Résumé : Au Canada, un tiers des enfants souffrent d’embonpoint ou sont obèses. Les événements survenant au cours de la vie intra-utérine jouent un rôle important dans la détermination de la susceptibilité des enfants à développer des maladies liées au métabolisme énergétique comme l’obésité et le diabète. L’épigénétique pourrait expliquer en partie ce risque à long terme. Mon projet de maîtrise s’appuie sur les résultats d’une approche à l’échelle du génome qui a pour objectif d’identifier les changements épigénétiques (méthylation de l’ADN), chez les nouveau-nés, associés à une exposition fœtale à l’obésité maternelle ou au diabète gestationnel (DG). Cette stratégie a permis d’identifier le gène PRDM16 qui joue un rôle clé dans le développement du tissu adipeux brun (BAT). Un déficit dans la fonction du BAT résulte en une accumulation de lipides dans le tissu graisseux ce qui est associé au développement de maladies métaboliques. Notre hypothèse est que le profil de méthylation de l’ADN de PRDM16 et d’autres gènes centraux impliqués dans la genèse du BAT est perturbé par une exposition aux débalancements métaboliques associés à l’obésité maternelle et au DG. Cette étude inclut 133 femmes et leur nouveau-né recrutés dans la région du Saguenay-Lac-St-Jean. Les données anthropométriques et métaboliques des femmes ont été récoltées à la fin de chaque trimestre de grossesse. Une hyperglycémie orale provoquée (HGOP, 75g), entre la 24e et la 28e semaine de grossesse, a permis d’établir le diagnostic de DG selon les critères de l’OMS. À la naissance, des biopsies de placenta ont été recueillies ainsi que les données anthropométriques et métaboliques pour chaque enfant. Les niveaux de méthylation de l’ADN des gènes PRDM16, PPARGC1α, BMP7 et CTBP2 dans le placenta fœtal ont été mesurés par pyroséquençage de l’ADN traité au bisulfite de sodium. Les résultats ont montré que le DG était associé à une méthylation de l’ADN du gène BMP7 plus faible dans le placenta des nouveau-nés exposés au DG contrairement à ceux non exposés. Les niveaux de méthylation des gènes BMP7, PPARGC1α et PRDM16 étaient corrélés au statut glycémique maternel au 2e trimestre de grossesse et expliquaient une partie des niveaux de leptine dans le sang de cordon ombilical. Ces résultats suggèrent que la méthylation des gènes associés à la genèse du BAT est affectée par le DG. Ces résultats suggèrent également que le métabolisme énergétique des enfants exposés au DG est altéré ce qui pourrait mener au développement de troubles métaboliques plus tard dans la vie comme l’obésité et le diabète de type 2. // Abstract : In Canada, one third of children are overweight or obese. The events occurring during the intrauterine life play an important role in determining the susceptibility of children to develop diseases related to energy metabolism such as obesity and diabetes. Epigenetic changes can to explain this long-term risk. My project is build on the results of an epigenome-wide aiming to identify epigenetic changes (DNA methylation) in newborns associated with fetal exposure to maternal obesity and gestational diabetes (GD). This strategy has identified the PRDM16 gene which plays a key role in the development of brown adipose tissue (BAT). A deficiency in the function of BAT results in accumulation of lipids in the adipose tissue that is associated with the development of metabolic diseases. Our hypothesis is that the profile of DNA methylation at PRDM16 gene and other genes involved in BAT genesis are disrupted by exposure to metabolic dysregulation associated with maternal obesity and GD. This study included 133 women and their newborn recruited in the Saguenay-Lac-St-Jean region. Anthropometric and metabolic data of the women were collected at the end of each trimester of pregnancy. An oral glucose tolerance test (OGTT, 75g), between the 24th and 28th week of pregnancy, resulted in the diagnosis of GD according to the WHO criteria. At birth, the fetal placenta biopsies were collected and anthropometric and metabolic data for each newborn. DNA methylation levels of PRDM16, PPARGC1α, BMP7 and CTBP2 genes in fetal placenta were measured by pyrosequencing of sodium bisulfite treated DNA. The results showed that GD was associated with lower DNA methylation of BMP7 gene in the placenta of newborns exposed to GD in contrast to those not exposed. DNA methylation levels of BMP7, PPARGC1α and PRDM16 genes were correlated with maternal glycemic status in the 2nd trimester of pregnancy and partially explained leptin level variability in cord blood . These results suggest that DNA methylation of genes associated with BAT genesis is affected by GD. Therefore, energy metabolism of children exposed to GD could be altered and thus leading to the development of metabolic disorders such as obesity and type 2 diabetes later in life .

