A Dual-Compartment Scaffolding Role for RACK1 in Hepatic Glucagon Signaling and Gluconeogenesis

Background & AimsThe hepatic glucagon-PKA-CREB signaling axis plays a central role in regulating gluconeogenesis and maintaining glucose homeostasis during fasting. However, the mechanisms that govern the spatial coordination and substrate specificity of this pathway remain incompletely understood. This study determines the role of the scaffolding protein RACK1 (Receptor for Activated C Kinase 1) in orchestrating glucagon signaling to regulate hepatic gluconeogenesis. MethodsRACK1 was acutely deleted in mouse liver and primary hepatocytes. Metabolic phenotypes were assessed by glucose, pyruvate, and insulin tolerance tests, and hepatocyte glucose production assays. Protein interactions were analyzed with co-immunoprecipitation, GST pulldown, and proximity ligation assays. Subcellular localization and signaling events were studied by Western blot, confocal microscopy, and fractionation. Functional rescue was performed by hepatic expression of a constitutively active PKA catalytic subunit (PKAcW196R). ResultsAcute hepatic RACK1 deficiency caused fasting hypoglycemia, impaired gluconeogenesis, and improved glucose and pyruvate tolerance without affecting insulin signaling. RACK1 directly bound GCGR, PKA regulatory (RII) and catalytic (PKAc) subunits, and CREB, functioning as a dual-compartment scaffold assembling GCGR-PKA complexes at the plasma membrane and PKAc-CREB complexes in the nucleus. Loss of RACK1 impaired PKAc translocation, CREB phosphorylation, and gluconeogenic gene expression. These defects were rescued by PKAcW196R expresson. Overexpression of RACK1 WD1-2 and WD3-4 domains, which mediate PKA and GCGR interactions, similarly disrupted PKA signaling and gluconeogenesis. ConclusionRACK1 spatially organizes the glucagon-PKA-CREB axis, ensuring precise signal propagation and efficient hepatic gluconeogenesis, revealing a novel mechanism of compartmentalized signal regulation in glucose metabolism.