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Studying molecular mechanisms underlying adipocyte differentiation is imperative to understanding adipocyte function and its role in obesity. However, the majority of research exploring adipogenesis is conducted with cell lines cultured directly on tissue culture plastic. Culturing cells on plastic may result in altered proliferation and differentiation, and subsequent change in pharmacological response. The extracellular matrix (ECM) plays a critical role in adipocyte development and survival. It is suggested that cells in vitro express high levels of ECM proteins to compensate for lack of an ECM. Differentiating preadipocytes on a substrate representative of the mature adipocyte extracellular environment may provide a more physiological response to drugs and environmental chemicals. The purpose of this study was to investigate the impact of Matrigel on 3T3-L1 cell growth, differentiation, lipid accumulation and responsiveness to Rosiglitazone. Matrigel decreased 3T3-L1 cell proliferation, enhanced lipid accumulation, and increased expression of adipogenic and lipogenic markers, including PPARγ, C/EBPα, SREBP1c, FAS, LPL, FABP4 and PLIN1. This was accompanied by a decrease in gene expression of ECM proteins, including fibronectin, collagen 1, collagen 3, collagen 4, laminin and collagen 6 in 3T3-L1 cells on Matrigel. Finally, Matrigel enhanced the response of 3T3-L1 cells to Rosiglitazone, which is a known PPARγ agonist and significantly increases lipid accumulation in 3T3-L1 cells. Our results suggest that enhanced lipid accumulation in 3T3-L1 cells on Matrigel is associated with decreased expression of ECM genes. Future studies require investigation of the cell-to-ECM interaction to confirm these findings. This study proposes that the nature of the ECM for cultured adipocytes alters temporal lipid accumulation patterns and response to various drugs as compared to 3T3-L1 cells grown on tissue culture plastic. / Thesis / Master of Science (MSc)

Dietary soy isoflavones have been shown to ameliorate insulin resistance and Type 2 diabetes. However, many in vitro studies used supra-physiological concentrations of individual isoflavones that make it difficult to interpret the results as potential mechanisms in vivo. Since the insulin-sensitizing effects of thiazolidinediones, anti-diabetic drugs, have been shown to be mediated through activation of peroxisome proliferators-activated receptor gamma (PPARγ), the key transcription factor for adipocyte differentiation, we examined the effects of the two main soy isoflavones genistein and daidzein either as individual compound or combined on adipocyte differentiation and PPARγ expression, as well as whether the Wnt/β-catenin signaling pathway is the underlying molecular mechanism. In 3T3-L1 cells, genistein and daidzein significantly enhanced adipocyte differentiation. Similarly the expression of PPARγ increased particularly at 20 µmol/L. The stimulatory effect is greater when the two isoflavones are combined, indicating a synergistic effect. Genistein and daidzein also increased the relative abundance of insulin-responsive glucose transporter 4 (GLUT4) mRNA with a greater effect when combined. Wnt10b expression was not affected by soy isoflavones treatments, while Wnt5b expression was only increased by the combination of genistein and daidzein. Our results suggest, that the combination of soy isoflavones has a greater effect in increasing the newly formation of adipocytes that are highly insulin-sensitive via an increase in PPARγ expression as well as increasing the expression of GLUT4. However, genistein and daidzein actions on Wnt signaling remain unclear. These data further support the epidemiological findings for the beneficial effect of soy consumption on insulin sensitivity.

Visando novos estudos com a proteína amilóide sérica A (SAA), propusemos a produção de seu recombinante humano em Escherichia coli, mais especificamente, a isoforma encontrada na fase aguda (A-SAA1) e da constitutivamente expressa (C-SAA4). Realizamos a expressão, identificação e purificação das proteínas recombinantes. Concomitantemente, também avaliamos o efeito da hipóxia na expressão e produção da proteína SAA nativa em linhagens de pré-adipócitos murinos 3T3-L1, não diferenciados e diferenciados e adipócitos humanos. Aparentemente quanto maior o grau de diferenciação celular, maior a expressão e produção da proteína. Para os adipócitos humanos, o perfil de expressão de mRNA da SAA mostra que SAA1>SAA2>SAA4 nas relações 500:150:1. Na hipóxia, há um aumento na expressão de SAA, entretanto não associamos esta expressão a um aumento da concentração da proteína. A importância do reconhecimento de que SAA pode ser umas das proteínas induzidas pela hipóxia em adipócitos é discutida em relação ao seu papel pró-inflamatório. / In order to provide more complex studies with the protein serum amyloid A (SAA), this study proposed the production of its human recombinant in bacteria (Escherichia coli), more specifically, the synthesis of the main isoform found in the acute phase (A-SAA1) and the constitutively expressed (C-SAA4). The expression, identification and purification of the recombinants proteins was performed. Concurrently, we also did a study evaluating the effect of hypoxia in the expression and protein production of native SAA in undifferentiated and differentiated murine preadipocytes 3T3-L1 and humans adipocytes. Apparently, the protein expression and production increases with the cell differentiation degree. For human adipocytes, we demonstrated the mRNA expression profile of SAA, in which SAA1 > SAA2 > SAA4 (500:150:1). In hypoxia, there is an increased expression of SAA, but we could not link it to an increase of protein concentration. The importance of recognizing that SAA may be one of the proteins induced by hypoxia in adipocytes is discussed in relation to the proinflammatory role of this protein.

