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.


Introduction
Glucose homeostasis is critical to animals. It requires the coordination between glucagon and insulin. In mammals such as humans, pigs, and rats, glucagon is a 29amino acid peptide encoded by the proglucagon gene (GCG) and produced by the pancreatic α-cells [1]. The action of glucagon is initiated by binding to the glucagon receptor (GCGR). GCGR couples to GTP-binding G protein and leads to the subsequent activation of adenylate cyclase (ADCY) to produce cyclic adenosine monophosphate (cAMP); the rise in cAMP activates protein kinase A (PKA), which in turn activates cAMP response element-binding (CREB) protein. The stimulation of CREB protein up-regulates the transcription of peroxisome proliferator -activated receptor-γ coactivator-1α (PGC-1α), thereby increasing gluconeogenesis by stimulating the gene expression of phosphoenolpyruvate carboxykinase (PCK) and glucose-6-phosphatase (G6PC) [2,3].
Type 2 diabetes (T2D) has become a worldwide metabolic disease. An uncontrolled increase in blood glucose levels is the hallmark of T2D [4,5]. Patients with T2D exhibit Table 1. Sequences of the PCR primers used for cloning, plasmid construction, and qPCR analysis.

Sequence Analysis and 3D Structure of GCG
All nucleotide sequences were blasted using the BlastX algorithm at the National Center for Biotechnology Information to determine gene identity. The Open Reading Frame of GCG was identified by ORF finder, and the cDNA sequence was translated. The protein domain was predicted by SMART. The GCG gene sequences of different species, including fish, were selected in the GenBank, and ClustalW software version 2.1 (University College Dublin, Dublin, Ireland) was used for multiple sequence comparison analysis. A phylogenetic tree was constructed based on multiple sequence alignments using the neighbor-joining method with the MEGA 5.0 program. The protein structure model was predicted using the SWILL-MODLE workspace.

Synthesis of Glucagon
According to the deduced amino acid sequence, the Japanese founder glucagon peptide with 29 amino acids was synthesized using the solid-phase synthetic peptide method by Sangon Biotech (Shanghai, China). The molecular weight of this peptide was detected by liquid chromatography-mass spectrometry, and its purity was detected by high-performance liquid chromatography.

Treatments with Glucagon and Inhibitors in Hepatocytes
Hepatocytes were isolated from the liver of Japanese flounder and cultured as described previously with some modifications [26]. Japanese flounder with an average weight of 38.52 ± 7.23 g were acclimated and kept in seawater containing 100 U mL −1 of penicillin (Gibco, Grand Island, NY, USA) and 100 µg mL −1 of streptomycin (Gibco, Grand Island, NY, USA) for 24 h. At the start of the experiment, the fish were anesthetized with eugenol (1:10,000 dilution with seawater) and then bloodletting for 5 min. After the fish surface was dried with a towel and disinfected with 75% (v/v) alcohol, the livers of five fish were excised and washed three times with cold phosphate-buffered saline (PBS) (Solarbio, Beijing, China) containing 100 IU mL −1 of penicillin-streptomycin (Gibco, Grand Island, NY, USA). The liver fragments were diced to 1 mm 3 in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) (Gibco, Grand Island, NY, USA) and then digested with 0.25% trypsin-EDTA (Gibco, Grand Island, NY, USA) for 15 min. During the digestion, the supernatant was collected every 5 min by a Pasteur pipette (Corning Inc., Corning, NY, USA) in 2 volumes of DMEM/F12 medium containing 15% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). Then, the isolated hepatocytes were purified through a sterile 40 µm mesh and harvested by centrifugation at 1000× g for 5 min at 4 • C. Red blood cell lysis buffer (Gibco, Grand Island, NY, USA) was used to eliminate the red blood cells. After washing twice with PBS for debris removal, the purified hepatocytes were re-suspended in the DMEM/F12 medium containing 15% FBS, 1% L-glutamine (Gibco, Grand Island, NY, USA), and 1% antibiotics. The hepatocytes were seeded in 6-well plates (Corning Inc., Corning, NY, USA) at a density of 1.0 × 10 6 cells/well and placed at 23 • C in a normal-atmosphere incubator overnight. The culture medium was refreshed every 2 days until the cells became 80-90% confluent. After the removal of the old culture medium, the test substances prepared in DMEM/F12 medium (the glucose concentration was 17.5 mM) were gently overlaid onto the hepatocytes for further studies.

