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The homeobox factor Irx3 maintains adipogenic identity.
Metabolism ( IF 10.8 ) Pub Date : 2019-11-18 , DOI: 10.1016/j.metabol.2019.154014
Jan-Inge Bjune 1 , Laurence Dyer 2 , Gro V Røsland 3 , Karl Johan Tronstad 4 , Pål R Njølstad 5 , Jørn V Sagen 2 , Simon N Dankel 1 , Gunnar Mellgren 1
Affiliation  

BACKGROUND Inhibition of Irx3 and Irx5 has been shown to reduce body weight and white adipose tissue (WAT) mass through cell-autonomous and sympathetic-induced increases in adipocyte beiging and thermogenesis in mice and humans. However, the underlying mechanisms of the Irx control over beiging are still largely unknown, as illustrated by recent reports showing divergent effects of Irx3 on adipocyte metabolism and function. Here, we investigated the role of Irx3 in controlling beige preadipocyte function and differentiation. METHODS Stable knock out of Irx3 in ME3 mouse preadipocytes capable of beiging was performed using a CRISPR-Cas9 system, and the effect on cell differentiation was assessed by qPCR, RNA-seq, Oil-red-O lipid staining and Alcian Blue staining of proteoglycans. Changes in cell identities were validated using cell type enrichment analysis from RNA-seq data. Proliferation and cell cycle progression in undifferentiated cells were measured by WST-1 and flow cytometry, reactive oxygen species (ROS) generation was determined by fluorescence spectrometry and mitochondrial respiration was investigated by Seahorse assay. RESULTS Irx3 was found to be essential for the identity, function and adipogenic differentiation of beige adipocyte precursors. Irx3-KO impaired proliferation, ROS generation and mitochondrial respiration in the preadipocytes. We further observed profound changes in numerous genes during both early and late stages of adipogenic differentiation, including genes important for adipocyte differentiation, cell cycle progression, oxidative phosphorylation (OXPHOS) and morphogenesis. Irx3-KO cells failed to accumulate lipids following adipogenic stimuli, and cell enrichment analysis revealed a loss of preadipocyte identity and a gain of chondrocyte-like identity in Irx3-KO cells during early differentiation. Finally, unlike the control cells, the Irx3-KO cells readily responded to chondrogenic stimuli. CONCLUSIONS Irx3 is required for preadipocyte identity and differentiation capacity. Our findings suggest that, while inhibition of Irx3 may be beneficial during later developmental stages to modulate adipogenesis in the beige direction, constitutive and complete absence of Irx3 in the embryonic fibroblast stage leads to detrimental loss of adipogenic differentiation capacity.

中文翻译:

同源框因子 Irx3 保持脂肪生成特性。

背景已显示抑制 Irx3 和 Irx5 可通过细胞自主和交感神经诱导的小鼠和人类脂肪细胞白化和产热增加来减轻体重和白色脂肪组织 (WAT) 质量。然而,Irx 控制白化的潜在机制在很大程度上仍是未知的,正如最近的报告所示,Irx3 对脂肪细胞代谢和功能的不同影响。在这里,我们研究了 Irx3 在控制米色前脂肪细胞功能和分化中的作用。方法 使用 CRISPR-Cas9 系统稳定敲除 ME3 小鼠前脂肪细胞中的 Irx3,并通过蛋白多糖的 qPCR、RNA-seq、油红-O 脂质染色和阿尔辛蓝染色评估对细胞分化的影响. 使用来自 RNA-seq 数据的细胞类型富集分析来验证细胞身份的变化。通过 WST-1 和流式细胞术测量未分化细胞的增殖和细胞周期进程,通过荧光光谱法测定活性氧 (ROS) 的生成,并通过 Seahorse 测定法研究线粒体呼吸。结果发现 Irx3 对米色脂肪细胞前体的身份、功能和脂肪形成分化至关重要。Irx3-KO 损害前脂肪细胞的增殖、ROS 生成和线粒体呼吸。我们进一步观察到许多基因在成脂分化的早期和晚期发生了深刻的变化,包括对脂肪细胞分化、细胞周期进程、氧化磷酸化 (OXPHOS) 和形态发生很重要的基因。Irx3-KO 细胞在脂肪形成刺激后未能积累脂质,细胞富集分析显示 Irx3-KO 细胞在早期分化过程中前脂肪细胞身份的丧失和软骨细胞样身份的获得。最后,与对照细胞不同的是,Irx3-KO 细胞很容易对软骨形成刺激做出反应。结论 Irx3 是前脂肪细胞身份和分化能力所必需的。我们的研究结果表明,虽然在发育后期抑制 Irx3 可能有利于调节米色方向的脂肪生成,但在胚胎成纤维细胞阶段 Irx3 的组成性和完全缺失会导致脂肪生成分化能力的有害丧失。和细胞富集分析显示,在早期分化过程中,Irx3-KO 细胞中前脂肪细胞身份的丧失和软骨细胞样身份的获得。最后,与对照细胞不同的是,Irx3-KO 细胞很容易对软骨形成刺激做出反应。结论 Irx3 是前脂肪细胞身份和分化能力所必需的。我们的研究结果表明,虽然在发育后期抑制 Irx3 可能有利于调节米色方向的脂肪生成,但在胚胎成纤维细胞阶段 Irx3 的组成性和完全缺失会导致脂肪生成分化能力的有害丧失。和细胞富集分析显示,在早期分化过程中,Irx3-KO 细胞中前脂肪细胞身份的丧失和软骨细胞样身份的获得。最后,与对照细胞不同的是,Irx3-KO 细胞很容易对软骨形成刺激做出反应。结论 Irx3 是前脂肪细胞身份和分化能力所必需的。我们的研究结果表明,虽然在发育后期抑制 Irx3 可能有利于调节米色方向的脂肪生成,但在胚胎成纤维细胞阶段 Irx3 的组成性和完全缺失会导致脂肪生成分化能力的有害丧失。结论 Irx3 是前脂肪细胞身份和分化能力所必需的。我们的研究结果表明,虽然在发育后期抑制 Irx3 可能有利于调节米色方向的脂肪生成,但在胚胎成纤维细胞阶段 Irx3 的组成性和完全缺失会导致脂肪生成分化能力的有害丧失。结论 Irx3 是前脂肪细胞身份和分化能力所必需的。我们的研究结果表明,虽然在发育后期抑制 Irx3 可能有利于调节米色方向的脂肪生成,但在胚胎成纤维细胞阶段 Irx3 的组成性和完全缺失会导致脂肪生成分化能力的有害丧失。
更新日期:2019-11-19
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