As células tronco mesenquimais (MSC) têm capacidade imunossupressora envolvendo diferentes mecanismos. No entanto, ainda não está claro o que desencadeia as diferentes vias de imunorregulação, ou mesmo o que determina a magnitude de sua capacidade supressora. A nossa hipótese é que a intensidade da atividade supressora das MSC esteja vinculada à sua capacidade de desencadear múltiplas vias moleculares, simultaneamente. Utilizamos uma estratégia experimental na qual testamos o efeito das MSC humanas do tecido adiposo (AdMSC), derivadas de diferentes indivíduos, sobre a proliferação de PBMC estimuladas, obtidas do mesmo indivíduo, visando relacionar o efeito supressor com a ocorrência simultânea de modificações imunomoleculares (expressão de mRNA e proteínas), tanto nos linfócitos T como nas AdMSC. Foram comparadas as correlações entre as modificações de expressão gênica e proteica nos ensaios com maior (> 50% inibição de proliferação de linfócitos T) ou menor (<50% de inibição) capacidade supressora. A primeira novidade do nosso trabalho é que durante a atividade supressora das AdMSC, múltiplas moléculas são acionadas, simultaneamente, nos linfócitos T (LT) e nas próprias AdMSC, indicando a importância da ação integrada de múltiplas vias moleculares. Várias dessas modificações de expressão gênica ocorreram de forma dominante (em todos os experimentos), como o aumento da expressão de IDO, ILB e MMP2 nos LT e de diversas moléculas nas AdMSC (entre elas: IDO, HLAG, IL10, PDL1, SEMAD4, MMP9, FASL e várias quimiocinas). Ademais, relatamos diversos novos mecanismos potencialmente envolvidos na atividade supressora das AdMSC humanas, entre eles, a diminuição do número de células T efetoras ativadas, CD4 e CD8 expressando ICOS e CD8 positivas expressando OX40, além do aumento do número de uma população específica de células T reguladoras (CD4+CD25hiFOXP3+) expressando a ectoenzima CD73, importante na geração de adenosina, molécula com atividade imunorreguladora. Descrevemos também outras novas moléculas possivelmente envolvidas nos mecanismos de imunossupressão das AdMSC como SEMA4D, MMP9, CCL22 e RUNX3, cuja expressão aumentou nas AdMSC após o cocultivo e GARP, CTNNB1, IL1B e MMP2, nos LT. Mostramos, pela primeira vez, um perfil imunomolecular diferencial nos ensaios de maior capacidade supressora, com correlações positivas específicas entre os aumentos da expressão gênica, nos dois tipos celulares, envolvendo moléculas como HLAG e CCR4, IL13, IL4, TLR10, IL1B, GARP, S1PR1 e CTNNB1 (-catenina), nos linfócitos T e, nas AdMSC, CCL22 com MMP9 e HLAG com FOXP3. Assim, sugerimos que a magnitude da atividade supressora das AdMSC depende da combinação de múltiplas vias moleculares mobilizadas, simultaneamente, e relacionadas com a indução de um perfil Th2, migração e sobrevida celular, bem como com a atividade imunorreguladora. Além de apontar novas moléculas ao repertório imunomolecular da atividade supressora das AdMSC humanas, trazemos uma contribuição importante, apresentando uma visão mais ampla da rede de interações imunomoleculares envolvida na atividade supressora das MSC / Mesenchymal stem cells (MSC) exhibit immunosuppressive activity operating by a variety of mechanisms. However, it is still unclear what triggers the different immunoregulatory pathways, or what determines the magnitude of their suppressive capacity. We hypothesized that the magnitude of MSC suppressive activity may be related to their capacity to activate multiple pathways, simultaneously. We used an approach in which we tested the effect of different human MSC derived from adipose tissue (AdMSC) over T cell proliferation, using PBMC obtained from a single donor. We determined the relationship between AdMSC suppressive activity and the simultaneous occurrence of immunomolecular changes (mRNA and protein expression) in both AdMSC and T cells, following AdMSC/PBMC interactions. Correlations of gene expression and protein modifications were compared in assays in which AdMSC displayed high (>50% of T cell proliferation inhibition) and low (<50% inhibition) immunosuppressive activity. The first novelty from our work is that multiple molecules are simultaneously activated, in