Overexpression of gcgr
The gcgr gene coding sequence (CDS) was amplified using specific primers (Table 1). The template gcgr was PCR-amplified using primers with homology arms (Table 1) to the BamHI region in pcDNA3.1-EGFP. The resulting amplicons were purified by gel electrophoresis and extracted using the SanPrep Column DNA Gel Extraction Kit (Sangon Biotech, Shanghai, China). The pcDNA3.1-EGFP plasmid (Biofeng, Shanghai, China) was digested with the enzyme BamHI. The purified resulting amplicons were then ligated into the pcDNA3.1-EGFP plasmid using T4 DNA ligase (TransGen, Beijing, China). The plasmid constructed was called pcDNA3.1-GCGR-EGFP. The construct was transformed into the Escherichia coli strain DH5α and then confirmed by DNA sequencing. The bacteria successfully transformed with the target gene were expanded by shaking the flask culture overnight, and adequate plasmid for transfection was collected using the EasyPure HiPure Plasmid MaxiPrep Kit (TransGen, Beijing, China).
Primary hepatocytes were seeded in 6-well plates at a density of 1.0 × 10 6 cells/well. When the hepatocytes were at 80-90% confluency, the cells were transfected with pcDNA3.1-GCGR-EGFP using 3.75 µL/well of Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. For each well, 5 µg of plasmid was used. The pcDNA3.1-EGFP plasmid served as a negative control for the experiment. No plasmid but an equal volume of PBS was added in the control group. After 48 h, the transfection efficiency was determined using a fluorescence microscope (Echo Laboratories, USA) and qPCR. The number of EGFP-positive cells was calculated using Image-Pro Plus 6.0 software (Media Cybernetics, USA). The transfection experiments were performed in triplicate.

Measurements of Metabolites
The medium of the hepatocytes was collected. The glucose concentration was determined using a glucose assay kit (F006-1-1) by the glucose oxidase method [27]. The concentration of lactate was determined using a lactate assay kit (A019-2) by the lactate dehydrogenase method [28]. The pyruvate concentration was measured with a pyruvate assay kit (A081-1-1) at an absorbance of 505 nm and calculated with reference to a pyruvate standard sample [29]. All these kits were purchased from Jiancheng Bioengineering Institute, Nanjing, China.
Glucose production was determined as previously described [30]. After incubation of the hepatocytes with appropriate concentrations of the test substances, the culture medium was replaced with 1 mL of glucose-free DMEM without phenol red (Gibco, Grand Island, NY, USA), supplemented with 0.5% BSA, 20 mM sodium lactate, and 2 mM sodium pyruvate. After 6 h of incubation, the medium was collected, and glucose concentration was measured using a glucose oxidation kit (Jiancheng Bioengineering Institute, Nanjing, China).

Real-Time Quantitative RT-PCR Analysis
The gene expression analysis was carried out using the SYBR Green Real-time PCR Master Mix (Q711-02, Vazyme Biotech, Nanjing, China) in a quantitative thermal cycler (Quant Studio 5, Applied Biosystems, Waltham, MA, USA) with the primers shown in Table 1. The mRNA levels were normalized to those of β-actin as a housekeeping gene. The data were analyzed by the ∆∆Ct method. Before the detection, the amplification efficiency of the primers for each gene was evaluated by serial dilutions to ensure that it was close to 100%.

Western Blot Analysis
The procedures of Western blot analysis were performed as previously described [31]. The following antibodies were used: antibodies against phospho-CREB (Ser 133 ) ( , and β-actin (dilution 1:5000, cat. No. bs-0061R, Bioss Antibodies, Beijing, China). All the antibodies were raised against orthologs that share amino acid similarities to the proteins in the Japanese flounder. The detection of Western blot bands of the correct sizes by these antibodies was confirmed. After antibody incubation, HRPconjugated goat anti-rabbit (dilution 1:5000, cat. No. A0208, Beyotime Biotechnology, Shanghai, China) or goat anti-mouse IgG (dilution 1:5000, cat. No. A0216, Beyotime Biotechnology, Shanghai, China) was added, and the ECL reagent (Beyotime Biotechnology, Shanghai, China) was used as an HRP substrate for signal development. The Western blot bands were quantified using NIH Image 1.63 software.

