 3 interventions including 12 weeks of exercise (n = 60), 6 months of calorie-restricted diet (n = 19) studies, and 12 months after bariatric surgery (n = 32). Chemerin mRNA is significantly higher expressed in adipose tissue of patients with type 2 diabetes mellitus and correlates with circulating chemerin, body mass index (BMI), percentage body fat, C-reactive protein, homeostasis model assessment of insulin resistance, and glucose infusion rate in euglycemic-hyperinsulinemic clamps. CMKLR1 mRNA expression was not significantly different between the 2 fat depots. Obesity surgery–induced weight loss causes a significant reduction on both OM and SC chemerin expression. All interventions led to significantly reduced chemerin serum concentrations. Decreased chemerin serum concentrations significantly correlate with improved glucose infusion rate and reduced C-reactive protein levels independently of changes in BMI. Insulin resistance and inflammation are BMI-independent predictors of elevated chemerin serum concentrations. Reduced chemerin expression and serum concentration may contribute to improved insulin sensitivity and subclinical inflammation beyond significant weight loss.

Au cours des dernières années, il est devenu évident que les sociétés des pays industrialisés sont à haut risque de maladies métaboliques. Une alimentation riche en énergie (lipide/glucide), combinée à une sédentarité accrue, est un facteur environnemental contribuant à l'augmentation de la prévalence de maladies reliées spécifiquement à des troubles endocriniens comme l'obésité et le diabète. Le traitement de ces désordres métaboliques doit donc passer par la connaissance et la compréhension des mécanismes moléculaires qui contrôlent ces désordres et le développement de traitements ciblés vers les facteurs responsables. Le tissu adipeux est une glande endocrine qui sécrète des substances, regroupées sous le terme d'adipokines, qui contrôlent l'homéostasie énergétique. L'augmentation de la masse adipeuse est responsable du développement de dérégulation hormonale qui mène à des dysfonctions physiologiques et métaboliques. Pour contrecarrer le développement démesuré du tissu adipeux, la signalisation insulinique ainsi que l’apport énergétique, responsables de la différenciation adipocytaire, doivent être inhibés. In vivo, la leptine, adipokine dont la concentration est corrélée à la masse adipeuse, présente des actions pro ou anti-insuliniques dans l’organisme pour réguler ce phénomène. Elle favorise l’effet inhibiteur de l’insuline sur la synthèse hépatique de glucose alors qu’elle s’oppose à son action sur l’expression des enzymes glucokinase et phosphoénol-pyruvate carboxykinase. La leptine influence aussi le taux circulant de triglycérides en diminuant sa concentration plasmatique. D'autre part, l'adiponectine, adipokine insulino- sensibilisante, voit sa sécrétion diminuée avec la prise de poids. La sensibilité à l'insuline est ainsi diminuée au fur et à mesure que le débalancement de ces deux adipokines s'accentue. La résistance à l'insuline s'installe alors pour s'opposer au stockage énergétique et à la prise illimitée de poids et la glycémie augmente. L'augmentation du glucose sanguin stimule la sécrétion d'insuline au niveau des cellules pancréatiques. C'est le diabète caractérisé par une hyperglycémie et une résistance à l'insuline. Le diabète, une des premières causes de mortalité dans le monde, est plus répandu sous sa forme non insulinodépendante (diabète de type 2, DT2) liée à l'obésité. Récemment, différents facteurs de transcription ont été identifiés comme régulateurs de l'expression d'une panoplie de gènes impliqués dans le métabolisme glucidique et lipidique. Parmi eux, les récepteurs des inducteurs de la prolifération des peroxysomes (PPAR, Peroxisome Proliferator-Activated Receptor), appartenant à la famille des récepteurs nucléaires. Les PPAR ont été démontrés comme ayant un rôle central dans le contrôle de la transcription des gènes codants pour des protéines impliquées dans le métabolisme : les adipokines. PPARg, en plus de son implication dans le contrôle de l'homéostasie glucidique et lipidique, est reconnu comme étant un facteur de transcription pivot régulant l'adipogenèse du fait de son expression majeure dans le tissu adipeux. D'autre part, il est bien établi maintenant que l'obésité et le diabète sont des facteurs contribuant au développement du processus inflammatoire vasculaire caractéristique de l’athérosclérose. En effet, les cellules endothéliales et musculaires lisses, principales composantes de la média de l’artère, sont très sensibles aux altérations métaboliques. Une diminution de la sensibilité à l’insuline entraine une réduction de la disponibilité du glucose et l’utilisation des acides gras comme alternatif par ces cellules. Ceci induit l’accumulation des acides gras oxydés dans l’intima et leur filtration dans la