both AdMSC and T cells, indicating the importance of an integrated action of several molecular pathways. Many of these gene expression modifications were dominantly (in all experiments) upregulated, namely: IDO, IL1B and MMP2 in T cells and several molecules in AdMSC such as: IDO, HLAG, IL10, PDL1, SEMAD4, MMP9, FASL, as well as many chemokines. We also report novel mechanisms potentially involved in human AdMSC suppressive activity, such as the decrease in the number of activated T cells, CD4 and CD8 cells expressing ICOS and CD8 positive cells expressing OX40. We also found an increase in the numbers of a specific Treg subpopulation (CD4+CD25hiFOXP3+) expressing the ectoenzime CD73, important in the generation of adenosine, a molecule displaying suppressive activity. Moreover, we report other novel molecules possibly involved in AdMSC suppressive activity, once their gene expression was upregulated in AdMSC (SEMA4D, MMP9, CCL22 and RUNX) and in T cells (GARP, CTNNB1, IL1B e MMP2), following AdMSC/PBMC interactions. We also show, for the first time, a differential immunomolecular profile in the assays with high suppressive activity, showing exclusive positive correlations among changes in gene expression, involving multiple molecules, such as HLAG with CCR4, IL13, IL4, TLR10, IL1B, GARP, S1PR1 and CTNNB1 (-catenin) in T cells, and CCL22 and MMP9, with HLAG and FOXP3, in AdMSC. We, therefore, suggest that the magnitude of AdMSC suppressive activity depends on a particular combination of multiple immunoregulatory pathways simultaneously activated and related with the induction of a Th2 profile, cell migration and survival, as well as with immunoregulatory activity. Besides adding new players to the scenario of MSC suppressive activity, we believe our data bring a major contribution to the field, by providing a broader view of the interactive immunoregulatory molecular networks involved in MSC immunologic activities

Les adipocytes (cellules spécialisées dans le stockage des graisses) sont formés à partir de cellules immatures appelées cellules progénitrices lors du processus d'adipogenèse. Chez l'homme, les différentes étapes de ce processus sont mal connues ainsi que les signaux qui le régulent. La première partie de mon travail de thèse a eu pour but de caractériser la cellule intermédiaire entre la cellule progénitrice et l'adipocyte : le préadipocyte. La deuxième partie a consisté à évaluer le rôle des protéines morphogénétiques de l'os (ou BMP), des inducteurs de l'adipogenèse décrits chez la souris, dans l'adipogenèse humaine. Nous avons montré que les BMP2, 4 et 7 sont produites dans le tissu gras humain et BMP7 est modulée par l'obésité. Les BMP2 et 4 induisent l'adipogenèse des cellules progénitrices humaines mais seule la BMP7 permet la production d'adipocytes particuliers " beiges " décrits pour consommer les lipides et produire de la chaleur. Ces travaux affinent nos connaissances sur les mécanismes impliqués dans l'expansion du tissu gras et permettront d'élaborer des stratégies pour lutter contre le développement des pathologies liées à l'obésité. / Adipocytes (cells specialized in fat storage) arise from immature cells, called progenitor cells, during the process of adipogenesis. In human, the different stages of adipogenesis are not well defined as well as the signals involved in adipogenic modulation. The first part of my thesis work aimed to characterize the intermediate cell state between progenitor cell and mature adipocyte: the preadipocyte. The second part aimed to evaluate the role of bone morphogenetic proteins (BMPs) in human adipogenesis. In mice, BMP2 and BMP4 induce classical adipogenesis whereas BMP7 leads to the production of "brite" adipocytes with the capacity to use lipids to produce heat. We have shown that BMP2, 4 and 7 are produced in human fat depots and BMP7 is modulated by obesity. BMP2 and 4 induce classical adipogenesis and BMP7 only induces brite adipogenesis from human progenitor cells. These works improve our knowledge about the mechanisms involved in the expansion of fat depot and may allow the identification of new strategies to fight against the development of obesity-associated pathologies.

