Ligand‐Activated Fluorescent Probe Reveals Mitochondrial Long‐Chain Saturated Fatty Acid Flux: An Effective Tool for Metabolic Disorder Studies

Real-time monitoring of long-chain saturated fatty acid (LCSFA) dynamics with high specificity at the subcellular level remains a major technical challenge. Herein, a high-efficiency long-chain saturated fatty acid-labeled sensor, termed HELLEN, based on the ligand binding domain (LBD) of peroxisome proliferator-activated receptor alpha (PPARα) and the "LXXLL" domain (LXD) of steroid receptor coactivator-1 (SRC-1) is generated via genetically encoded expression. HELLEN exhibits a pronounced fluorescent increase in response to intracellular stearic acid (SA, C18:0), one of the major LCSFAs, and this response demonstrates subcellular resolution and excellent selectivity. Using HELLEN, we detect the uptake of exogenous SA under physiological conditions and visualize SA translocation to mitochondria in hepatocytes under inflammatory stress. Furthermore, in an inflammatory mouse model, excessive LCSFAs, including SA, are trafficked to hepatic mitochondria. Together, HELLEN provides a novel tool for mapping SA metabolism and offers valuable insights into LCSFA-related metabolic pathways.