média pour former un core lipidique. Bien que l’induction de la dysfonction endothéliale soit impliquée très précocement, certaines études pointent l’accumulation lipidique dans les cellules musculaires lisses vasculaires (CML) et leur dysfonction comme déclencheurs de l’athérosclérose. Ce travail visait donc, dans un premier temps, à développer un modèle d'altérations métaboliques liées à la modulation de l'activité du tissu adipeux via une alimentation riche en lipides. Dans un second temps, cette étude tentait d'évaluer l’impact des adipocytes de souris sur les CML vasculaires et sur la modulation de leurs fonctions dans ce modèle d'altérations métaboliques et DT2 liés à l'alimentation et à l'obésité. Ainsi, par le biais de deux diètes pauvres en cholestérol à profil lipidique différent, nous avons développé un modèle murin présentant divers stades d'altérations du métabolisme allant jusqu'au DT2 en lien avec l'obésité chez les mâles et chez les femelles. D’autre part, des signes de cardiomyopathie ainsi qu’une modulation du taux des adipokines sont reliés à ces mêmes diètes. Parallèlement, l’activité de PPAR!2 est modulée chez les souris sous diètes enrichies en gras. Ensuite, nous avons démontré que les adipocytes, provenant de souris alimentées avec une diète enrichie en gras, modulaient la migration et la prolifération des CML comparativement au groupe contrôle. Ces modulations dépendaient en grande partie de la nature de la diète consommée, mais également du sexe de la souris. Par ailleurs, les altérations fonctionnelles des CML, couplées à des modulations géniques, sont associées aux changements du profil de sécrétion des adipokines mesurées chez les adipocytes. L’ensemble de ces travaux suggère une action directe de la nature de la stimulation du tissu adipeux blanc dans la modulation du profil de sécrétion des adipokines et l'induction du DT2 in vivo. Ces altérations de la physiologie adipocytaire se reflètent in vitro où le tissu adipeux contribue aux altérations physiopathologiques des CML liées au DT2. Ainsi, cette étude est l'une des premières à établir un lien direct entre les modulations adipocytaires et les effets de leurs sécrétions sur la physiologie des CML. Ces observations peuvent être exploitées cliniquement dans un développement futur d’outils thérapeutiques visant à prévenir et à traiter les troubles métaboliques et le DT2, en ciblant le tissu adipeux comme entité métabolique et endocrine.
Obesity is recognized as a risk factor to a variety of chronic diseases linked to the metabolic syndrome like atherosclerosis and type 2 diabetes (T2D), and is a major cause of increased risk of morbidity and mortality worldwide. High fat diets (HFD) coupled with sedentarity in the industrialized societies contribute to the raise of metabolic alterations prevalence specifically linked to endocrine troubles. Treatment of these latter should include the comprehension of the molecular mechanisms underlying these disorders in order to appropriately target factors responsible for the disease establishment. Adipose tissue is no longer considered as a passive organ which only stores lipids, but also works as an active gland that secretes several bioactive substances called adipokines. Among them, there are key factors known to play a pivotal role in the regulation of glucose and lipid homeostasis, lipid storage, adipogenesis. They are also recognized for their control of a wide range of cell type like adipocytes, hepatocytes and skeletal myocytes. Accumulation of adipose tissue in obesity, linked with the type as much as the amount of dietary lipids, is due to hyperplasia and hypertrophy of adipocytes. These changes are associated with modification in their secretion and inflammatory profile. To counteract excessive fat tissue development, insulin signalling known for its role in adipogenesis is inhibited. Thus, leptin is secreted by adipocytes to inhibit insulin action and the insulin sensitizer adipokine, adiponectin, is down regulated. The two factors are correlated to weight gain and their respective secretion profile is upregulated for leptin and down regulated for adiponectin. Insulin resistance is developed to prevent energetic storage and unlimited weight gain but glycemic control fails and glycaemia raises. Hyperglycaemia stimulates more insulin secretion, a characteristic of T2D linked to obesity. An estimated 80% of those who develop T2D are obese. Obesity induces important and complex changes, not only in glycemic homeostasis but also in the adipocytes. Following fatty acids (FA) stimulation, the main ligand-activated transcriptional factor that controls adipose tissue metabolism and adipokine secretion, peroxisome proliferator-activated receptor gamma (PPARg), is activated. This nuclear receptor subtype regroups two isoforms: PPARg1, expressed in many tissues (adipose tissue, muscle, heart and liver) where it controls