Le tissue adipeux (TA) est un organe complexe specialisé dans le stockage et la libération d'énergie sous forme de lipides. Cet organe adipeux est essentiel pour le maintien de l'homéostasie énergétique. Les adipocytes sont les cellules prototypiques du TA. Elles se forment durant la différenciation de précurseurs, un processus appelé adipogenèse. L'adipogenèse est intimement associée à la synthèse des acides gras et de triglycérides lors de la lipogenèse. Néanmoins, divers facteurs peuvent perturber l'adipogénèse et la lipogenèse, contribuant au dysfonctionnement du TA et au développement des maladies métaboliques. Le but de cette thèse a été d'étudier la lipogenèse dans le contexte du stress du réticulum endoplasmiques (SRE), de la restriction calorique et du vieillissement. Dans le projet A, nous avons montré que l'exposition d'adipocytes à un SRE aigu inhibe l'expression des gènes liés à la lipogenèse et empêche l'incorporation du glucose dans les lipides. En plus, l'exposition des préadipocytes à un SRE chronique, détériore à la fois la lipogenèse et l'adipogenèse. Par contre, pour les adipocytes, un SRE chronique mais modéré n'a pas d'effet évident sur la lipogenèse. Ces effets du SRE pourraient contribuer à la détérioration de la fonction de TA vue dans l'obésité. La capacité du TA à stocker des lipides diminue avec l'âge, probablement à cause de l'accumulation de cellules sénescentes ou un SRE plus élevé. Dans le projet B, nous avons étudié la capacité lipogénique du TA humain en relation à la sénescence et aux marqueurs du SRE au sein d'une cohorte de femmes obèses jeunes ou âgées. Tandis que l'expression des principaux marqueurs de la sénescence était augmentée dans le TA des femmes âgées, l'expression génique des enzymes de lipogenèse et des chaperonnes était diminuée dans le TA des personnes âgées. Ces résultats étaient partiellement retrouvés dans les adipocytes différenciés in vitro des mêmes individus ce qui suggère une moins bonne capacité à faire face au SRE lors du vieillissement. Le régime à très basses calories (VLCD) est souvent prescrit en première intention pour une rapide perte de poids. L'amélioration de la sensibilité à l'insuline se voit dès 2 jours de VLCD. Néanmoins, on ne sait quasiment rien des modifications métaboliques du TA survenant durant les premiers jours. Dans le projet C, nous avons donc comparé les réponses métaboliques et inflammatoires du TA sous-cutané précocément (2 jours) et plus tardivement (28 jours) lors d'un VLCD. A 2 jours de régime, l'expression des gènes lipolytiques était augmentée, alors que l'expression des gènes lipogéniques était diminuées. Les marqueurs d'inflammation n'étaient pas changés dans le TA. Néanmoins, les changements d'expression dans le TA lors de la phase précoce du régime ne pouvait pas expliquer l'effet de ce régime court à l'amélioration de la sensibilité à l'insuline. Dans la phase tardive, l'expression des gènes impliqués dans la lipogenèse et la ß-oxydation était largement réduite, tandis que l'expression des marqueurs inflammatoires était augmentée. Nous avons donc montré que les réponses métaboliques et inflammatoires du TA sous-cutané à 2 jours et 28 jours de VLCD sont différentes. Dans le projet D, nous avons comparé et défini les effets de la restriction calorique modérée sur la physiologie des préadipocytes et des adipocytes différenciés in vitro chez des jeunes obèses ou des personnes âgées obèses. De façon surprenante, on n'a observé aucun effet de l'intervention sur le métabolisme des préadipocytes dans les deux groupes. Par contre, un effet bénéfique de l'intervention sur le métabolisme adipocytaire n'a été observé que chez les personnes âgées. Nos données montrent donc qu'une restriction calorique modérée peut avoir un effet positif sur