The role of secreted factors in white and brown adipogenesis

Abstract

Obesity is a global epidemic and the main risk factor for cardiovascular diseases which is the number one cause of death worldwide. Current treatment methods for obesity are very limited and often fail in the long term. In the last decade it has been demonstrated that not only human newborns but also adults have functional brown adipose tissue mass. Furthermore, there is evidence for uncoupling protein 1 (UCP1)-expressing adipocytes within the white adipose tissue (WAT) of humans. White adipocyte browning is defined by a significant increase in UCP1 expression in the adipose tissue depot and has been shown to significantly increase overall metabolic health inter alia by higher glucose uptake and fatty acid uptake, thus providing a strategy for obesity treatment. An approach for implementation, is to stimulate human adipose stromal cells (hASCs) within the WAT to undergo adipogenesis into UCP1-expressing adipocytes. However, the mechanism how these cells differentiate either into adipocytes with a white or with a brown phenotype is not fully understood. Our group has shown that hASCs isolated from deep neck (dn) adipose tissue differentiated in vitro into adipocytes with a brown phenotype and hASCs derived from the subcutaneous (sc) adipose tissue differentiated into adipocytes with a white phenotype. Microarray analysis revealed different expression patterns of these hASCs dependent on their adipose tissue origin. Therefore, we hypothesized, that hASCs secrete factors which stimulate or inhibit the adipogenesis into a brown adipocyte phenotype in an auto- or paracrine manner. After target ranking of these differentially regulated genes and literature screening the latent transforming growth factor beta binding proteins (LTBP1-4) were identified as potential factors involved in generating a brown adipocyte phenotype. Except for LTBP2, they are known to play a crucial role in the bioavailability of transforming growth factors (TGFβ). These TGFβ cytokines in turn are known to be involved in regulating UCP1 expression in vitro and WAT browning in vivo. The SMAD2/SMAD3 mediated TGFβ pathway has already been shown to inhibit white adipocyte browning and TGFβ2 was reported to increase UCP1 expression in murine BAT. The aim of this study was to investigate the impact of the LTBP isoforms on brown marker genes and the metabolic function in vitro. LTBP1, LTBP3 and LTBP4 were significantly higher expressed in the dn hASCs and all four LTBPs were expressed during the adipogenesis of a human sc preadipocyte cell strain (SGBS cells). LTBP1 and LTBP4 were not involved in the generation of a brown phenotype in SGBS cells. However, LTBP2 deficiency showed a reduced UCP1 mRNA expression and altered metabolic function. Moreover, deficiency of LTBP3 in SGBS adipocytes led to a repression of UCP1 expression on mRNA and protein level. Furthermore, the metabolic function of these cells was altered as well. Inhibition of the TGFβ-pathway in human SGBS cells led in three different approaches to an adipogenesis towards a white phenotype. TGFβ deficiency, TGFβ receptor1 kinase inhibition and knockdown of SMAD4 led to a significant decrease in UCP1 expression, whereby the differentiation rate was not affected in these cells. These results are in accordance with the observed UCP1 expression in LTBP2- and LTBP3-deficient cells. Measuring the TGFβ content in the supernatant of LTBP2- and LTBP3-deficient preadipocytes revealed a significant lower level for TGFβ2 compared to the control. The TGFβ2 knockout in turn resulted in a decreased UCP1 expression and metabolic function as well. Moreover, the LTBP3 and TGFβ2 axis seems to play also a relevant role in human WAT browning. LTBP3 expression correlated positively with the TGFβ2 expression in human sc WAT samples. Furthermore, both LTBP3 and TGFβ2 correlated significantly positive with the UCP1 expression in the same patient samples. As a result, one can hypothesize that LTBP3 regulates the adipogenesis towards a brown phenotype via TGFβ2 bioavailability. The findings of this thesis revealed LTBP3 as a new factor involved in generating a brown adipocyte phenotype. Furthermore, the inhibition of TGFβ pathway demonstrated new insights of TGFβ and the regulation of UCP1 expression. The specific pathway how LTBP3 and TGFβ2 mediate a shift towards a brown adipocyte phenotype should be addressed in the future, thereby identifying a new mechanism to induce UCP1 expression in human WAT. As a consequence, LTBP3 or TGFβ2 may become new promising targets for the treatment of obesity.