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

Wnt/ß-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/ß-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 ß-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. Overall design: We took advantage of the Wnt/ß-catenin signaling specific reporter mouse line TCF/Lef:H2B-GFP that allows real-time monitoring of Wnt/ß-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 /ß-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.

Wnt/β-连环蛋白（Wnt/β-catenin）信号通路已被证实为脂肪生成的强效抑制剂。本研究在胚胎与成年小鼠脂肪垫中，通过Tcf/Lef-GFP报告等位基因的荧光信号，鉴定出一群持续激活Wnt/β-连环蛋白信号通路的脂肪细胞，并将其命名为Wnt+脂肪细胞。我们证实，该群细胞中β-连环蛋白介导的信号激活不依赖Wnt配体与受体，但依赖AKT/mTOR信号通路，且对细胞存活至关重要。这群脂肪细胞在转录组与基因组特征上均与经典脂肪细胞存在显著差异，且可从包括人类细胞在内的多种间充质基质细胞来源诱导获得。遗传谱系示踪与靶向细胞清除实验显示，这群脂肪细胞可直接转化为米色脂肪细胞，同时在热应激下的米色脂肪募集过程中发挥必需作用，表明其在适应性产热中同时具备细胞自主性与非细胞自主性功能。此外，靶向清除这群脂肪细胞的小鼠会表现出葡萄糖耐受不良，而外源移植该群细胞的小鼠则展现出更强的葡萄糖利用能力。本研究揭示了一类独特的脂肪细胞群，其可调控脂肪组织的米色化进程与系统性葡萄糖稳态。

实验整体设计：本研究利用Wnt/β-连环蛋白信号通路特异性报告小鼠品系TCF/Lef:H2B-GFP，可在单细胞分辨率下实时监测Wnt/β-连环蛋白的活性。我们体外培养Wnt+与Wnt-脂肪细胞，并以绿色荧光蛋白（GFP）作为检测标记，通过荧光激活细胞分选（FACS）对两类细胞进行富集。为区分Wnt+与Wnt-脂肪细胞，并从分子与基因组层面探究其全局多样性，我们对成年小鼠脂肪组织来源的体外培养Wnt+与Wnt-脂肪细胞进行了单细胞RNA测序（scRNA-seq）与单细胞转座酶可及性测序（scATAC-seq）。将永生化GFPpos-1前体细胞进行3天的促脂肪生成诱导以获得Wnt+脂肪细胞，随后用LF3处理细胞1天以抑制细胞内β-连环蛋白介导的信号通路，未接受LF3处理的Wnt+脂肪细胞作为对照组。分别从对照组与处理组（每组n=3）中提取总RNA，按照制造商说明书，使用NEBNext Ultra II Directional RNA Library Prep Kit for Illumina（NEW ENGLAND BioLabs）构建测序文库。将标准化的双链DNA文库混合后，在杜兰大学医学院感染与炎症转化研究中心使用Illumina NextSeq 1000、NextSeq 2000测序仪及P2（100个循环）测序试剂盒进行测序。使用HISAT2、featureCounts与DESeq2软件完成测序读段比对至小鼠参考基因组（版本mm10）、转录本丰度定量及差异基因表达分析。