le métabolisme adipocytaire des séniors. Pour conclure, cette thèse montre que la lipogenèse dans le TA humain peut être inhibée par le SRE, la restriction calorique sévère et le vieillissement. / Adipose tissue (AT) is a complex organ specialised in safe storage and release of energy as lipids. The adipose organ is therefore essential for the maintenance of energy homeostasis. The prototypical cells of AT are adipocytes, emerging from the precursors in a process called adipogenesis. Adipogenesis itself is tightly connected with lipogenesis, i.e. with the synthesis of fatty acids and triglycerides. Various stimuli can disturb adipocyte differentiation and lipogenesis and thus contribute to AT dysfunction and development of associated metabolic diseases. This thesis was focused on the investigation of lipogenesis in the context of endoplasmic reticulum stress (ERS), calorie restriction and aging. In Project A, we showed that exposition of adipocytes to high acute ERS inhibits expression of lipogenic genes and glucose incorporation into lipids. Moreover, chronic exposure of preadipocytes to ERS impaired both, lipogenesis and adipogenesis. On the other hand, chronic low ERS had no apparent effect on lipogenesis in adipocytes. These effects of ERS could therefore contribute to the worsening of AT function seen in obesity. The capacity of AT to store lipids decreases in aging, possibly due to the accumulation of senescence cells or higher ERS. In Project B, we investigated lipogenic capacity of human AT in relation to senescence and markers of ERS. AT and adipose cells from young and elderly women were investigated. While mRNA expression of major senescent markers was increased in AT from the elderly compared to young individuals, mRNA expression of lipogenic enzymes and chaperones was decreased in AT from elderly individuals. These results were also partly observed in vitro in differentiated adipocytes from AT of the same individuals suggesting the reduced capability to cope with ERS in aging. Very-low calorie diet (VLCD) is first line lifestyle intervention to achieve rapid weight loss. The improvement of whole body insulin sensitivity can be seen as soon as after 2 days of VLCD. However, little is known about AT metabolic changes in those early days. Thus, in Project C, we compared metabolic and inflammation-related characteristics of subcutaneous AT in the early (2 days) and later (28 days) phase of a VLCD. In the early phase of VLCD, the expression of lipolytic genes was increased, whereas the expression of lipogenic genes was suppressed. The inflammatory markers remained unchanged in AT. The changes in AT gene expression in the early phase of VLCD could not explain the effect of short calorie restriction on the improvement of insulin sensitivity. At the later phase, expression of genes involved in lipogenesis and ß-oxidation was markedly suppressed, whereas the expression of inflammatory markers was increased. Thus, we found that the early and later phases of VLCD differ with respect to metabolic and inflammatory responses in subcutaneous AT. In Project D, we compared and defined the effects of moderate calorie restriction on preadipocytes and in vitro differentiated adipocytes in two groups of obese men: juniors and seniors. We did not observe any effect of the intervention on metabolism of preadipocytes in either group. However, we observed an intervention-driven improvement in adipocyte metabolism selectively in the group of seniors. Therefore, our data suggest that moderate calorie restriction could initiate positive changes in metabolism of adipocytes in seniors. In conclusion, this thesis brought several pieces of evidence that lipogenesis in human AT can be inhibited by ER stress, severe caloric restriction and aging.

