Background: Brown adipose tissue (BAT) is a specialized organ that dissipates chemical energy to protect against hypothermia and obesity through nonshivering thermogenesis. BAT has been identified in humans, and its activation may reduce body weight and improve glucose homeostasis.
Independently of BAT, thermogenic fat cells (called ‘beige’ adipocytes) have also been identified in depots of white adipose tissue (WAT). This ‘browning’ of WAT occurs following cold exposure, chronic sympathetic stimulation, or thiazolidinedione treatment. In mice, we have previously reported that iron chelation causes ‘browning’ of WAT, concomitantly increasing energy expenditure and attenuating high-fat diet (HFD)-induced weight gain.
WAT is highly heterogeneous, being composed of adipocytes, preadipocytes, vascular endothelial cells, and immune cells. In a WAT depot, therefore, it is difficult to determine changes in gene expression specifically in beige adipocytes. We have used translating ribosome affinity purification (TRAP) to purify ribosomal RNA from genetically-defined beige adipocytes within WAT.
Methods: TRAP mice are transgenic for a rosa26-lox-stop-lox-EGFP-ribosome fusion construct. When crossed with mice expressing Cre, the ‘stop’ is excised, and the ribosomes of Cre-expressing cells are labeled with EGFP. RNA is then isolated using a GFP antibody. TRAP mice were crossed with Prdm16-Cre mice, to generate Prdm16-TRAP mice, which express Cre specifically in beige adipocytes (from Bruce Spiegelman). These mice were fed HFD±iron chelator (30 mg/kg/day) for two weeks. Beige adipocyte RNA was isolated from inguinal subcutaneous WAT using TRAP.
Results: Iron chelation increased the expression of Ucp1 mRNA >80-fold, and expression levels of other regulators of thermogenesis (Ppargc1a, Prdm16, and Cidea) were increased 8 to 24-fold vs. untreated mice. Leptin, a marker of white adipocytes, was reduced 15-fold following iron chelation treatment, and expression of the beige adipocyte marker Tbx1 was increased 8-fold.
Conclusions: TRAP is a valuable molecular tool for studying gene expression changes specifically in beige adipocytes.