GDD, a first-in-class APPL1 activator, restores mitochondrial homeostasis and insulin sensitivity with regulation of LKB1-AMPK axis.

Type 2 diabetes mellitus (T2DM) is a prevalent metabolic disorder with increasing morbidity and mortality, and current pharmacotherapies are limited by adverse effects and an inability to reverse the underlying metabolic decline. Tussilago farfara L., a traditional Chinese medicine historically used for diabetes treatment, contains the highly abundant sesquiterpenoid GDD with favorable anti-diabetic properties. Insulin sensitivity was assessed by 2-NBDG uptake in C2C12 myotubes and in high-fat diet (HFD)-induced mice, while mitochondrial content and function were evaluated via Mito-Tracker staining, ATP content, mitochondrial membrane potential, and mitochondrial ROS. Lip-MS, CETSA, DARTS, SPR, molecular docking, and molecular dynamics simulations were applied to identify and validate the direct target of GDD. GDD dose-dependently enhanced insulin-stimulated glucose uptake in C2C12 myotubes, an effect attributed to the clearance of lipotoxic intermediates through mitochondrial biogenesis and functional enhancement, with the LKB1-AMPK cascade participating in this process, as observed in Ampkα1 silencing assay. Critically, GDD bound the PTB domain of APPL1, inhibited its ubiquitination and degradation, and stabilized a conformation favoring APPL1-LKB1 interaction, thereby activating AMPK-related and AMPK-unrelated arms of insulin action. The insulin-sensitizing activity of GDD was abolished in Appl1-silenced cells. In HFD-fed mice, GDD improved insulin sensitivity, reduced fat mass gain, alleviated hyperlipidemia and hepatic steatosis, and restored mitochondrial function in skeletal muscle, with additional protective effects in liver, adipose tissue, pancreas, and kidney. These findings establish GDD as a first-in-class APPL1 activator that reprograms mitochondrial homeostasis and reinstates insulin signaling, providing proof of concept for pharmacological targeting of APPL1 as a novel anti-diabetic strategy.