Células-tronco mesenquimais derivadas de medula óssea (CTMs-MO) e de tecido adiposo (CTMs-TA) são uma ferramenta atrativa para a reparação do tecido ósseo baseada na terapia celular. No presente estudo, foi investigado o potencial osteogênico e adipogênico de CTMs-MO e CTMs-TA, assim como o efeito da intercomunicação entre osteoblastos e adipócitos na expressão do fenótipo celular. CTMs-MO e CTMs-TA de ratos foram cultivadas em meios de crescimento, osteogênico e adipogênico para avaliar a diferenciação osteoblástica e adipocítica. Adicionalmente, osteoblastos e adipócitos foram cocultivados de forma indireta para investigar o efeito dos adipócitos sobre os osteoblastos e vice versa. CTMs-MO e CTMs-TA apresentaram potencial tanto osteogênico quanto adipogênico em condições não indutoras de diferenciação. No entanto, quando expostas ao meio osteogênico, as CTMs-MO exibiram maior expressão gênica de RUNX2, fosfatase alcalina e osteocalcina, expressão proteíca de RUNX2 e maior formação de matriz extracelular mineralizada comparadas às CTMs-TA. Por outro lado, em condições adipogênicas, as CTMs-TA apresentaram maior expressão gênica de PPARγ, proteína adipocítica 2 e resistina, expressão proteíca de PPARγ e maior formação de acúmulo lipídico comparadas às CTMs-MO. A presença de adipócitos em coculturas indiretas inibiu a expressão do fenótipo osteoblástico, enquanto os osteoblastos não apresentaram um efeito marcante sobre a expressão do fenótipo adipocítico. Em conclusão, o presente estudo mostrou que as CTMs-MO são mais osteogênicas enquanto as CTMs-TA são mais adipogênicas. Adicionalmente, foi observado que a intercomunicação entre osteoblastos e adipócitos pode afetar negativamente o reparo ósseo. Assim, postulamos que o maior potencial osteogênico das CTMs-MO as tornam a escolha mais adequada para a indução do reparo ósseo baseado na terapia celular. / Mesenchymal stem cells from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are attractive tools for cell-based therapies to repair bone tissue. In the present study, we investigated the osteogenic and adipogenic potential of BM-MSCs and AT-MSCs as well as the effect of crosstalk between osteoblasts and adipocytes on cell phenotype expression. Rat BM-MSCs and AT-MSCs were cultured either in growth, osteogenic or adipogenic medium to evaluate osteoblast and adipocyte differentiation. Also, osteoblasts and adipocytes were indirectly cocultured to investigate the effect of adipocytes on osteoblast differentiation and vice versa. BM-MSCs and AT-MSCs exhibit osteogenic and adipogenic potential under non-differentiation-inducing conditions. However, when exposed to osteogenic medium, BM-MSCs exhibited higher gene expression of RUNX2, alkaline phosphatase and osteocalcin, RUNX2 protein expression and more extracellular matrix mineralization compared with AT-MSCs. Conversely, under adipogenic conditions, AT-MSCs displayed higher gene expression of PPARγ, fatty acid binding protein 4 and resistin, PPARγ protein expression and more lipid accumulation compared with BM-MSCs. The presence of adipocytes as indirect coculture repressed the expression of osteoblast phenotype while osteoblasts did not exert remarkable effect on adipocyte phenotype expression. In conclusion, the present study showed that BM-MSCs are more osteogenic while AT-MSCs are more adipogenic. Also, we observed that the crosstalk between osteoblasts and adipocytes may negatively impact bone repair. Thus, we postulate that the higher osteogenic potential of BM-MSCs makes them the first choice for inducing bone repair in cell-based therapies.

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.

