Crocetin, the key hypoglycemic agent in Gardenia jasminoides identified by spectrum-effect relationship, targets GRK5 to activate AKT2 and improve glucose homeostasis

Abstract Natural products are often used as stand-alone treatments or as complementary approaches alongside modern medicine to manage type 2 diabetes mellitus. Gardenia jasminoides, a substance used as both food and medicine, has been used in traditional Chinese medicine to manage hyperglycemia. Here, we combined spectrum-effect analysis, bioactivity-guided component knockout, untargeted metabolomics, and network pharmacology to identify crocetin as a major glucose-regulating constituent of G. jasminoides and G protein-coupled receptor kinase 5 (GRK5) as a candidate molecular target. Cellular thermal shift assay (CETSA) and surface plasmon resonance (SPR) analyses demonstrated binding between crocetin and GRK5. In an insulin-resistant HepG2 cell model, crocetin increased GRK5 protein abundance and enhanced phosphoinositide 3-kinase (PI3K)/AKT2 signaling and glucose transporter 4 (GLUT4) expression; GRK5 knockdown attenuated these effects and the associated increase in glucose consumption. Reciprocal co-immunoprecipitation further demonstrated an association between GRK5 and AKT2. Collectively, these findings support a model in which crocetin binds GRK5 and promotes GRK5-dependent AKT2 signaling, thereby improving cellular glucose homeostasis.