Intra-adipose cortisol is derived from the systemic circulation via the hypothalamic-pituitaryadrenal axis (HPAA) and generated locally through conversion of inactive cortisone to cortisol by the intra-cellular enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1). This thesis addresses the relative contributions of the HPAA and adipose tissue 11βHSD1 to the adipose tissue glucocorticoid pool and describes development and validation of a novel stable isotope tracer, 1,2 [2H]2-cortisone (d2-cortisone), to measure 11βHSD1- dehydrogenase activity in adipose tissue and skeletal muscle in vivo. In otherwise healthy females (n=6) undergoing hysterectomy for a benign indication, an intravenous infusion of d4-cortisol was administered and subcutaneous and omental adipose tissue biopsies were obtained along with concomitant peripheral venous blood, to measure the rate of exchange of cortisol between plasma and adipose tissue for comparison with rates of intra-cellular cortisol generation by 11βHSD1. Cortisol concentrations and enrichment with d4-cortisol were lower in adipose tissue than in plasma. The rate of accumulation of d4-cortisol in adipose tissue depots was ~0.5nmol/kg/h despite the infusion contributing 1.9μmol/h d4-cortisol into the circulation, and the proportion of the intra-adipose cortisol pool replaced each hour was ~10%. The contribution of 11βHSD1 to this turnover could not be quantified since very little substrate d3-cortisone accumulated in adipose during infusion. Method development for d2-cortisone included optimising LC-MS/MS conditions, confirming that d2-cortisone was a substrate for human 11βHSD1 and that no significant primary isotope effect existed. The pharmacokinetics of d2-cortisone were assessed in vivo in healthy male volunteers (n=3). The method was validated by measuring whole body cortisone production in healthy volunteers (n=3) before and after eating liquorice which resulted in a ~50% fall in cortisone production. 11βHSD1-dehydrogenase activity was measured in adipose tissue and skeletal muscle in healthy volunteers (n=6) using d2- cortisone and substantial 11β-dehydrogenase activity was present in both tissues (~1.5-fold higher 11β-dehydrogenase activity than 11β-reductase activity in adipose tissue and approximately equal 11β-reductase and 11β-dehydrogenase activity in skeletal muscle). 11βHSD1-reductase activity was also assessed using a 9,11,12,12 [2H]4-cortisol infusion (d4-cortisol). Skeletal muscle and adipose tissue displayed 11β-reductase activity. In adipose tissue this activity was of a similar magnitude to previous reports. Insulin increased whole body 11β-reductase activity, but did not switch 11βHSD1 direction in muscle or adipose tissue, indicating the predominant effect of insulin may be on hepatic 11βHSD1. Therefore, turnover of the intra-adipose tissue glucocorticoid pool is slow and it is unlikely that rapid acute fluctuations in circulating cortisol are reflected in adipose tissue, although this has not been confirmed under normal physiological conditions. Secondly, 11βHSD1 may be bidirectional in human subcutaneous adipose tissue and skeletal muscle in vivo, and insulin does not regulate the balance of activities. However, in this study blood sampling occurred from blood vessels which express 11βHSD2, and thus some of the measured dehydrogenase activity in this study may reflect endothelial 11βHSD2 activity. Together these findings further our understanding of adipose tissue cortisol physiology in health, suggesting that 11βHSD1 may play a relatively important role in modulating activation of glucocorticoid receptors in adipose tissue, and that dysregulation or inhibition of 11βHSD1 may affect cortisol inactivation as well as regeneration.

