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. 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.