Glucagon receptor-mediated regulation of gluconeogenic gene transcription is endocytosis-dependent in primary hepatocytes

A number of G protein-coupled receptors (GPCRs) are now thought to use endocytosis to promote cellular cAMP signaling that drives downstream transcription of cAMP-dependent genes. We tested if this is true for the glucagon receptor (GCGR), which mediates physiological regulation of hepatic glucose metabolism via cAMP signaling. We show that epitope-tagged GCGRs undergo clathrin- and dynamin-dependent endocytosis in HEK293 and Huh-7-Lunet cells after activation by glucagon within 5 min and transit via EEA1-marked endosomes shown previously to be sites of GPCR/Gs-stimulated production of cAMP. We further show that endocytosis potentiates cytoplasmic cAMP elevation produced by GCGR activation and promotes expression of phosphoenolpyruvate carboxykinase 1 (PCK1), the enzyme catalyzing the rate-limiting step in gluconeogenesis. We verify endocytosis-dependent induction of PCK1 expression by endogenous GCGRs in primary hepatocytes and show similar control of two other gluconeogenic genes (PGC1α and G6PC). Together, these results implicate the endosomal signaling paradigm in metabolic regulation by glucagon.