Project description:We depicted a genome-wide integrative view of DNA methylome by reduced representation bisulfite sequencing (RRBS) and transcriptome by RNA-seq during brown and white adipocyte differentiation. Our analysis demonstrated that DNA methylation is a stable epigenetic signature for brown and white cell lineage before, during and after differentiation. When comparing white to brown adipocytes at all three time points of differentiation, we identified five members of the Hox family whose expression levels were anti-correlated with promoter methylation, suggesting a regulatory role of DNA methylation in transcription.
Project description:We depicted a genome-wide integrative view of DNA methylome by reduced representation bisulfite sequencing (RRBS) and transcriptome by RNA-seq during brown and white adipocyte differentiation. Our analysis demonstrated that DNA methylation is a stable epigenetic signature for brown and white cell lineage before, during and after differentiation. When comparing white to brown adipocytes at all three time points of differentiation, we identified five members of the Hox family whose expression levels were anti-correlated with promoter methylation, suggesting a regulatory role of DNA methylation in transcription.
Project description:Brown adipose tissue (BAT) is a central thermogenic organ that enhances energy expenditure (EE) and cardiometabolic health. However, regulators that specifically increase the number of thermogenic adipocytes are still an unmet need. Here, we show by phosphoproteomics that cAMP activates distinct signaling pathways in brown progenitors, with the cAMP-EPAC1 axis enhancing proliferation and differentiation of thermogenic but not white adipocytes. Further analysis revealed that a specific subpopulation of preadipocytes that are PDGFRα-positive express EPAC1. In vivo, pharmacological activation of EPAC1 enhances BAT growth and browning of white fat, leading to increased EE and reduced diet-induced adiposity. In contrast, mice lacking EPAC1 in PDGFRα-positive preadipocytes show the opposite phenotype. Importantly, EPAC1 activation enhances proliferation and differentiation of human brown adipocytes and human brown fat organoids. Interestingly, a coding variant in EPAC1 that positively correlates with BMI abolishes norepinephrine-induced proliferation of brown adipocytes. Thus, EPAC1 might be an attractive target to enhance thermogenic adipocyte number and EE to combat metabolic diseases.
Project description:Brown adipose tissue is specialized to burn lipids for heat generation as a natural defense against cold and obesity. Previous studies established microRNAs as essential regulators of brown adipocyte differentiation, but it remains unknown whether microRNAs are required for the feature maintenance of mature brown adipocytes. To address this question, we ablated Dgcr8, a key regulator of the microRNA biogenesis pathway, in mature brown as well as white adipocytes. The adipose tissue -specific Dgcr8 knockout mice displayed enlarged but pale interscapular brown fat with decreased expression of genes characteristic of brown fat, and the mice were intolerant to cold exposure. In vitro primary brown adipocyte cultures confirmed that microRNAs are required for marker gene expression in mature brown adipocytes. We also demonstrated that microRNAs are essential for the browning of subcutaneous white adipocyte both in vitro and in vivo. Using this animal model, we performed microRNA expression profiling analysis and identified a set of BAT-specific microRNAs that are up-regulated during brown adipocyte differentiation and enriched in brown fat compared to other organs. We identified miR-182 and miR-203 as new regulators of brown adipocyte development. Taken together, our study demonstrates an essential role of microRNAs in the maintenance as well as the differentiation of brown adipocytes.
Project description:The adipose organ, including white and brown adipose tissues, is an important player in systemic energy homeostasis, storing excess energy in form of lipids while releasing energy upon various energy demands. Recent studies have demonstrated that white and brown adipocytes also function as endocrine cells and regulate systemic metabolism by secreting factors that act locally and systemically. However, a comparative proteomic analysis of secreted factors from white and brown adipocytes and their responsiveness to adrenergic stimulation has not been reported yet. Therefore, we studied and compared the secretome of white and brown adipocytes, with and without norepinephrine (NE) stimulation. Our results reveal that in the absence of NE, carbohydrate metabolism-regulating proteins are preferably secreted from white adipocytes, while brown adipocytes predominantly secrete integrin signaling proteins. Upon NE stimulation, white adipocytes secrete more proteins involved in lipid metabolism, while brown adipocytes secrete more proteins with specific anti-inflammatory properties. In conclusion, our study provides a comprehensive catalogue of novel adipokine candidates secreted from white and brown adipocytes with many of them responsive to NE.
Project description:Adipose tissue abundance relies partly on the factors that regulate adipogenesis, i.e. proliferation and differentiation of adipocytes. While the transcriptional program that initiates adipogenesis is well-known, the importance of microRNAs in adipogenesis is less well studied. We thus set out to investigate whether miRNAs would be actively modulated during adipogenesis and obesity. Several models exist to study adipogenesis in vitro, of which the cell line 3T3-L1 is probably the most well known, albeit not the most physiologically appropriate. We used a microarray strategy to provide a global profile of miRNAs in brown and white primary murine adipocytes (prior to and following differentiation) and evaluated the similarity of the responses to non-primary cell models, through literature data-mining. We found 65 miRNAs regulated during in vitro adipogenesis in primary adipocytes. When we compared our primary adipocyte profiles with those of cell lines reported in the literature, we found a high degree of difference in adipogenesis-regulated miRNAs. We evaluated the expression of 10 of our adipogenesis-regulated miRNAs using real-time qPCR and then selected 5 miRNAs that showed robust expression levels and profiled these by qPCR in subcutaneous adipose tissue of 20 humans with a range of body mass indices (BMI, range=21-48). Of the miRNAs tested, mir-21 was both highly expressed in human adipose tissue and positively correlated with BMI (R2=0.49, p<0.001). In conclusion, we provide the preliminary analysis of miRNAs important for primary cell in vitro adipogenesis and find that the inflammation-associated miRNA, mir-21, is up-regulated in subcutaneous adipose tissue in human obesity. 3 samples of pre adipocytes isolated from brown adipose tissue examined pre and post differentiation to brown adipocytes. 3 samples of pre-adipocytes isolated from white adipose tissue and examined pre and post differentiation to adipocytes.
Project description:The experiment was designed to determine the gene expression changes cultured brown adipocytes in response to the inflammatory stimulus of LPS treatment. Both wild type and TLR4 knockout cells were applied to enable assessment of the contribution of TLR4 to the response.