Inflammation plays a key role in the underlying pathogenesis of obesity and its associated health risks, with increased markers of inflammation evident in both liver and adipose tissue. In parallel, there is dysregulation of glucocorticoid metabolism in obesity, with increased adipose levels of the glucocorticoid-regenerating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and increased hepatic levels of 5α-reductase type 1 (5αR1), which catalyses the reduction of glucocorticoids. Both the mechanisms and consequences of this glucocorticoid metabolism dysregulation remain unclear, however, there is evidence that it may be related to inflammation. In vitro studies have demonstrated that pro-inflammatory markers upregulate 11βHSD1 expression in adipocytes, potentially explaining increased expression of this enzyme in obesity. Previous work has also demonstrated that the glucocorticoid metabolites produced by 5αR1 lack the metabolic effects of the parent glucocorticoid, but retain its anti-inflammatory properties, indicating that increased expression of hepatic 5αR1 may serve to dampen down inflammation in the liver. The hypotheses addressed in this thesis are that in obesity, inflammation regulates adipose glucocorticoid metabolism through 11βHSD1, and that hepatic glucocorticoid metabolism regulates the inflammatory state of the liver through 5αR1. The role of inflammation in the regulation of 11βHSD1 was assessed in vivo in mice treated with the anti-inflammatory compound sodium salicylate (salicylate). In diet-induced obese mice, salicylate downregulated 11βHSD1 expression and activity selectively in visceral adipose tissue, alongside improved glucose tolerance, reduced plasma non-esterified fatty acids, and changes in adipose lipid metabolism. 11βHSD1-deficient mice fed a high-fat diet were resistant to the insulin sensitising effects of salicylate treatment. These results indicate a novel role for 11βHSD1 down-regulation in mediating the insulin sensitising effect of anti-inflammatory treatment. The mechanisms underpinning the anti-inflammatory properties of 5α-reduced glucocorticoids were explored in vitro and in vivo. In lipopolysaccharide-stimulated murine macrophages, both 5α-reduced glucocorticoid metabolites tested, namely 5α-dihydrocorticosterone (5αDHB) and 5α-tetrahydrocorticosterone (5αTHB), suppressed tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) release, although to a lesser extent than corticosterone (B). Similar to B, both 5αDHB and 5α THB suppressed phosphorylation of intra-cellular inflammatory signalling mitogen-activated protein kinases (MAPK) proteins c-Jun N-terminal kinase (JNK) and p38, as well as increasing protein expression of MAPK phosphatase-1 (MKP-1). Treatment of phorbol ester-stimulated HEK293 kidney cells with these 5α-metabolites revealed that 5αDHB suppressed nuclear factor κB (NFκB) and activator protein-1 (AP-1) activation to a similar extent to that of B, whilst 5αTHB increased activation of these pro-inflammatory transcription factors, indicating cell-specific effects of 5αTHB. In conclusion, reduced intra-adipose glucocorticoid regeneration by 11βHSD1 mediates the insulin sensitising effects of salicylate, suggesting that altered glucocorticoid metabolism may reflect altered intra-adipose inflammation in obesity. Furthermore, these data support the concept that this enzyme provides a therapeutic target in obesity-related metabolic disorders. 5α-reduced metabolites of glucocorticoids have similar anti-inflammatory properties to the parent glucocorticoid, indicating that the elevated hepatic levels of 5α-reductase in obesity may be a protective mechanism to limit the adverse metabolic effects of glucocorticoids upon the liver, but maintain the beneficial anti-inflammatory properties. These 5α-reduced glucocorticoid metabolites may provide a potential therapeutic treatment as selective glucocorticoid receptor modulators for inflammatory conditions.

