Glucagon Promotes Gluconeogenesis through the GCGR/PKA/CREB/PGC-1α Pathway in Hepatocytes of the Japanese Flounder Paralichthys olivaceus

In order to investigate the mechanism of glucagon regulation of gluconeogenesis, primary hepatocytes of the Japanese flounder (Paralichthys olivaceus) were incubated with synthesized glucagon, and methods based on inhibitors and gene overexpression were employed. The results indicated that glucagon promoted glucose production and increased the mRNA levels of glucagon receptor (gcgr), guanine nucleotide-binding protein Gs α subunit (gnas), adenylate cyclase 2 (adcy2), protein kinase A (pka), cAMP response element-binding protein 1 (creb1), peroxisome proliferator-activated receptor-γ coactivator 1α (pgc-1α), phosphoenolpyruvate carboxykinase 1 (pck1), and glucose-6-phosphatase (g6pc) in the hepatocytes. An inhibitor of GCGR decreased the mRNA expression of gcgr, gnas, adcy2, pka, creb1, pgc-1α, pck1, g6pc, the protein expression of phosphorylated CREB and PGC-1α, and glucose production. The overexpression of gcgr caused the opposite results. An inhibitor of PKA decreased the mRNA expression of pgc-1α, pck1, g6pc, the protein expression of phosphorylated-CREB, and glucose production in hepatocytes. A CREB-targeted inhibitor significantly decreased the stimulation by glucagon of the mRNA expression of creb1, pgc-1α, and gluconeogenic genes, and glucose production decreased accordingly. After incubating the hepatocytes with an inhibitor of PGC-1α, the glucagon-activated mRNA expression of pck1 and g6pc was significantly down-regulated. Together, these results demonstrate that glucagon promotes gluconeogenesis through the GCGR/PKA/CREB/PGC-1α pathway in the Japanese flounder.