Statistics Analysis
All data are presented as means ± standard error and were analyzed using the software SPSS 25.0. Two-tailed t-test, one-way ANOVA with Tukey post-hoc test, or two-way ANOVA with Tukey post-hoc test was used to compare the means between groups, as indicated in the Figure legends. p < 0.05 was considered significant.

Glucagon Induces Gene Expression in Hepatocytes and Increases the Glucose Concentration in the Medium
As shown in Figure 2A, the mRNA levels of gcgr, guanine nucleotide-binding protein Gs subunit alpha (gnas), adcy2, pka, creb1, and pgc-1α were stimulated by glucagon incubation in a time-dependent manner. The mRNA levels of gnas and adcy2 were up-regulated significantly at 3 h compared to 0 h (p < 0.05) and reached their maximum values at 9 h and 24 h, respectively. Compared with 0 h, the gcgr, pka, and creb1 mRNA levels were significantly up-regulated at 9 h (p < 0.05). The maximal effect of glucagon on pgc-1α mRNA levels was observed at 24 h after treatment. As shown in Figure 2B, the mRNA levels of pck1 and g6pc in the glucagon group were higher than those in the control group after treatment. Both genes were up-regulated as the time increased, and the maximal stimulatory responses for them following glucagon incubation were observed at 24 h. Then, the mRNA levels of pck1 and g6pc declined. The glucose concentration in the medium of the glucagon group was higher than that in the medium of the control group after incubation. It was significantly higher than that in the medium of the control group at 48 h (p < 0.05) ( Figure 2C).

Glucagon Induces Gene Expression in Hepatocytes and Increases the Glucose Concentration in the Medium
As shown in Figure 2A, the mRNA levels of gcgr, guanine nucleotide-binding protein Gs subunit alpha (gnas), adcy2, pka, creb1, and pgc-1α were stimulated by glucagon incubation in a time-dependent manner. The mRNA levels of gnas and adcy2 were up-regulated significantly at 3 h compared to 0 h (p < 0.05) and reached their maximum values at 9 h and 24 h, respectively. Compared with 0 h, the gcgr, pka, and creb1 mRNA levels were significantly up-regulated at 9 h (p < 0.05). The maximal effect of glucagon on pgc-1α mRNA levels was observed at 24 h after treatment. As shown in Figure 2B, the mRNA levels of pck1 and g6pc in the glucagon group were higher than those in the control group after treatment. Both genes were up-regulated as the time increased, and the maximal stimulatory responses for them following glucagon incubation were observed at 24 h. Then, the mRNA levels of pck1 and g6pc declined. The glucose concentration in the medium of the glucagon group was higher than that in the medium of the control group after incubation. It was significantly higher than that in the medium of the control group at 48 h (p < 0.05) ( Figure 2C).   Figure 3A shows that increasing concentrations of glucagon could elevate the mRNA levels of glucagon pathway-related genes in a dose-dependent manner. Compared with the control group, the gcgr mRNA levels were significantly up-regulated when the glucagon concentration was 1 nM (p < 0.05). Treatment with 10 nM glucagon significantly increased the pka and pgc-1α mRNA levels (p < 0.05). The mRNA levels of adcy2 were up-regulated markedly in the presence of 100 nM glucagon (p < 0.05). The mRNA levels of gnas and creb1 were up-regulated significantly by 1 µM glucagon (p < 0.05). The maximal response of all measured genes to glucagon was observed using a dose of 10 µM glucagon. As shown in Figure 3B, compared with the control group, the pck1 mRNA level was up-regulated markedly by 100 nM glucagon (p < 0.05) and reached the maximum value at 10 µM. For g6pc, its mRNA level was significantly up-regulated by 1 nM glucagon (p < 0.05) and peaked at 10 µM glucagon. The glucose concentration in the medium showed an upward trend with the increase of glucagon concentration, but no significant difference was observed (p > 0.05) ( Figure 3C). treatment (1 µM) or DMSO. All data are expressed as mean ± SE, n = 3, two-way ANOVA with Tukey post-hoc test. Values with different letters mean statistical differences (p < 0.05). T: time points; I: reagents for incubation; T × I: interaction between time and reagents. Figure 3A shows that increasing concentrations of glucagon could elevate the mRNA levels of glucagon pathway-related genes in a dose-dependent manner. Compared with the control group, the gcgr mRNA levels were significantly up-regulated when the glucagon concentration was 1 nM (p < 0.05). Treatment with 10 nM glucagon significantly increased the pka and pgc-1α mRNA levels (p < 0.05). The mRNA levels of adcy2 were up-regulated markedly in the presence of 100 nM glucagon (p < 0.05). The mRNA levels of gnas and creb1 were up-regulated significantly by 1µM glucagon (p < 0.05). The maximal response of all measured genes to glucagon was observed using a dose of 10 µM glucagon. As shown in Figure 3B, compared with the control group, the pck1 mRNA level was up-regulated markedly by 100 nM glucagon (p < 0.05) and reached the maximum value at 10 µM. For g6pc, its mRNA level was significantly up-regulated by 1 nM glucagon (p < 0.05) and peaked at 10 µM glucagon. The glucose concentration in the medium showed an upward trend with the increase of glucagon concentration, but no significant difference was observed (p > 0.05) ( Figure 3C).