Image-based quantifications of visceral adipose tissue (VAT) volumes from segmented VAT areas are increasingly considered for risk assessment in obese patients. The goal of this study was to determine the power of partial VAT areas to predict total VAT volume in morbidly obese patients (BMI > 40 kg/m2) as a function of gender, age and anatomical landmarks. 130 morbidly obese patients (mean BMI 46.5 kg/m2; 94 females) underwent IRB-approved MRI. Total VAT volumes were predicted from segmented VAT areas (of single or five adjacent slices) at common axial landmark levels and compared with the measured ones (VVAT-T, about 40 slices between diaphragm and pelvic floor). Standard deviations σ1 and σ5 of the respective VAT volume differences served as measures of agreement. Mean VVAT-T was 4.9 L for females and 8.1 L for males. Best predictions were found at intervertebral spaces L3-L4 for females (σ5 = 688 ml, σ1 = 832 ml) and L1-L2 for males (σ5 = 846 ml, σ1 = 992 ml), irrespective of age. In conclusion, VAT volumes in morbidly obese patients can be reliably predicted by multiplying the segmented VAT area at a gender-specific lumbar reference level with a fixed scaling factor and effective slice thickness.

The aim of this thesis was to investigate the effects of dietary fatty acids on body composition and ectopic fat in humans, with emphasis on the role of the omega-6 polyunsaturated fatty acid (PUFA) linoleic acid (18:2n-6) and the saturated fatty acid (SFA) palmitic acid (16:0). The overall hypothesis was that linoleic acid would be beneficial compared with palmitic acid during overfeeding, as previously indicated in animals. Papers I, II and IV were double-blinded, randomized interventions in which different dietary fats were provided to participants and Paper III was a cross-sectional study in a community-based cohort (PIVUS) in which serum fatty acid composition was assessed as a biomarker of dietary fat intake. In Paper I, overfeeding with sunflower oil (n-6 PUFA) for 7 weeks caused less accumulation of liver fat, visceral fat and total body fat (as assessed by MRI) compared with palm oil (SFA) in young and lean subjects despite similar weight gain among groups. Instead, sunflower oil caused a larger accumulation of lean tissue. In Paper II, plasma from Paper I was analyzed with NMR-based metabolomics, aiming to identify metabolites differentially affected by the two dietary treatments. Acetate decreased by PUFA and increased by SFA whereas lactate increased by PUFA and decreased by SFA. In Paper III, the proportion of linoleic acid in serum was inversely associated with contents of visceral-, subcutaneous- and total body adipose tissue whereas the proportion of palmitic acid was directly associated with visceral- and total body adipose tissue in 70-year old men and women. In Paper IV, overfeeding with sunflower oil for 8 weeks caused less accumulation of liver fat compared with palm oil also in overweight and obese subjects. SFA increased visceral fat in men only. Accumulation of lean tissue was similar between groups. In conclusion, SFA (palmitic acid) from palm oil promotes marked liver fat accumulation in both normal-weight and overweight/obese subjects during overeating, whereas n-6 PUFA (linoleic acid) from sunflower oil prevents such liver fat accumulation. Diverging effects of SFA and PUFA on visceral adipose tissue and lean tissue may only be applicable in some groups and/or circumstances. These results imply that negative effects associated with weight gain (e.g. fatty liver) may be partly counteracted by the type fat in the diet, overall supporting a beneficial role of diets higher in unsaturated fat compared with saturated fat for preventing liver fat accumulation.