<p>The timing of cell differentiation is important for development and renewal of well functioning organs and tissues. One protein involved in this process is Preadipocyte factor 1 (Pref-1). Most likely, the role of this protein is to maintain cells in an undifferentiated state. </p><p>The work presented in this thesis, has employed the rat as an animal model for the studies of Pref-1. Rat models of obesity (Zucker, ZO) and type II diabetes (Goto-Kakizaki, GK) were used to determine metabolic influence on Pref-1 and adipokine mRNA expression in adipose tissues.</p><p>The Pref-1 cleavage product was purified from rat amniotic fluid and physicochemically characterised. Concentration of Pref-1 in serum, amniotic fluid and urine was determined by ELISA. Soluble Pref-1 and the compartmentalisation of the protein were highly similar to what had previously been demonstrated in mice and humans.</p><p>Immunohistochemistry studies displayed similar staining patterns of Pref-1 in adrenal glands, ovaries and pituitary glands of non-pregnant and pregnant rats. This suggests that pregnancy do not influence the protein expression of Pref-1 in these organs.</p><p>In the GK rats, Pref-1 mRNA was altered and a decrease in the visceral compared to subcutaneous adipose depots was demonstrated, in contrast to the ZO rats. Additionally, adiponectin, leptin, IL-6 and TNF-α mRNA levels were altered in the diabetic strain, indicating that this animal model expresses many of the typical features of type II diabetes.</p><p>In conclusion, the rat is an appropriate model for studies of FA1/Pref-1/dlk. Pref-1 is highly elevated in fetal and maternal serum during pregnancy. However, the expression of Pref-1 in some endocrine tissues did not alter due to pregnancy. The mRNA expression of Pref-1 was altered between adipose depots and demonstrated to be affected by metabolic disturbances in the animals.</p>

Adipose tissue is an endocrine organ, and its homeostatic mechanisms in normal weight, overweight and obese subjects must be elucidated. We sought to determine the basal adipose tissue biology of visceral (VIF) and subcutaneous (SQF) fat depots in 8 month old Sinclair minipigs, an animal that has been shown to be physiologically similar to humans. Metabolic analysis showed a decrease in LDL, white blood cells (WBC), and lymphocyte percentages as the minipigs aged from 6 to 8 months (p <0.0001 and = 0.0046 and 0.0165 respectively). There were no significant changes in triglycerides, HDL, VLDL, and neutrophil percentages. There was a trend in insulin increase (P=0.0722). Microarray analysis was performed to determine transcriptome differences between VIF and SQF. When VIF was compared to SQF, expression of a total of 788 transcript ID’s differed: were 240 up-regulated and 548 down-regulated. Examples included hydroxysteroid 11-beta dehydrogenase 2, fatty acid synthase, IL-18, and platelet factor 4 which were all up-regulated in VIF vs. SQF. The down-regulated transcripts included estrogen receptor 1, insulin-like growth factor binding protein 5, and platelet derived growth factor D. When SQF was compared to VIF, a total of 598 transcript IDs were up or down-regulated by more than a 2 fold difference (P<0.05). From this subset of the transcriptome, we found 471 IDs were up-regulated in SQ fat, and 127 were down- regulated. Interestingly, the up-regulated genes included prostaglandin F2 receptor negative regulator, estrogen receptor 1, thrombospondin 1, lipoprotein related receptor protein 2, and platelet derived growth factor D. Down-regulated genes in SQF compared to VIF included IL-18, platelet factor 4, cyclooxygenase, and fatty acid synthase. We found no significant difference in gene expression between SQF and VIF TNF alpha, TLR 4, and adiponectin in our. Immunofluorensce (IF) assay revealed that SQF expressed more CD 163 positive (alternatively activated) macrophages than VIF, and little to no CD 68 (classically activated) positive macrophages. Additionally, VIF expressed more CD 68 positive macrophages compared to SQF. The data from this study is consistent with the human and rodent literature which states that VIF is more metabolically active and pro-inflammatory compared to SQF.

