Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis.

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.

Both proliferation and lipogenesis of brown adipocytes contribute to postnatal brown adipose tissue growth in mice.

Brown adipose tissue (BAT) is the primary non-shivering thermogenesis organ in mammals, which plays essential roles in maintaining the body temperature of infants. Although the development of BAT during embryogenesis has been well addressed in rodents, how BAT grows after birth remains unknown. Using mouse interscapular BAT (iBAT) as an example, we studied the cellular and molecular mechanisms that regulate postnatal BAT growth. By analyzing the developmental dynamics of brown adipocytes (BAs), we found that BAs size enlargement partially accounts for iBAT growth. By investigating the BAs cell cycle activities, we confirmed the presence of proliferative BAs in the neonatal mice. Two weeks after birth, most of the BAs exit cell cycle, and the further expansion of the BAT was mainly due to lipogenesis-mediated BAs volume increase. Microscopy and fluorescence-activated cell sorting analyses suggest that most BAs are mononuclear and diploid. Based on the developmental dynamics of brown adipocytes, we propose that the murine iBAT has two different growth phases between birth and weaning: increase of BAs size and number in the first two weeks, and BAs size enlargement thereafter. In summary, our data demonstrate that both lipogenesis and proliferation of BAs contribute to postnatal iBAT growth in mice.