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction: Nitric oxide (NO) is an important chemical messenger in the cardiovascular system. Despite considerable progress in this field, there remains an on-going need for affordable and user-friendly NO measurement techniques. Therefore, in this study we aimed to develop and characterise NO-detection techniques not previously used in our laboratory, and, in addition, characterise an ex vivo method to measure the functional effects of the endothelium and NO production in the vasculature. Methods: Three different NO-detection techniques were compared: (i) Amperometric NO sensors. Here, NO-increasing effects of known NO synthase (NOS) activators were investigated (insulin, acetylcholine and biosynthetic human insulin). Three different NO sensors were evaluated on cultured endothelial cells and aortic tissue. Putative NOincreasing effects of shear stress were also investigated; (ii) Nitrite (NO2 -) + nitrate (NO3 -) sensors. Here, I aimed to measure NO release from cultured endothelial cells; (iii) Colorimetric NO2 - measurement assay with the Griess reagent. Here, NO2 - production by endothelial cells was measured with a plate reader. In the second part of the study an organ bath - isometric tension technique was established to measure endothelium-dependent function of aortic rings. Functional differences in aortic rings isolated from diet-induced obese rats compared to lean rats were investigated. Ring contraction was induced with phenylephrine and relaxation with acetylcholine. These investigations were further supported by western blot analyses of selected critical proteins. Lastly, the effects of perivascular adipose tissue (PVAT) on contraction and relaxation were investigated in endothelium-containing or denuded aortic ring segments. Results: Although some success was achieved with the amperometric sensors regarding calibration, any experimental results obtained were difficult to repeat due to instability of the sensors. With the NO2 -/NO3 - sensor we were not able to carry out any planned experiments due to failure to properly calibrate and standardise the sensors. Success was achieved with the Griess method. All the drugs used as positive controls (DEA/NO, fenofibrate, oleanolic acid and IL-1ß) proved to be potent inducers of NO2 - release from endothelial cells. Interestingly, the isometric tension studies showed a higher % relaxation in high fat (HF) diet aortic rings compared to those from lean animals. Western blot data showed downregulation of eNOS activation and iNOS expression in obese groups, which was suggestive of endothelial dysfunction. Interestingly, proteins associated with oxidative stress (p22phox and nitrotyrosine) were downregulated in obese groups. The presence of PVAT exerted anti-contractile effects on the rings from HF rats, however in denuded aortic rings, PVAT showed a significant pro-contractile response in both lean and HF groups. PVAT also exerted anti-relaxation effects in aortic rings from both lean and HF rats. Conclusion: We managed to successfully establish two new techniques for our laboratory (Griess method and the organ bath – isometric tension method) which can complement the more established techniques in our laboratory in order to aid us in future vascular research. Finally, the isometric tension technique used in the obese rat studies generated interesting data, which further assisted in characterising the dietinduced obesity rat model in our laboratory. / AFRIKAANSE OPSOMMING: Inleiding: Stikstofoksied (NO) is ‘n belangrike chemiese boodskapper in die kardiovaskulêre sisteem. Ondanks vordering in die veld, bestaan daar ‘n aangaande behoefte aan bekostigbare en gebruikersvriendelike NO-metingstegnieke. Gevolglik het ons in hierdie studie daarna gemik om NO-metingstegnieke wat nie vantevore in ons laboratorium beskikbaar was nie, te ontwikkel en karakteriseer. Verder het ons ten doel gehad om ‘n ex vivo model te karakteriseer om die funksionele effekte van vaskulêre endoteel en NO produksie te meet. Metodes: Drie verskillende NO-metingstegnieke was ondersoek: (i) Amperometriese NO sensors. Hier het ons die verhogende effekte op NO van bekende aktiveerders van NO sintetase (NOS) ondersoek (Insulien, asetielcholien en biosintetiese menslike insulien). Drie verskillende NO-sensors was ge-evalueer in gekultuurde endoteelselle en aortaweefsel. Die vermeende NO verhogende effekte van die wrywingskragte opgewek deur laminere vloei (“shear stress”) is ook ondersoek. (ii) Nitriet (NO2 -) + nitraat (NO3 -) sensors. Hier het ons beplan om NO-vrystelling deur gekultuurde endoteelselle te meet. (iii) Kolorimetriese meting van NO2 - met die Griess reagens. Hier het ons m.b.v. ‘n mikroplaat leser die NO2 - - vrystelling deur endoteelselle gemeet. In die tweede deel van die studie het ons ‘n orgaan bad–isometriese spanningstegniek opgestel om endoteelafhanklike funksie van aortaringe te meet. Funksionele verskille in aortaringe van vetsugtige rotte is vergelyk met kontrole rotte. Ringkontraksie is met fenielefrien geïnduseer en verslapping met asetielcholien. Hierdie ondersoeke is verder ondersteun deur Western blot analises van sleutelproteïene in die aortaweefsel. Laastens het ons die effekte van perivaskulêre vetweefsel (PVAT) op kontraksie en verslapping in aortaringe met of sonder intakte endoteel ondersoek. Resultate: Alhoewel ‘n mate van sukses behaal was met die kalibrasie van die amperometriese sensors, was eksperimentele resultate moeilik om te herhaal a.g.v. sensor-onstabiliteit. Geen eksperimente kon met die NO2 -/NO3 - sensors uitgevoer word nie weens ‘n onvermoë om ordentlike kalibrasie en standardisering uit te voer. Ons het egter wel sukses behaal met die Griess-metode. Al die middels wat as positiewe kontroles gebruik was (DEA/NO, fenofibraat, oleanoliese suur and IL-1ß) het geblyk kragtige induseerders van NO2 - produksie vanaf endoteelselle te wees. Die isometriese spanningsstudies het ‘n hoer % verslapping getoon in die hoë vet (HF) dieet aortaringe in vergelyking met die kontroles. Western blot data het ‘n afregulering van eNOS en iNOS getoon in die HF diere, wat aanduidend is van endoteel disfunksie, terwyl proteïene geassosieer met oksidatiewe stress (p22phox en nitrotirosien) afgereguleer was in die HF groep. Die aanwesigheid van PVAT het ‘n anti-kontraktiele effek gehad op die ringe van die HF groep. Toe die endoteel egter verwyder was, het PVAT in beide kontrole en HF ringe ‘n beduidende pro-kontraktiele effek gehad. Verder het PVAT ook anti-verslappingseffekte op aortaringe beide kontrole en HF rotte uitgeoefen. Gevolgtrekking: Ons het daarin geslaag om twee nuwe tegnieke vir ons laboratorium suksesvol te vestig (Griess metode en die orgaanbad-isometriese spanningstegniek) wat in die toekoms die meer gevestigde tegnieke in ons laboratorium kan komplementeer. Laastens het die isometriese spanningstegniek wat in die dieetstudies gebruik is, data opgelewer wat ons verder sal help om die vetsug model in ons laboratorium te karakteriseer.