There is an increased risk for cardiovascular (CV) comorbidity among patients with rheumatoid arthritis (RA), with premature atherosclerosis, and a higher incidence of CV events, compared with the general population. Disease related factors add to the CV risk, and interact with the traditional CV risk factors. The underlying mechanism for this is not completely understood. In active RA there is a loss of muscle mass and an increase in body fat content. Production of cytokines, i.e., adipokines, in the adipose tissue could link the inflammation with the CV system. Control of the inflammation has been suggested to modify the CV risk in RA, and the recently introduced biological drugs, such as the tumor necrosis factor inhibitors (TNFi), have opened up new treatment opportunities. The aim of this thesis was to evaluate aspects of the interaction between inflammation and CV comorbidity in RA using biochemical and epidemiological methods. Methods In the first two studies, patients with established RA were examined for clinical disease activity, and blood samples were analysed for cytokines and adipokines using ELISAs and multiplex technology. In Study I (n RA=23) anthropometric measurements were assessed and in Study II (n RA=51) measurements of intima-media thickness (IMT), and endothelial function (FMD). From a subgroup of patients (Study II, n RA=13) samples of abdominal subcutaneous adipose tissue (SAT) were analysed for content of adipokines. In study III and IV associations between treatment with TNFi and acute coronary syndromes (ACS) were analysed using data from the Swedish Rheumatology Register; in Study III regarding early RA (n TNFi exposed=1,271, n bionaïve RA=4,729), and in Study IV comprising patients with RA of all stages (n TNFi exposed=7,213, n bionaïve RA=17,769) and with a matched general population comparator cohort (n=32,161). Associations between response to TNFi therapy and risk for ACS in the early RA cohort were evaluated in a nested case-control design (cases n=24, controls n=81). Results Serum levels of the cytokines/adipokines interleukin-1 receptor antagonist (IL-1Ra), IL-6, osteopontin, visfatin and TNF were increased in patients compared with controls (p≤0.001-0.036). The amount of TNF receptor II extracted from SAT was greater in patients (p=0.006). The serum (s-) levels of IL-1Ra correlated with s-leptin (r=0.71, p≤0.001) and s-haptoglobin in RA patients (r=0.56, p≤0.01). The result from a factor analysis indicated IL-1Ra to be associated with both adipose tissue and inflammation. Levels of s-visfatin (p=0.019) and s-IL-1Ra (p=0.023), respectively, were positively associated with IMT independently of inflammatory activity and CV risk factors. PAI-1 and MCP-1 extracted from SAT showed inverse associations with IMT. Patients with RA, whether exposed to TNFi or bio-naïve, had a doubled risk for ACS compared with the general population; HR 2.09 (95%CI 1.58-2.76) and 1.80 (1.49-2.17), respectively. No significant associations between risk for ACS and TNFi exposure were detected after adjustments; HR 0.80 (0.52-1.24) in early RA and HR 1.08 (0.82-1.41) in RA of any duration. Furthermore, no association between the risk for ACS and response to TNFi treatment in patients with early RA was observed, OR 1.5 (0.3-6.9). Conclusions The results indicate that cytokines/adipokines may have a role in the development of atherosclerosis in RA patients. A continuing increase in the risk of ACS in RA compared with the general population, despite modern therapeutic strategies, was noted. Neither exposure nor response to treatment with TNFi was associated with any modification of the risk for ACS.

Obesity and specifically central obesity is related to insulin resistance, type 2 diabetes and other components of the so-called metabolic syndrome. The aim of this study was to elucidate the interplay between hormones, nutrients and adipose depots in normal and insulin-resistant fat cell metabolism. High levels of free fatty acids (FFAs) induce insulin resistance in muscle and liver in vivo. In the present study, rat adipocytes were treated with high physiological levels of oleic or palmitic acid in vitro for 4-24 h. This treatment had no effect on basal or insulin-stimulated glucose uptake capacity in these cells, neither did it affect the levels of the insulin signalling proteins; insulin receptor substrate (IRS)-1 or –2, phosphatidylinositol 3-kinase (PI3-K), protein kinase B (PKB) or glucose transporter (GLUT) 4, or the regulation of lipolysis rate. Visceral adiposity is considered to be more harmful than peripheral adiposity with respect to metabolic and cardiovascular complications. In adipose biopsies from subjects undergoing abdominal surgery, we found that glucose uptake capacity was elevated in omental as compared to subcutaneous adipocytes. The sensitivity (EC50) or maximum relative response to insulin, measured as % of basal, did however not differ between the depots. In women, subcutaneous adipocytes displayed a higher