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.

An 8-week feeding experiment aimed to investigate the effects of fermented soybean residue in diets on the growth performance, serum antioxidant parameters, intestinal digestive capacity, and microbial composition of the hindgut of Furong crucian carp. The feeds were formulated to contain 0%, 6%, 12%, 18%, and 24% fermented soybean residue (CON, FSR6, FSR12, FSR18, and FSR24, respectively), respectively, to form five diets (crude protein: 33%; crude lipid: 5%). The results showed that incorporating 6% fermented soybean residue into the diet significantly increased the weight gain rate (WGR) and specific growth rate (SGR) and decreased the feed coefficient rate (p < 0.05). Through regression analysis of WGR and SGR, the optimal levels of the dietary fermented soybean residue of Furong crucian carp were determined to be 6.78% and 7.06%. Incorporating fermented soybean residue into the diet decreased the lipid content of the whole body and the levels of glucose, total cholesterol, and triglyceride in the serum of Furong crucian carp. The inclusion of 6% and 12% fermented soybean residue in the diet markedly increased the antioxidant capacity, intestinal amylase activity, and intestinal villous height of Furong crucian carp (p < 0.05). At the 6% level, fermented soybean residue significantly increased the abundance of Romboutsia and Clostridium_sensu_stricto_1 while significantly decreasing the abundance of Vibrio (p < 0.05), indicating that a 6% level of fermented soybean residue was beneficial for intestinal health. In conclusion, incorporating 6–7% fermented soybean residue into the diet of Furong crucian carp was recommended.

Glucagon is a key hormone regulating gluconeogenesis and glucose homeostasis in mammals, yet its regulatory mechanisms in glucose metabolism in carnivorous fish remain incompletely understood. To systematically investigate glucagon-mediated glucose metabolism in Japanese flounder ( Paralichthys olivaceus ), liver samples were collected before glucagon injection (0 h) and at 1 h and 6 h post-injection for transcriptome sequencing (RNA-seq). Transcriptome analysis identified numerous differentially expressed genes involved in glucose and energy metabolism. In total, 507, 1458, and 709 differentially expressed genes were detected in the comparisons of 0 h vs 1 h, 1 h vs 6 h, and 0 h vs 6 h, respectively. KEGG enrichment analysis showed that glucagon activated pathways related to glucagon signaling, insulin resistance, FoxO signaling, and energy metabolism, including AMPK and PPAR pathways, suggesting that glucagon rapidly stimulates gluconeogenesis. At 6 h post-injection, genes involved in glycolysis and glucose transport were upregulated, whereas key gluconeogenic genes were downregulated, indicating attenuation of glucagon-induced metabolic responses. Further analysis showed that glucagon suppressed the insulin-mediated PI3K/AKT signaling pathway. Among the candidate genes, SOCS3 and TRIB3 were upregulated and may serve as key regulators linking glucagon and insulin signaling. Functional experiments further showed that knockdown of TRIB3 reduced glucose levels in hepatocyte culture medium and increased the expression of insulin signaling-related genes. Overall, glucagon regulates glucose metabolism in Japanese flounder by promoting gluconeogenesis while suppressing insulin signaling, providing transcriptomic insights into endocrine regulation in carnivorous fish.