glucose and lipid homeostasis, and PPARg2, the adipocyte’s specific form, which further governs preadipocyte differentiation, up-regulation of genes involved in lipogenesis and the expression of adipokines. Recent advances showed that increased FA and glycaemia trigger vascular alterations that lead to atherosclerosis. In fact, endothelial cells (EC) and smooth muscle cells (SMC), the main arterial components, are sensitive to metabolic alterations. A lack in insulin sensitivity, leading to lower glucose availability, forces arterial cells to use FA as alternative energy source. Thus, in atheroprone regions susceptible to plaque formation, EC and SMC are subjected to metabolic modifications that lead to oxidized low-density lipoprotein (oxLDL) accumulation in the intima and the progression of vascular disease. Many studies confirmed that the presence of SMC in the atherosclerotic plaque originates from the vascular wall but are showing a distinct phenotype. Even if the role of these cells in atherogenesis is not clear, trans-differentiation of SMC into foam cells has been reported in vitro. Thus, the present study aims at studying a HFD-induced obesity mouse model, developed to evaluate the impact of FA nature on the adipokine secretion profile of adipocytes. We also intended to determine gender-specific impact on modulation of metabolic disorders in response to those diets. On the other hand, we aim to determine the role of adipocytes in the development of obesity-linked atherosclerosis. For that, the second part of this study targeted the effect of adipocytes isolated from mice fed with HFD on SMC physiology. We focused our investigation on the effects of adipocytes regardless of the impact of other cell types in the adipose tissue. To reach our goal, we developed a HFD-fed mouse preparation demonstrating different stages of metabolic disorders leading to T2D. This model allowed us to generate adipocytes with different alteration status, reflected by the modulation of their adipokine secretion profile. Modifications in adipokine secretions were associated with PPAR!2 modulation. These results, reported in both genders, were delayed in female who expressed higher levels of estrogen receptor alpha (ER"). Then, the adipocytes were used to produce conditioned cultured media. To decipher the mechanistic contributions of HFD in adipokines modulation, the potential of adipocytes to induce SMC pathophysiologic disorders was evaluated in SMC stimulated by conditioned cultured media. This protocol enables the transposition of diet-induced fat cell modifications into extended alterations in the physiology of vascular SMC. These results strongly support pro-atherogenic effects of abdominal adipocytes on an important vascular component function through paracrine actions. Thus, adipocytes can be recognized as a link between the pathogenic potential of obesity and the impairments of SMC functions. A better understanding of the pathogenic effects of the adipose tissue on other tissues and organ systems might assist to develop better strategies in treating obesity- induced cardiovascular disease and metabolic syndrome.

(AJ) Adipose tissue produces numerous adipokines, pro- and antiinflammatory cytokines and hormones which may influence the development of insulin resistance, type 2 diabetes mellitus and other comorbidities of the metabolic syndrome. The ability of adipose tissue to store lipids and thus protect other organs and tissues from ectopic lipid accumulation and development of insulin resistance (IR) is largely dependent on the adipogenic potential of preadipocytes. The amount and function of these cells may be the key factor in creating "healthy" adipose tissue or on the contrary "unhealthy" adipose tissue eventually leading to metabolic derangements. The regulation of the amount of body fat by converting preadipocytes into mature adipocytes may be crucial in the prevention and treatment of obesity and its comorbidities. One of the reasons for development of insulin resistance can be the inhibition of the differentiation process of preadipocytes into mature adipocytes with consequent ectopic lipid accumulation caused by the secretion of preadipocyte factor - 1 (Pref - 1). Pref - 1 has been discovered recently as a protein produced by preadipocytes but not by mature adipocytes. Pref - 1 is a member of the protein family sharing similarity with epidermal growth factors which regulate the differentiation of...