Résumé : Une dysrégulation de la lipolyse des tissus adipeux peut conduire à une surexposition des tissus non-adipeux aux acides gras non-estérifiés (AGNE), qui peut mener à un certain degré de lipotoxicité dans ces tissus. La lipotoxicité constitue, par ailleurs, l’une des causes majeures du développement de la résistance à l’insuline et du diabète de type 2. En plus de ses fonctions glucorégulatrices, l’insuline a pour fonction d’inhiber la lipolyse et donc de diminuer les niveaux d’AGNE en circulation, prévenant ainsi la lipotoxicité. Il n’y a pas d’étalon d’or pour mesurer la sensibilité de la lipolyse à l’insuline. Le clamp euglycémique hyperinsulinémique constitue la méthode étalon d’or pour évaluer la sensibilité du glucose à l’insuline mais il est aussi utilisé pour mesurer la suppression de la lipolyse par l’insuline. Par contre, cette méthode est couteuse et laborieuse, et ne peut pas s’appliquer à de grandes populations. Il existe aussi des indices pour estimer la fonction antilipolytique de l’insuline dérivés de l’hyperglycémie provoquée par voie orale (HGPO), un test moins dispendieux et plus simple à effectuer à grande échelle. Cette étude vise donc à : 1) Étudier la relation entre les indices de suppressibilité des AGNE par l’insuline dérivés du clamp et ceux dérivés de l’HGPO; et 2) Déterminer laquelle de ces mesures corrèle le mieux avec les facteurs connus comme étant reliés à la dysfonction adipeuse : paramètres anthropométriques et indices de dysfonction métabolique. Les résultats montrent que dans le groupe de sujets étudiés (n=29 femmes, 15 témoins saines et 14 femmes avec résistance à l’insuline car atteintes du syndrome des ovaires polykystiques), certains indices de sensibilité à l’insuline pour la lipolyse dérivés de l’HGPO corrèlent bien avec ceux dérivés du clamp euglycémique hyperinsulinémique. Parmi ces indices, celui qui corrèle le mieux avec les indices du clamp et les paramètres anthropométriques et de dysfonction adipeuse est le T50[indice inférieur AGNE] (temps nécessaire pour diminuer de 50% le taux de base – à jeun – des AGNE). Nos résultats suggèrent donc que l’HGPO, facile à réaliser, peut être utilisée pour évaluer la sensibilité de la lipolyse à l’insuline. Nous pensons que la lipo-résistance à l’insuline peut être facilement quantifiée en clinique humaine. / Abstract : It has been shown that a dysfunctional regulation of adipose-tissue lipolysis could conduct to non-adipose tissues overexpos ure to non exterified fatty acids (NEFA), leading to lipotoxicity. Lipotoxicity is considered as a key factor in the development of insulin resistance and type 2 diabetes. Insulin regulates glucose metabolism but also NEFA storage and release. To our knowledge, there is no gold standard for evaluating insulin sensitivity for lipolysis. The gold standard to measure insulin sensitivity for glucose is the euglycemic-hyperinsulinemic clamp. This method is simple to interpret because it achieves static levels of metabolic parameters at the end of each step of the clamp. The major limit of the clamp is that it is time-consuming, expensive and cannot be used on large population. On the other hand, the oral glucose tolerance test (OGTT) consists in a dynamic test also used to estimate insulin mediated glucose disappearance after ingestion of 75 g of glucose. Since the OGTT is easier to use, less expensive and can be suggested in large cohort studies, its potential use has been suggested to estimate insulin sensitivity for lipolysis, as well. T his work is the first to validate the use of simple indices derived from OGTT to estimate insulin sensitivity for lipolysis against the euglycemic clamp and adipose-tissue dysfunction in women. The results of this study clearly show in a group of 29 women (15 normal and 14 with polycystic ovary syndrome, who are used to increase the range of insulin resistance) that T50[subscript NEFA] (time to suppress 50% of NEFA baseline levels) during OGTT is the best index associated with glucose insulin clamp indices and clinical markers related to adipose tissue dysfunction and metabolic parameters. T50[subscript NEFA] (OGTT) was also better associated with central adiposity and metabolic parameters than clamp-derived indices. Since the OGTT is much easier to perform and is less expensive than the clamp technique, the use of OGTT to calculate T50[subscript NEFA] seems to be a valid method to assess antilipolytic action of insulin in large cohorts.

L'obésité est depuis quelques années reconnue comme un des fléaux du XXIe siècle en affectant environ 10% de la population adulte mondiale. Le passage de l'obésité au stade de pandémie est le résultat de multiples facteurs dont une mauvaise hygiène alimentaire et une sédentarité croissante. La découverte récente de l'existence d'un état inflammatoire chronique au cours de l'obésité pouvant intervenir dans la physiopathologie de la maladie et de ses nombreuses complications fait aujourd'hui l'objet d'intenses investigations. Or, le tissu adipeux (TA) qui, parallèlement à son activité métabolique, possède également une activité sécrétoire intense, est un acteur majeur de ce syndrome inflammatoire. L'autotaxine (ATX), l'adiponectine et la résistine sont les médiateurs de l'inflammation sécrétés par le TA. Si le rôle de ces facteurs dans la