Abstract
Background & aims: The hepatic glucagon-protein kinase A (PKA)-cAMP response element-binding protein (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. Methods: RACK1 was acutely deleted in mouse liver and primary hepatocytes. Metabolic phenotypes were assessed by glucose, pyruvate, glucagon and insulin tolerance tests, as well as hepatocyte glucose production assays. Protein interactions were examined by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and miniTurbo-ID-mediated proximity labeling. Subcellular localization and signaling events were assessed by Western blotting, confocal microscopy, and cellular fractionation. Functional rescue was achieved by hepatic expression of a constitutively active PKA catalytic subunit (PKAcαW196R). Results: Acute hepatic RACK1 deficiency caused fasting hypoglycemia, impaired gluconeogenesis, and improved glucose, pyruvate, and glucagon tolerance without affecting insulin signaling. RACK1 directly bound glucagon receptor (GCGR) and PKA regulatory (RIIα) and catalytic (PKAcα) subunits, as well as CREB, functioning as a dual-compartment scaffold assembling GCGR-PKA complexes at the plasma membrane and PKAcα-CREB complexes in the nucleus. Loss of RACK1 impaired PKAcα translocation, CREB phosphorylation, and gluconeogenic gene expression. These defects were rescued by PKAcαW196R expression. Overexpression of RACK1 WD1-2 and WD3-4 domains, which mediate PKA, GCGR, and CREB interactions, similarly disrupted PKA signaling and gluconeogenesis. Conclusions: RACK1 functions as a dual-compartment scaffold, assembling GCGR-PKA at the plasma membrane and PKAcα-CREB in the nucleus, enabling precise glucagon signaling and gluconeogenesis while sparing insulin pathways, thereby ensuring compartmentalized regulation of hepatic glucose homeostasis.