Le cycle glycérolipides/acides gras libres (GL/FFA) est une voie métabolique clé qui relie le métabolisme du glucose et des acides gras et il est composé de deux processus métaboliques appelés lipogenèse et lipolyse. Le cycle GL/FFA, en particulier la lipolyse des triglycérides, génère diverses molécules de signalisation pour réguler la sécrétion d'insuline dans les cellules bêta pancréatiques et la thermogenèse non-frissonnante dans les adipocytes. Actuellement, les lipides provenant spécifiquement de la lipolyse impliqués dans ce processus sont mal connus. L’hydrolyse des triglycérides dans les cellules β est réalisée par les actions successives de la triglycéride lipase adipocytaire pour produire le diacylglycérol, ensuite par la lipase hormono-sensible pour produire le monoacylglycérol (MAG) et enfin par la MAG lipase (MAGL) qui relâche du glycerol et des acides gras. Dans les cellules bêta, la MAGL classique est très peu exprimée et cette étude a démontré que l’hydrolyse de MAG dans les cellules β est principalement réalisée par l'α/β-Hydrolase Domain-6 (ABHD6) nouvellement identifiée. L’inhibition d’ABHD6 par son inhibiteur spécifique WWL70, conduit à une accumulation des 1-MAG à longues chaines saturées à l'intérieur des cellules, accompagnée d’une augmentation de la sécrétion d'insuline stimulée par le glucose (GSIS). Baisser les niveaux de MAG en surexprimant ABHD6 dans la lignée cellulaire bêta INS832/13 réduit la GSIS, tandis qu’une augmentation des niveaux de MAG par le « knockdown » d’ABHD6 améliore la GSIS. L'exposition aiguë des monoacylglycérols exogènes stimule la sécrétion d'insuline de manière dose-dépendante et restaure la GSIS supprimée par un inhibiteur de lipases appelé orlistat. En outre, les souris avec une inactivation du gène ABHD6 dans tous les tissus (ABHD6-KO) et celles avec une inactivation du gène ABHD6 spécifiquement dans la cellule β présentent une GSIS stimulée, et leurs îlots montrent une augmentation de la production de monoacylglycérol et de la sécrétion d'insuline en réponse au glucose. L’inhibition d’ABHD6 chez les souris diabétiques (modèle induit par de faibles doses de streptozotocine) restaure la GSIS et améliore la tolérance au glucose. De plus, les résultats montrent que les MAGs non seulement améliorent la GSIS, mais potentialisent également la sécrétion d’insuline induite par les acides gras libres ainsi que la sécrétion d’insuline induite par divers agents et hormones, sans altération de l'oxydation et l'utilisation du glucose ainsi que l'oxydation des acides gras. Nous avons démontré que le MAG se lie à la protéine d’amorçage des vésicules appelée Munc13-1 et l’active, induisant ainsi l’exocytose de l'insuline. Sur la base de ces observations, nous proposons que le 1-MAG à chaines saturées agit comme facteur de couplage métabolique pour réguler la sécrétion d'insuline et que ABHD6 est un modulateur négatif de la sécrétion d'insuline. En plus de son rôle dans les cellules bêta, ABHD6 est également fortement exprimé dans les adipocytes et son niveau est augmenté avec l'obésité. Les souris dépourvues globalement d’ABHD6 et nourris avec une diète riche en gras (HFD) montrent une faible diminution de la prise alimentaire, une diminution du gain de poids corporel et de la glycémie à jeun et une amélioration de la tolérance au glucose et de la sensibilité à l'insuline et ont une activité locomotrice accrue. En outre, les souris ABHD6-KO affichent une augmentation de la dépense énergétique et de la thermogenèse induite par le froid. En conformité avec ceci, ces souris présentent des niveaux élevés d’UCP1 dans les adipocytes blancs et bruns, indiquant le brunissement des adipocytes blancs. Le phénotype de brunissement est reproduit dans les souris soit en les traitant de manière chronique avec WWL70 (inhibiteur d’ABHD6) ou des oligonucléotides anti-sense ciblant l’ABHD6. Les tissus adipeux blanc et brun isolés de souris ABHD6-KO montrent des niveaux très élevés de 1-MAG, mais pas de 2-MAG. L'augmentation des niveaux de MAG soit par administration exogène in vitro de 1-MAG ou par inhibition ou délétion génétique d’ABHD6 provoque le brunissement des adipocytes blancs. Une autre évidence indique que les 1-MAGs sont capables de transactiver PPARα et PPARγ et que l'effet de brunissement induit par WWL70 ou le MAG exogène est aboli par les antagonistes de PPARα et PPARγ. L’administration in vivo de l’antagoniste de PPARα GW6471 à des souris ABHD6-KO inverse partiellement les effets causés par l’inactivation du gène ABHD6 sur le gain de poids corporel, et abolit l’augmentation de la thermogenèse, le brunissement du tissu adipeux blanc et l'oxydation des acides gras dans le tissu adipeux brun. L’ensemble de ces observations indique que ABHD6 régule non seulement l’homéostasie de l'insuline et du glucose, mais aussi l'homéostasie énergétique et la fonction des tissus adipeux. Ainsi, 1-MAG agit non seulement comme un facteur de couplage métabolique pour réguler la sécrétion d'insuline en activant Munc13-1 dans les cellules bêta, mais régule aussi le brunissement des adipocytes blancs et améliore la fonction de la graisse brune par l'activation de PPARα et PPARγ. Ces résultats indiquent que ABHD6 est une cible prometteuse pour le développement de thérapies contre l'obésité, le diabète de type 2 et le syndrome métabolique.