lipolysis rate following cAMP-stimulation than omental adipocytes, whereas there was a tendency towards the opposite in adipocytes from men. No differences were found between depots or sexes in the ability of insulin to inhibit lipolysis or in the levels of the lipolysis regulating proteins, i.e. protein kinase A (PKA), hormone sensitive lipase (HSL) and perilipin. Glucocorticoids, e.g. cortisol, exert pronounced insulin-antagonistic effects and are associated with redistribution of fat from peripheral to central fat depots in humans. Treatment of human subcutaneous and omental adipocytes in vitro, with the cortisol analogue dexamethasone, resulted in a dose dependent down-regulation of basal and insulin-stimulated glucose uptake capacity in omental, but not in subcutaneous cells. Concomitantly, the levels of IRS-1 and PKB were decreased only in omental adipocytes after dexamethasone treatment. The relative effect of insulin to stimulate glucose uptake was however not altered by dexamethasone treatment. The cAMP-stimulated lipolysis rate was elevated by dexamethasone treatment in cells from the subcutaneous depot in women and tended to be elevated in omental cells from men. No alterations however, were seen in the levels of the assessed lipolysis regulating proteins. Subcutaneous as well as omental fat cell size correlated negatively to insulin action in subcutaneous fat cells in vitro after adjusting for age, sex and body fat parameters in non-diabetic, but not in type 2 diabetic, subjects. Large subcutaneous fat cell size was strongly related to plasma leptin levels in non-diabetic and in type 2 diabetic subjects. We conclude that 1) adipocytes seem to be less vulnerable to elevated levels of fatty acids than muscle and liver cells, 2) the interactions between glucocorticoids and insulin in the regulation of glucose uptake differ between adipose depots, 3) depot specific hormonal lipolysis regulation differs between sexes and 4) fat cell size is related to insulin action in subcutaneous fat cells and to circulating levels of leptin.

Background: Cardiovascular disease (CVD) is the leading cause of death among women in Sweden. The risk of CVD increases rapidly after the menopause. A major contributing factor may be the redistribution of adipose tissue, from the peripheral to central depots, associated with menopause. This change in body composition is commonly attributed to declining estrogen levels but may also be affected by tissue-specific alterations in exposure to other steroid hormones, notably glucocorticoids – mainly cortisol in humans. Indeed, adipose tissue-specific overexpression of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) induces central obesity, insulin resistance and hypertension in mice. Interestingly, estrogen may regulate this enzyme. The aim of this thesis was to investigate putative links between estrogen and glucocorticoid activation by 11βHSD1. Materials and Methods: 11βHSD1 expression and/or activity in adipose tissue and liver, and adipose estrogen receptor α and β (ERα and ERβ) gene expression, were investigated in lean pre- and postmenopausal women and ovariectomized rodents with and without estrogen supplementation. In lean women measures of 11βHSD1 were correlated to risk markers for CVD. The association between adipose 11βHSD1 and ER mRNA expression was investigated in both lean women and rats and in an additional cohort of obese premenopausal women. In vitro experiments with adipocyte cell lines were used to explore possible pathways for estrogen regulation of 11βHSD1. Results: Subcutaneous adipose tissue transcript levels and hepatic activity of 11βHSD1 were higher in postmenopausal vs. premenopausal women. In rodents, estrogen treatment to ovariectomized rats decreased visceral adipose tissue 11βHSD1, resulting in a shift towards higher subcutaneous (vs. visceral) 11βHSD1 mRNA expression/activity. Increased adipose and hepatic 11βHSD1 were associated with increased blood pressure and a disadvantageous blood lipid profile in humans. We found significant positive associations between 11βHSD1 and ERβ transcript levels in adipose tissue. The in vitro experiments showed upregulation of 11βHSD1 mRNA expression and activity with estrogen or ERβ-agonist treatment at low (corresponding to physiological) concentrations. Conclusions: Our studies show for the first time increased local tissue glucocorticoid activation with menopause/age in women. This may contribute to an increased risk of CVD. Estrogen treatment in rodents induces a shift in 11βHSD1 activity towards the subcutaneous adipose tissue depots, which may direct fat accumulation to this metabolically “safer” depot. The in vitro studies suggest that low-dose estrogen treatment upregulates 11βHSD1 via ERβ. In summary, estrogen - glucocorticoid metabolism interactions may be key in the development of menopause-related metabolic dysfunction and in part mediate the beneficial effects of postmenopausal estrogen treatment on body fat distribution.