The aim of the present thesis was to further increase our understanding of mechanisms contributing to and maintaining cellular insulin resistance in type 2 diabetes (T2D). For this reason, the effects of high glucose and insulin levels on glucose transport capacity and insulin signaling, with emphasis on insulin receptor substrate 1 (IRS-1) were assessed in fat cells. Altered levels of IRS-1 have previously been observed in adipose tissue from insulin-resistant and T2D subjects. A high glucose level (≥15 mM) for 24 h exerted only a minor impairment on glucose transport capacity in human adipocytes, as opposed to rat adipocytes. However, when combined with a high insulin level (104 µU/ml), basal and insulin-stimulated glucose transport was significantly impaired in both human and rat adipocytes. This was associated with a depletion of IRS-1 and IRS-2 protein levels in rat adipocytes, as a result of post-translational changes and altered gene transcription, respectively. In human adipocytes was only IRS-1 protein levels reduced. The high glucose/high insulin setting achieved maximal impairment of glucose transport within 6 h. Subsequent incubations of rat adipocytes under physiological conditions could partially restore insulin sensitivity. Interestingly, in both human and rat fat cells, decreased levels of IRSs occurred after the establishment of impaired glucose transport, suggesting that the observed depletion of IRSs is a consequence rather than a cause of insulin resistance. Nonetheless, IRS depletion is likely to further aggravate insulin resistance. Tyrosine phosphorylation of IRS-1 upon insulin stimulation activates the signaling pathway that mediates glucose transport. Pre-treatment of human adipocytes with high glucose and insulin levels was not associated with any alterations in the total IRS-1 Tyr612 phosphorylation following 10 min insulin stimulation. However, a significant increase in basal Tyr612 phosphorylation was observed. Furthermore, a rise in basal IRS-1 Ser312 phosphorylation was found. This is associated with reduced IRS-1 function and is considered to target IRS-1 to degradation pathways, and thus could potentially explain the observed decrease in IRS-1 protein levels. Our results imply an enhanced activation of insulin’s negative-feedback control mechanism that inhibit IRS-1 function. This could potentially have contributed to the observed impairment of insulin action on glucose transport in these cells. Accordingly, we have also shown that the downstream activation of protein kinase B upon insulin-stimulation is significantly impaired in human adipocytes exposed to the high glucose/high insulin setting, indicating a defect in the signaling pathway mediating glucose transport. We also investigated whether there are humoral factors in the circulation of T2D patients that contribute to peripheral insulin resistance. Human adipocytes cultured for 24 h in medium supplemented with 25% serum from T2D subjects, as compared to serum from non-diabetic subjects, displayed significantly reduced insulin-stimulated glucose uptake capacity. The effect could neither be attributed to glucose, insulin, FFA, TNF-α or IL-6 levels in the serum, but other circulating factor(s) seem to be of importance. In conclusion, chronic conditions of elevated glucose and/or insulin levels all impair insulin action on glucose turnover, but to different extents. A clear distinction between rat and human fat cells in the response to these different milieus was also observed. Alterations in the function of the key insulin signaling protein IRS-1 might be involved in the mechanisms underlying the impaired glucose uptake capacity. IRS-1 reduction however, occurs after but probably aggravates the existing insulin resistance. The effects of high glucose and/or insulin levels may be of importance in T2D, but additional novel factors present in the circulation of T2D patients seem to contribute to cellular insulin resistance.

Les cavéolines et les protéines cavines forment le manteau d'invaginations membranaires appelées cavéoles. Dans le tissu adipeux (TA), ces structures sont très abondantes dans les adipocytes et les cellules endothéliales. Une perte d'expression de cavéoline-1 a pour conséquence une lipoatrophie sévère chez les souris invalidées pour ce gène (Cav1(-/-)), ce qui a conduit à suggérer un rôle important de Cav1 pour le contrôle de la capacité de stockage du TA. Par ailleurs, les souris Cav1(-/-) présentent des défauts vasculaires, phénotype totalement corrigé par une réexpression spécifique de Cav1 au niveau de l'endothélium (souris Cav1 RC). Afin de déterminer les rôles respectifs de la cavéoline-1 adipocytaire et endothéliale dans l'établissement de la lipoatrophie observée chez les souris Cav1(-/-), nous avons comparé les phénotypes adipeux de souris contrôles, Cav1(-/-) et Cav1 RC et établi ainsi le rôle majeur de la cavéoline-1 adipocytaire dans l'apparition de ce phénotype. Pour caractériser les mécanismes impliqués, nous avons surexprimé Cav1 et les cavines dans la lignée adipocytaire 3T3-L1. Nous montrons que ces surexpressions augmentent le nombre de cavéoles à la membrane plasmique des adipocytes. En revanche, seule la surexpression de Cav1 induit la croissance des gouttelettes lipidiques (GL), suggérant que cet effet de Cav1 s'exerce en dehors des cavéoles. En accord, la surexpression de Cav1 exclusivement à la surface des GLs augmente la taille de ces organites dans les adipocytes. Ces résultats démontrent un rôle spécifique de la cavéoline-1 à la surface de la GL, indépendant des cavéoles, dans le contrôle de la capacité de stockage lipidique adipocytaire