The glycerolipid/ free fatty acid (GL/FFA) cycle is a key metabolic pathway that links glucose and fatty acid metabolism and it consists of lipogenesis and lipolysis. GL/FFA cycling, especially in its lipolysis arm, generates various lipid signaling molecules to regulate insulin secretion in pancreatic ß-cells and non-shivering thermogenesis in adipocytes. Currently, the lipolysis-derived lipid signals involved in this process are uncertain. Triglyceride hydrolysis in mammalian cells is accomplished by the sequential actions of adipose triglyceride lipase to produce diacylglycerol, by hormone sensitive lipase to produce monoacylglycerol (MAG) and by MAG lipase (MAGL) that releases free fatty acid and glycerol. Our work shows that in pancreatic ß-cell, the classical MAGL is poorly expressed and that MAG hydrolysis is mainly conducted by the newly identified α/β-Hydrolase Domain-6 (ABHD6). Inhibition of ABHD6 by its specific inhibitor WWL70, leads to long-chain saturated 1-MAG accumulation inside the cells, accompanied by enhanced glucose-stimulated insulin secretion (GSIS). Decreasing the MAG levels by overexpression of ABHD6 in the ß-cell line INS832/13 reduces GSIS, while increasing MAG levels by ABHD6 knockdown enhances GSIS. Acute exposure of INS832/13 cells to various MAG species dose-dependently stimulates insulin secretion and restores GSIS suppressed by the pan-lipase inhibitor orlistat. Also, various biochemical and pharmacological experiments show that saturated 1-MAG levels species rather than unsaturated or 2-MAG species best correlate with insulin secretion. Furthermore, whole-body and β-cell-specific ABHD6-KO mice exhibit enhanced GSIS in vivo, and their isolated islets show elevated MAG production and GSIS. Inhibition of ABHD6 in low dose streptozotocin diabetic mice restores GSIS and improves glucose tolerance. Results further show that ABHD6-accessible MAGs not only enhance GSIS, but also potentiate fatty acid and non-fuel-induced insulin secretion without alteration in glucose oxidation and utilization as well as fatty acid oxidation. We have identified that MAG binds and activates the vesicle priming protein Munc13-1, thereby inducing insulin exocytosis. Based on all these observations, we propose that lipolysis-derived saturated 1-MAG acts as a metabolic coupling factor to regulate insulin secretion and ABHD6 is a negative modulator of insulin secretion. Besides its role in ß-cells, ABHD6 is also highly expressed in adipocytes and its level is increased with obesity. Mice globally lacking ABHD6 on high fat diet (HFD) show modestly reduced food intake, decreased body weight gain, insulinemia and fasting glycemia and improved glucose tolerance and insulin sensitivity and enhanced locomotor activity. In addition, ABHD6-KO mice display increased energy expenditure and cold-induced thermogenesis. In accordance with this, these mice show elevated UCP1 level in white and brown adipocytes, indicating browning of white adipocytes. The browning phenotype is reproduced in the mice either chronically treated with the ABHD6 inhibitor WWL70 or an antisense oligonucleotides targeting ABHD6. White and brown adipose tissues isolated from whole body ABHD6 KO mice show greatly elevated levels of 1-MAG, but not 2-MAG. Increasing MAG levels by either exogenous administration of 1-MAG or ABHD6 inhibition or genetic deletion induces browning of white adipocytes in a cell-autonomous manner. Further evidence indicates that 1-MAGs can transactivate PPARα and PPARγ and the browning effect induced by WWL70 or exogenous MAG is abolished by PPARα and PPARγ antagonists. In vivo administration of the PPARα antagonist GW6471 to ABHD6 KO mice partially reversed the ABHD6-KO effects on body weight gain, and abolishes the enhanced thermogenesis, white adipose browning and fatty acid oxidation in brown adipose tissue. All these observations indicate that ABHD6 regulates not only insulin and glucose homeostasis but also energy homeostasis and adipose tissue function. Thus, ABHD6-accessible 1-MAG not only acts as a metabolic coupling factor to regulate fuel and non-fuel induced insulin secretion by activating Munc13-1 in beta cells, but also regulates glucose, insulin and energy homeostasis. The latter effects are mediated at least in part via browning of white adipocytes and enhanced brown fat function through the activation of PPARα and PPARγ. Collectively these findings suggest that ABHD6 is a promising target for developing therapeutics against obesity, type 2 diabetes and metabolic syndrome.

Innate lymphoid cells (ILCs) are a lymphocyte subset which lack adaptive antigen-specific receptors, being the innate counterparts of T helper lymphocytes. ILCs act early in the immune response by producing effector cytokines in response to tissue-derived inducer cytokines. ILC2s, a subtype of ILCs, produce type 2 cytokines in response to helminthic infection, allergens, and epithelial injury. Besides their role in immune defense, ILC2s also contribute to metabolic homeostasis by maintaining an anti-inflammatory protective environment in the adipose tissue (AT). In obesity, excessive lipid accumulation results in chronic low-grade inflammation, causing a shift in immune cell populations that can lead to a systemic metabolic imbalance, known as metabolic syndrome. ILC2-derived molecules act on immune cells and on adipocytes, limiting obesity-induced inflammation and lipid accumulation, respectively. However, how ILC2s perceive environmental cues and integrate signals to maintain AT homeostasis remains poorly understood. Here, we hypothesize that neuroimmune interactions can control ILC2 function in the AT downstream from sympathetic nervous system innervation. Using pharmacological, genetic and imaging approaches, we show that AT ILC2s can integrate neuroregulatory molecules to control AT physiology.