régulation du métabolisme est bien établi, leurs effets sur d'autres organes ne sont pas toujours connus. Il est notamment intéressant d'étudier leurs effets dans le système nerveux central (SNC), les récepteurs de certains y étant présent. Dans ce contexte, nous avons dans un premier temps étudié l'effet de l'ATX sur le stress oxydant généré par le H2O2 (peroxyde d'hydrogène) et sur l'inflammation induit par le lipopolysaccharide (LPS) ou le trimethylétain (TMT) dans les cellules microgliales murines, cellules immunitaires résidentes du SNC. Nos résultats montrent pour la première fois, l'effet anti-oxydant et anti-inflammatoire de l'ATX sur les cellules microgliales. Chez la souris, le TMT affecte la région hippocampique du SNC et nous avons montré qu'il induit la production d'ATX après 5 jours d'une injection i.p. de TMT. Ce qui suggère un rôle important de l'ATX dans la régulation de l'homéostasie des microglies et du SNC ainsi que dans la neuroinflammation. L'effet du TMT sur l'inflammation produite par le TA a également été étudié et les résultats montrent que le TMT induit une réponse inflammatoire dans deux types cellulaires du TA : adipocytes et macrophages ainsi que dans le TA de souris obèse ob/ob. Ensuite, nous avons sous-cloné les ADNc de l'adiponectine et de la résistine dans des vecteurs d'expression eucaryote afin d'étudier leurs effets sur les cellules microgliales dans des conditions normales ou inflammatoires. Cette étude devrait permettre une meilleure compréhension de l'influence du TA sur le SNC et donc la relation entre l'obésité les maladies neurodégénérative afin de proposer de nouvelles approches thérapeutiques. / In the last years, Obesity has been considered a major health issue with around 10% of the world population affected. The causes of this pandemy are multiple, but include bad alimentary habits and an increasing settle way of life. The recent discovery that chronic inflammation during obesity was associated with this disease physiopathology and others complications is the center of many investigations. Adipose tissue (AT), in addition to its metabolic activity, has an intense secretory activity and is a main player in this inflammatory syndrome. Autotaxin (ATX), adiponectin and resistin are such mediators secreted by AT. These factors functions in metabolism regulation are well described, but their effects on others organs are not always known. As it has been shown that some of these factors receptors are present in the central nervous system (CNS), it is interesting to study their effects in this organ. In this context, we initially studied the effect of ATX on the oxidative stress generated by H2O2 (hydrogen peroxide) and inflammation induced by lipopolysaccharide (LPS) or trimethyltin (TMT) in murine microglial cells, resident immune cells of the CNS. Our results show for the first time, the anti-oxidant and anti-inflammatory effects of ATX in microglial cells. In mice, TMT affects hippocampal region of CNS and we showed that ATX RNA is up regulated in the hippocampus 5 days post TMT treatment. Our results suggest that ATX could be involved in the regulation of microglia homeostasis and in neuroinflammation. The effect of TMT on the inflammation produced by the AT has also been studied and the results show that TMT induces an inflammatory response in two cell types of AT: adipocytes and macrophages and in fat tissue from ob/ob mice.We then subcloned the cDNA of adiponectin and resistin in eukaryotic expression vectors to study their effects on microglial cells under normal or inflammatory conditions. This study should lead to better understanding of adipose tissue influence on the CNS and therefore the relationship between obesity and neurodegenerative diseases to develop new therapeutic approaches.

Les aldose réductases (AKR1B) sont des oxydoréductases dépendantes du NADPH initialement décrites pour leurs fonctions de détoxication cellulaire et de réduction du glucose. La découverte de l’expression d’Akr1b7 dans le tissu adipeux murin ainsi que l’activité prostaglandine F2α synthase (PGFS) spécifique de certaines isoformes suggèrent des rôles biologiques inédits pour ces enzymes. La prostaglandine F2α (PGF2α) inhibant l’adipogenèse, cette fonction PGFS met en avant l’implication des AKR1B dans la physiologie du tissu adipeux blanc (TAB). L’objectif de ces travaux était de caractériser l’expression de l’ensemble des AKR1B au sein des TAB murins et humains et de comprendre leur impact sur l’homéostasie du tissu adipeux et en particulier sur l’adipogenèse et la lipolyse. Nous avons montré que l’ensemble des AKR1B était exprimé dans le TAB murin. Akr1b3, Akr1b8 et Akr1b16 sont exprimées à la fois dans les fractions stroma‑vasculaires (contenant des cellules immunitaires, vasculaires, progénitrices…) et adipocytaires. A l’inverse, Akr1b7 n’est pas exprimé par les adipocytes. Les analyses réalisées in vitro indiquent qu’à l’exception d’Akr1b16, les isoformes murines des AKR1B voient leur expression augmenter précocement et transitoirement au cours de l’adipogenèse. Chez l’homme, l’isoforme AKR1B1 est exprimée dans le TAB sous‑cutané de patients obèses alors qu’AKR1B10 est difficilement détectable (western blot, RT‑qPCR). In vitro, l’expression d’AKR1B1 augmente tout au long de la différenciation