Discovery and functional assessment of a novel adipocyte population driven by intracellular Wnt/β-catenin signaling in mammals

Wnt/β-catenin signaling has been well established as a potent inhibitor of adipogenesis. Here, we identified a population of adipocytes that exhibit persistent activity of Wnt/β-catenin signaling, as revealed by the Tcf/Lef-Gfp reporter allele, in embryonic and adult mouse fat depots, named as Wnt+ adipocytes. We showed that this β-catenin-mediated signaling activation in these cells is Wnt ligand- and receptor-independent but relies on AKT/mTOR pathway and is essential for cell survival. Such adipocytes are distinct from classical ones in transcriptomic and genomic signatures and can be induced from various sources of mesenchymal stromal cells including human cells. Genetic lineage-tracing and targeted cell ablation studies revealed that these adipocytes convert into beige adipocytes directly and are also required for beige fat recruitment under thermal challenge, demonstrating both cell autonomous and non-cell autonomous roles in adaptive thermogenesis. Furthermore, mice bearing targeted ablation of these adipocytes exhibited glucose intolerance, while mice receiving exogenously supplied such cells manifested enhanced glucose utilization. Our studies uncover a unique adipocyte population in regulating beiging in adipose tissues and systemic glucose homeostasis. We took advantage of the Wnt/β-catenin signaling specific reporter mouse line TCF/Lef:H2B-GFP that allows real-time monitoring of Wnt/β-catenin activity at single-cell resolution. We cultured Wnt+ and Wnt- adipocytes in vitro and enriched them based on FACS separation, using GFP as a readout. To distinguish Wnt+ adipocytes from Wnt- fat cells and explore the global diversity at molecular and genomic levels, we profiled cultured Wnt+ and Wnt- adipocytes derived from adult mice adipose tissues by scRNA-seq and scATAC-seq. Wnt+ adipocytes from immortalized GFPpos-1 precursor cells, after 3-day pro-adipogenic induction, were subject to LF3 treatment for one day to suppress the intracellular /β-catenin-mediated signaling. Wnt+ adipocytes without LF3-treated were included as control groups. Total RNAs were extracted from control and treatment groups (n = 3 each) and prepared using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEW ENGLAND BioLabs) according to the manufacturer?s instruction. The normalized dsDNA libraries were pooled and sequenced on Illumina NextSeq 1000 and NextSeq 2000 with P2 (100 cycles) kit at the Center for Translation Research in Infection and Inflammation in Tulane School of Medicine. HISAT2, featureCounts, and DESeq2 softwares were used for reads alignment to mouse reference genome (version mm10), quantification of transcript abundances, and differential gene expression analysis.