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
A diabetes tipo 2 é um distúrbio metabólico multifatorial que se caracteriza por uma deficiência absoluta ou relativa da insulina associada ao fator insulino-resistência, o que conduz a complicações vasculares e a sua prevalência está a aumentar em todo o mundo. Esta patologia surge associada à obesidade e há evidências de uma relação recíproca entre a resistência à insulina e a disfunção endotelial. A função do endotélio vascular encontra-se comprometida nas doenças cardiovasculares, na obesidade e na diabetes tipo 2, o que leva à disfunção endotelial que se traduz na incapacidade do endotélio manter a homeostase vascular e é caracterizada principalmente pela biodisponibilidade insuficiente de óxido nítrico (NO).O tecido adiposo perivascular (PVAT) liberta uma grande variedade de moléculas biologicamente ativas que têm influência na vasculatura. Em condições normais, o PVAT exerce funções vasodilatadoras e anti-inflamatórias, porém, na obesidade, devido à inflamação do PVAT, caracterizada pelo desequilíbrio entre as células pró e anti-inflamatórias, há indução da secreção anormal de adipocinas e produção de espécies reativas de oxigénio (ROS).O propagermanio possui uma potente atividade moduladora imune associada a propriedades anti-inflamatórias e sabe-se que este anti-inflamatório inibe o recetor de quimiocinas C-C 2 (CCR2) e suprime a infiltração de macrófagos/monócitos. Contudo, não existe nenhum estudo a abordar as potencialidades deste composto em termos vasculares e perivasculares. Este trabalho teve como objetivo estudar o papel do tecido adiposo perivascular e a eficácia terapêutica do propagermanio na disfunção endotelial associada à diabetes tipo 2, bem como o seu impacto perivascular.Para isso foram avaliados dois modelos animais diferentes: como modelo de diabetes tipo 2, ratos machos Goto-Kakizaki (GK) e como modelo controlo, ratos machos saudáveis Wistar (W). Os ratos GK foram divididos em quatro grupos de estudo: grupo controlo (GK C0); grupo controlo tratado com 50 mg/kg de propagermanio (GK CT); grupo de animais alimentados com uma dieta rica constituída por 50 Kcal % de gordura (GK H0) e ainda um grupo de animais alimentados com uma dieta rica em gordura e tratado com 50mg/kg de propagermanio (GK HT). O propagermanio foi administrado por via oral, durante 3 meses. Foram avaliados diversos parâmetros in vivo como o peso corporal, o comprimento nasoanal (para o cálculo do Índice de Lee), o perfil lipídico (níveis sistémicos de colesterol total e triglicerídeos), os níveis de glicose em jejum, a tolerância à glicose e a sensibilidade à insulina (através de provas de tolerância à glicose e à insulina, respetivamente). Ao nível vascular foi estudado ex vivo o relaxamento dependente e independente do endotélio e a contração do endotélio em resposta à endotelina-1. Por fim, foram calculados os níveis de hemoglobina A1c.Verificámos que a administração do propagermanio, por via oral, não interferiu no peso dos animais e não alterou o índice de Lee, no entanto, melhorou os níveis da glicose em jejum e a resistência à insulina, não alterou os níveis de colesterol total e triglicerídeos, não teve um efeito significativo na prova de tolerância à glicose intraperitoneal, melhorou a sensibilidade à insulina e a disfunção endotelial e recuperou o efeito anti-contrátil do tecido adiposo perivascular.A presença do propagermanio melhorou a disfunção endotelial e recuperou o fenótipo vasodilatador do tecido adiposo perivascular muito provavelmente devido à sua ação anti-inflamatória. Assim, o tecido adiposo perivascular está envolvido na regulação da função endotelial e surge como um potencial alvo terapêutico para a disfunção vascular relacionada com a diabetes tipo 2.
Type 2 diabetes is a multifactorial metabolic disorder characterized by an absolute or relative insulin deficiency associated with insulin resistance factor, leading to vascular complications and its prevalence is increasing worldwide. This pathology is associated with obesity and there is evidence of a reciprocal relationship between insulin resistance and endothelial dysfunction. Vascular endothelial function is compromised in cardiovascular diseases, obesity and type 2 diabetes, leading to endothelial dysfunction resulting in the inability of the endothelium to maintain vascular homeostasis and is mainly characterized by insufficient bioavailability of nitric oxide (NO).Perivascular adipose tissue (PVAT) releases a wide variety of biologically active molecules that have influence on the vasculature. In normal conditions, PVAT exerts vasodilatory and anti-inflammatory functions, but in obesity due to inflammation of PVAT, characterized by imbalance between pro and anti-inflammatory cells, there is induction of abnormal adipokine secretion and reactive oxygen species (ROS) production.Propagermanium has potent immune modulating activity associated with anti-inflammatory properties and it is known that this anti-inflammatory inhibits the C-C 2 chemokine receptor (CCR2) and suppresses macrophage/monocyte infiltration. However, there is no study addressing the potential of this compound at vascular and perivascular level. This work aimed to study the role of perivascular adipose tissue, the therapeutic efficacy of propagermanium in endothelial dysfunction associated with type 2 diabetes and its perivascular impact.Two different animal models were evaluated: as a type 2 diabetes model, Goto-Kakizaki (GK) male rats and as a control model, Wistar (W) healthy male rats. GK rats were divided into four study groups: control group (GK C0); group treated with 50 mg/kg of propagermanium (GK CT); group of animals fed a rich diet consisting of 50 Kcal% of fat (GK H0) and a group of animals fed a high fat diet and treated with 50 mg/kg of propagermanium (GK HT). The propagermanium was given orally for 3 months. Several in vivo parameters such as body weight, nasoanal length (for the Lee Index), lipid profile (total cholesterol and triglyceride systemic levels), fasting glucose levels, glucose tolerance and insulin sensitivity (through glucose and insulin tolerance tests, respectively). At the vascular level, independent and endothelium-dependent relaxation and endothelial contraction were studied ex vivo in response to endothelin-1. Finally, the levels of hemoglobin A1c were calculated.We found that oral administration of propagermanium didn’t interfere with animal weight and didn’t change Lee's index, however, it improved fasting glucose levels and insulin resistance, it didn’t change total cholesterol and triglyceride levels, it didn’t have a significant effect on the intraperitoneal glucose tolerance test, improved insulin sensitivity and endothelial dysfunction, and recovered the anti-contractile effect of perivascular adipose tissue.The presence of propagermanium improved endothelial dysfunction and recovered the vasodilating phenotype of perivascular adipose tissue probably due to its anti-inflammatory features. So, perivascular adipose tissue is involved in the regulation of endothelial function and appears as a potential therapeutic target for vascular dysfunction related to type 2 diabetes.
