The long noncoding RNA ADIPINT is a gatekeeper of pyruvate carboxylase function regulating human fat cell metabolism. The long noncoding RNA ADIPINT is a gatekeeper of pyruvate carboxylase function regulating human fat cell metabolism

The pleiotropic function of long noncoding RNAs (lncRNAs) is well recognized, but their direct role in governing metabolic homeostasis is less understood. Here, we describe a human adipocyte-specific lncRNA, ADIPINT, that regulates pyruvate carboxylase (PC), a pivotal enzyme in energy metabolism. With a novel approach, Targeted RNA-protein identification using Orthogonal Organic Phase Separation (TROOPS), and validation with electron microscopy, we show that ADIPINT binds to PC. ADIPINT knockdown alters the interactome and decreases the abundance and enzymatic activty of PC in the mitochondria. Reduced ADIPINT or PC expression lowers adipocyte lipid synthesis, breakdown, and lipid content. Overall design: Knockdown of ADIPINT was carried out on day 8 of differentiation (when the cells showed adipocyte features), using a Neon™ transfection system MPK5000 (Invitrogen) with 2 pulses of 710 ms at 1300 V. Details of the Antisense LNA GapmeRs (Qiagen) used for ADIPINT knockdown and control conditions are following: GapmeR_1: TCTTGATTGCTGCAGA; GapmeR_2: TACTTTGCCTCTTAGA; GapmeR_3: CGAAGATTCATGGTCA; GapmeR NC: Negative Control A (Cat# LG00000002). Cell viability was assessed using Alamar blue fluorescence (ThermoFisher) as per manufacturers’ recommendations. All hADSC analyses were performed at day 13 of differentiation for RNA extraction. 4 replicates for each Gapmer and 7 replicates for the sicontrol group. Total RNA was extracted for microarray analysis.长链非编码RNA（long noncoding RNAs，lncRNAs）的多效性功能已得到广泛认可，但其在调控代谢稳态中的直接作用仍有待深入阐明。本研究报道了一种人脂肪细胞特异性长链非编码RNA ADIPINT，其可调控丙酮酸羧化酶（pyruvate carboxylase，PC）——能量代谢中的关键酶。我们借助一种全新的基于正交有机相分离的靶向RNA-蛋白质鉴定技术（Targeted RNA-protein identification using Orthogonal Organic Phase Separation，TROOPS），并结合电子显微镜验证，证实ADIPINT可与PC相结合。ADIPINT敲低会改变细胞的相互作用组，并降低线粒体中PC的丰度及酶促活性。ADIPINT表达下调或PC表达降低，均会降低脂肪细胞的脂质合成、分解能力及细胞内脂质含量。实验整体设计如下：在细胞分化第8天（此时细胞已呈现脂肪细胞特征），使用Neon™ 转染系统MPK5000（Invitrogen），以1300 V电压、710 ms时长的脉冲参数进行2次电转，以此敲低ADIPINT的表达。本研究用于敲低ADIPINT的反义锁核酸（Locked Nucleic Acid，LNA）GapmeRs（Qiagen）及对照试剂的具体信息如下：GapmeR_1: TCTTGATTGCTGCAGA; GapmeR_2: TACTTTGCCTCTTAGA; GapmeR_3: CGAAGATTCATGGTCA; GapmeR NC: 阴性对照A（货号LG00000002）。细胞活力检测采用Alamar blue荧光法（ThermoFisher），严格按照厂商推荐的操作流程进行。所有成人脂肪干细胞（human adipose-derived stem cells，hADSC）相关实验均在分化第13天开展，以提取总RNA。每个GapmeR组设置4个生物学重复，阴性对照组设置7个生物学重复。提取总RNA用于基因芯片分析。