Obesity is a growing disorder in the United States, affecting over 60% of the population. We previously defined sympathetic nervous system (SNS) outflow from brain to white adipose tissue (WAT) using a viral transneuronal tract tracer. SNS innervation of WAT is the principle initiator of lipolysis, whereas decreases in sympathetic drive promote lipid accumulation. Which of the many origins of SNS outflow from brain to WAT results in SNS-mediated changes in lipid mobilization (increases in drive) or accumulation (decrease in drive) is unknown. Previous research indicates that sympathetic denervation blocks lipid mobilization; thus, rostral sites in the neuroaxis connected to WAT via the SNS may promote WAT lipid mobilization. The hypothalamic paraventricular nucleus (PVN) may play a role via its descending projections to the intermediolateral horn of the spinal cord. Therefore, the consequences of PVN lesions (PVNx) on WAT mobilization or accumulation were tested. PVNx resulted in increased lipid accumulation, indicated by increases in retroperitoneal (RWAT) , epididymal (EWAT) , and inguinal WAT (IWAT) pad masses, in fed hamsters, but PVNx did not block fasting (56 h)-induced lipid mobilization. Because adrenal medullary catecholamines, especially epinephrine, also play a minor role in lipid mobilization, we tested the contribution of catecholamine release on lipid mobilization through adrenal demedullation (ADMEDx), with and without PVNx, and found fastinginduced lipid mobilization was not blocked. There was, however, a suggestion that distal denervation of IWAT, with and without ADMEDx, partially blocked lipid mobilization. In addition, evidence suggests SNS also may be an important controller of fat cell proliferation. Surgical denervation of WAT triggers increases in fat cell number (FCN), but have not determined if this FCN increase is due to preadipocyte proliferation or differentiation of preadipocytes into mature fat cells. We also have not demonstrated what role sensory innervation may have in regulating white adipocyte proliferation. Therefore, the role of WAT sympathetic or sensory innervation on adipocyte proliferation was tested. The SNS but not sensory denervation triggered bona fide proliferation as indicated by bromodeoxyuridine plus AD3, a specific adipocyte membrane protein, colabeling. These and previous data suggest that the SNS plays a role in regulating adiposity.

Obesity is America’s fastest growing health threat. Although a primary health risk factor, obesity increases the probability of secondary health consequences such as stroke, diabetes mellitus and heart disease. Siberian hamsters offer a convenient model to study obesity, as they exhibit a photoperiod-driven reversal of obesity during the fall-winter months (i.e., short-days-SD). SD responses in the Siberian hamster, amongst others, include decreased adiposity and gonadal regression. The duration of the dark period is faithfully transmitted into a neuroendocrine melatonin (MEL) signal; that codes seasonal information. The brain communicates with body fat (white adipose tissue- WAT) via the sympathetic nervous system (SNS). The central MEL binding sites, however, necessary for the body fat/lipid mobilization responses during SDs are not precisely known, although melatonin receptor (MEL 1aR) mRNA has been co-localized with sympathetic outflow neurons of WAT in several forebrain areas. This dissertation aims to identify and to characterize the contribution of central sites that are important in seasonal responses in Siberian hamsters. Thus, I asked: Which specific brain sites are both sufficient and necessary to stimulate SD-like decreases in body, WAT and testes mass? Furthermore, I tested if SD-induced decreases in body fat mass are accompanied by increased energy expenditure, specifically brown adipose tissue (BAT) thermogenesis. It is hoped that the identification of brain sites and mechanisms involved in the effortless reversal of obesity in these animals can be applied to treatment opportunities of human obesity.