Outro - COMPETE: POCI-01-0145-FEDER-016784
Outro - FCT: PTDC/BIM-MET/4447/2014

(EN) GH/IGF-1 axis components (GH, growth hormone receptor (GH-R), IGF-1, IGF-1 receptor (IGF-1R), IGF-binding proteins (IGFBPs)) participate in the control of glucose metabolism, inflammatory processes as well as cell proliferation and differentiation, including adipocytes and monocytes. The aim of the present study was to evaluate the role of local mRNA expression of GH/IGF-1 axis components in subcutaneous adipose tissue (SCAT) and peripheral monocytes (PM) in the development of insulin resistance and differences of adipose tissue mass in following groups of patients: obese females with and without type 2 diabetes mellitus and subjects with active untreated acromegaly. A total number of 66 subjects were included in the study: obese females without type 2 diabetes mellitus (OB), obese females with type 2 diabetes mellitus (T2DM), acromegalic patients (AC) and healthy lean control subjects (C). T2DM underwent 2 weeks of very-low- calorie diet (VLCD - energy content 2500 kJ/day). According to our results we suggest that decreased mRNA expression of IGF-1, IGF-1R, IGFBP-2 and IGFBP-3 in adipose tissue of T2DM subjects may contribute to changes of fat differentiation capacity and the increased IGF-1R mRNA expression in peripheral monocytes in these patients may play a role in the regulation of...

Le diabète de type 2 (DT2) est une maladie chronique affectant 3 millions de canadiens. Une augmentation progressive de la résistance à l'insuline (RI) et de la sécrétion d'insuline est observée chez des sujets normoglycémiques bien avant la survenue du DT2. La moindre fonction du tissu adipeux blanc (TAB) est centrale dans le développement du DT2 car elle accroît le flux d'acides gras vers les tissus périphériques, y induisant la RI, l'hyperinsulinémie et l’inflammation chronique. Durant ma maîtrise, nous avions démontré que les lipoprotéines de basses densité (LDL) natifs réduisent la différentiation et la fonction des adipocytes et induisent la dysfonction du TAB humain. De plus, nous avions montré qu'un taux plasmatique élevé d'apolipoprotéine B (apoB), indiquant un nombre élevé d'apoB-lipoprotéines dont principalement les LDL, est associé à la RI, la sécrétion d'insuline gluco-stimulée (SIGS) élevée, la clairance plasmatique retardée des gras alimentaires et la moindre fonction du TAB chez 81 sujets obèses non diabétiques. Afin de déterminer si l'apoB plasmatique permet aussi d'identifier les sujets obèses répondant le mieux à une diète hypocalorique en termes de réduction des facteurs de risque du DT2, nous avons testé l'effet d'une intervention hypocalorique de six mois. Parmis 59 sujets qui ont terminés l’intervention, nous avons mesuré une diminution de la SIGS et une amélioration de la fonction du TAB seulement chez les sujets avec apoB plasmatique élevée. Toutefois, les mécanismes de ces effets délétères possibles des apoB-lipoprotéines n'avaient pas été explorés. Des évidences suggèrent que l'activation chronique de l'inflammasome Nucleotide- binding domain and Leucine-rich repeat Receptor containing a Pyrin domain 3 (NLRP3) et la sécrétion d'interleukine-1b (IL-1b) promeuvent la dysfonction du TAB et la RI systémique. Cependant, les signaux métaboliques induisant l'inflammasome NLRP3 dans le TAB humain sont inconnus. Afin de tester si l'activation de l'inflammasome NLRP3/système IL-1b participe au mécanisme précédemment identifié liant les apoB-lipoprotéines et les facteurs de risque du DT2, nous avons investigué l'association et l'effet direct des apoB-lipoprotéines sur le système IL-1b. Nous avons démontré chez 81 sujets obèses non-diabétiques que les individus avec apoB plasmatique élevée montrent un taux élevé d'antagoniste du récepteur à l'IL-1 (IL-1Ra) plasmatique, un marqueur de l'activation systémique de la voie IL-1b. Aussi, les associations entre l'apoB plasmatique élevée et la RI et SIGS étaient statistiquement dépendantes des niveaux d'IL-1Ra plasmatique. Dans une autre population de 32 sujets, nous avons démontrés que ceux avec apoB plasmatique élevée ont une sécrétion augmentée d'IL-1b par le TAB