adipocytaire contrairement à AKR1B10 qui est préférentiellement exprimé dans les cellules indifférenciées. L’utilisation d’un inhibiteur spécifique des AKR1B montre que l’activité PGFS d’AKR1B1 constitue un frein à l’adipogenèse. Nous montrons aussi que les mécanismes régulant l’action de la PGF2α diffèrent en fonction des espèces. Chez l’homme, l’expression du récepteur FP est régulée dans le temps alors que dans les cellules murines, c’est l’expression des PGFS et donc la synthèse de PGF2α qui définit, au cours de l’adipogenèse, la fenêtre d’action de cette prostaglandine. Les souris invalidées pour la PGFS Akr1b7 présentent une diminution des quantités intra‑tissulaires en PGF2α associée à une expansion accrue de leurs tissus adipeux due à une augmentation de l’adipogenèse et à une hypertrophie adipocytaire sans modification de l’expression des enzymes impliquées dans la lipogenèse (Volat et al., 2012). Ces données en accord avec le rôle anti‑adipogénique de la PGF2α suggèrent aussi une action sur la lipolyse. Nous démontrons ici que la perte d’Akr1b7 entraîne une diminution de l’activité lipolytique du TAB. L’utilisation de cellules murines (3T3‑L1) et humaines (hMADS) différenciées en adipocytes, nous a permis de montrer que la stimulation de l’activité lipolytique suite à l’activation du récepteur FP résultait en partie d’une augmentation de la phosphorylation de HSL (forme active) et de l’accumulation de la lipase ATGL. Le troisième volet de ce travail de thèse a consisté à caractériser un modèle de souris transgénique surexprimant AKR1B1 dans le TAB (souris aP2‑AKR1B1) afin d’étudier le rôle biologique de cette isoforme humaine. / Aldose reductases are NADPH-dependent oxydoreductases described for their involvement in cellular detoxification and glucose reduction. The discovery of Akr1b7 expression in murine adipose tissue together with the prostaglandin F2α Synthase (PGFS) activity of some isoforms suggest unreleased biological roles for these enzymes. Prostaglandin F2α (PGF2α) inhibiting adipogenesis, this PGFS function highlights AKR1B potential involvement in white adipose tissue (WAT) physiology. This work aimed at characterising the expression of all AKR1B in both murine and human WAT and understanding their impact on adipose tissue homeostasis and especially on adipogenesis and lipolysis. We showed that all AKR1B were expressed in murine WAT. Akr1b3, Akr1b8 and Akr1b16 were both expressed in the stromal vascular fraction (containing immune cells, vascular cells, progenitors…) and in the adipose fraction. In contrast, Akr1b7 was not expressed in adipocytes. In vitro analyses indicated that, except for Akr1b16, murine AKR1B isoform expression increased early and transiently during adipogenesis. In human, AKR1B1 was expressed in human subcutaneous WAT from obese patients whereas AKR1B10 was hardly detectable (western blot, RT‑qPCR). In vitro, AKR1B1 expression increased throughout adipocyte differentiation unlike AKR1B10, which was preferentially expressed in undifferentiated cells. Using an AKR1B specific inhibitor, we demonstrated that AKR1B1 PGFS activity was a dampen to adipogenesis. We also showed that mechanisms regulating PGF2α action differed according to the species. In human cells, the expression of FP receptor was time-regulated whereas, in murine cells, PGFS expression and thus, PGF2α synthesis, limited PGF2α activity during adipogenesis. Akr1b7 knockout mice have decreased PGF2α intratissular levels associated with an expansion of adipose tissue resulting from an increase of adipogenesis and an adipocyte hypertrophia without any modification of lipogenic enzymes expression (Volat et al., 2012). These data, in agreement with PGF2α anti-adipogenic action, suggest an impact on lipolysis. We demonstrated that loss of Akr1b7 led to a decrease of WAT lipolytic activity. The use of murine (3T3‑L1) and human (hMADS) differentiated cells allowed us to show that the stimulation of lipolysis in response to FP activation was, in part, due to an increase of HSL phosphorylation (active form) and an increase of ATGL accumulation. The third part of this work consisted in characterizing the phenotype of transgenic mice overexpressing AKR1B1 in WAT (aP2‑AKR1B1 mice) in order to study the biological role of this human isoform.

Alcoholic Fatty Liver Disease (AFLD) is caused by excessive alcohol consumption and is a leading cause of liver related mortalities, with currently no treatments available. The goal of this project was to establish the effect of alcohol consumption on adipose tissue-derived secreted factors, adiponectin and C1q TNF Related Proteins 1-3 (CTRP1-3). We propose that excessive alcohol consumption will reduce circulating levels of adiponectin and CTRPs 1-3. Mice were fed a Lieber-Decarli control or alcohol diet for 10-days with a gavage (NIAAA model) or 6-weeks with no gavage (chronic model). Serum and adipose tissue were collected and CTRPs 1-3 and adiponectin levels were examined by immunoblot analysis. Our results indicate that long-term alcohol consumption effects adipokine secretion in a sex specific manner. Further research will be needed to explore the physiological relevance of these findings, to determine if these changes are beneficial to combat the negative effects of excessive alcohol consumption.