ex vivo. Les relations entre l'apoB plasmatique, la clairance plasmatique retardée des gras alimentaires et la sécrétion de C-peptide glucostimulée étaient statistiquement dépendantes de la sécrétion d'IL- 1b du TAB. Puis, les LDL natifs ajoutés au TAB ex vivo induisaient la sécrétion d'IL-1b, y agissant en tant que signaux d'amorçage (1er signal de l'inflammasome NLRP3/système IL-1b). En conclusion, ces résultats suggèrent que les LDL natifs, forme principale d'apoB- lipoprotéines, régulent positivement l'inflammasome NLRP3 du TAB humain. Ceci pourrait expliquer la dysfonction du TAB, l'hyperinsulinémie et l'incidence élevée du DT2 présents chez les sujets avec apoB plasmatique élevée. En outre, ils suggèrent que l'apoB plasmatique élevée pourrait être un biomarqueur permettant d'identifier les sujets obèses qui répondraient le mieux à la diète hypocalorique afin de réduire le risque de DT2.
Type 2 diabetes (T2D) is chronic disease affecting 3 million Canadians and a new case is diagnosed every 3 minutes in Canada. Long before the onset of T2D, a progressive increase in insulin resistance (IR) and insulin secretion is observed in normoglycemic subjects. A decreased white adipose tissue (WAT) function is central to the development of T2D as it promotes an increased fatty acid flux to peripheral tissues, inducing IR, hyperinsulinemia and chronic inflammation. During my MSc, we reported that low density lipoproteins (LDL) reduce the differentiation and function of adipocytes and induce the dysfunction of human WAT. Moreover, we showed that elevated plasma apolipoprotein B (apoB), indicating high numbers of circulating apoB-lipoproteins mainly in the form of LDL, is associated to IR, elevated glucose-induced insulin secretion (GIIS), delayed postprandial plasma clarance of fat and reduced WAT function in 81 non-diabetic obese subjects. To explore whether apoB also identifies obese subjects who best respond to weight loss to reduce risk factors for T2D, we tested the effect of a 6 months hypocaloric diet. We showed in the 59 completers of the hypocaloric intervention that the decrease in GIIS and increase in WAT function were significant in subjects with high plasma apoB but not in subjects with low plasma apoB. However, the mechanism underlying the negative effects apoB-lipoproteins was yet unexplored. Chronic activation of the Nucleotide-binding domain and Leucine-rich repeat Receptor containing a Pyrin domain 3 (NLRP3) inflammasome and secretion of interleukin-1b (IL-1b) promote WAT dysfunction and systemic IR. However, endogenous metabolic signals that induce the activation of WAT NLRP3 inflammasome are unknown. To test if the activation of the NLRP3 inflammasome/ IL-1b system is an underlying mechanism linking apoB- lipoproteins to risk factors for T2D, we examined the association and direct effect of apoB- lipoproteins on the IL-1b system. We observed in our cohort of 81 non-diabetic obese subects that subjects with high plasma apoB have higher plasma IL-1 receptor antagonist (IL-1Ra), which is an marker of systemic activation of the Il-1b pathway. Furthermore, the associations between high plasma apoB and IR and GIIS were statistically dependent on plasma IL-1Ra. Additionnaly, in a separate population of 32 subjects, we demonstrated that subjects with high plasma apoB have higher ex vivo WAT IL-1b secretion. The relation between plasma apoB and delayed postprandial plasma fat clearance and elevated glucose-induced C-peptide secretion were statistically dependent on WAT IL-1b secretion. Finally, native LDLs directly induce IL- 1b secretion from ex vivo WAT, acting primarily as priming signals (i.e. the first signal leading to activation of the NLRP3 inflammasome/ IL-1b system). In conclusion, the findings from this thesis suggest that native LDL, the main form of apoB-lipoproteins, upregulate human WAT NLRP3 inflammasome. This may explain WAT dysfunction, hyperinsulinemia and higher incidence of T2D in subjects with high plasma apoB. Moreover, they suggest that high apoB may serve as biomarker to identify obese subjects who best respond to a hypocaloric-intervention to reduce the risk of T2D.