Adomeglivant Inhibits the Glucagon Pathway in Hepatocytes by Targeting GCGR
As shown in Figure 4A, compared with the control group, adomeglivant significantly reduced the gcgr mRNA levels (p < 0.05) and abolished the increase of gcgr mRNA

Adomeglivant Inhibits the Glucagon Pathway in Hepatocytes by Targeting GCGR
As shown in Figure 4A, compared with the control group, adomeglivant significantly reduced the gcgr mRNA levels (p < 0.05) and abolished the increase of gcgr mRNA levels induced by glucagon (p < 0.05). The mRNA levels of adcy2, pka, creb1, and pgc-1α showed similar trends as those of gcgr. The gnas mRNA levels were significantly lower in the group treated with both glucagon and adomeglivant than in the group only incubated with glucagon (p < 0.05). No significant difference was observed between the control group and the adomeglivant group (p > 0.05). As shown in Figure 4B, the mRNA levels of pck1 showed similar trends as those of gnas. Adomeglivant significantly decreased the g6pc mRNA levels compared with the control group (p < 0.05). Following pre-incubation with adomeglivant, the stimulatory effects of glucagon on the g6pc mRNA levels were abolished (p < 0.05). As shown in Figure 4C, glucagon was effective at triggering the phosphorylation of CREB (p < 0.05). The glucagon-induced phosphorylated CREB protein expression could be blocked by pre-treatment with the adomeglivant (p < 0.05). Compared with the control group, PGC-1α protein expression showed an upward trend following glucagon incubation and a downward trend following adomeglivant treatment (p > 0.05). It also tended to decrease following glucagon-plus-adomeglivant treatment compared with glucagon treatment (p > 0.05). As shown in Figure 4D, compared with the control group, glucagon significantly increased the glucose production in hepatocytes (p < 0.05), but the glucose concentration in the medium was not significantly affected (p > 0.05). Incubation with adomeglivant significantly decreased the glucose production in hepatocytes (p < 0.05) and did not significantly increase the glucose concentration in the medium (p > 0.05). As for the gluconeogenic substrates in the medium, the lactate and pyruvate concentrations significantly increased folllowing adomeglivant incubation (p < 0.05).

Overexpression of Gcgr Activates the Glucagon Pathway in Hepatocytes
Images of the hepatocytes 48 h after transfection are shown in Figure 5A. The percentage of EGFP-positive cells was 31.01 ± 1.52% in the pcDNA3.1-EGFP group and 30.51 ± 1.07% in the pcDNA3.1-MSTN-1-EGFP group. Compared with the control group, transfection with the pcDNA3.1-EGFP plasmid did not influence the mRNA expression of gcgr (p > 0.05), while gcgr overexpression via pcDNA3.1-GCGR-EGFP transfection significantly increased the mRNA levels of gcgr by about 10 times (p < 0.001) ( Figure 5B). Compared with the control group, 48 h after transfecting with pcDNA3.1-GCGR-EGFP, the mRNA levels of adcy2, pka, creb1, pgc-1α, pck1, and g6pc ( Figure 5C) were up-regulated significantly (p < 0.01), while the gnas mRNA levels were up-regulated but not significantly (p > 0.05). The protein expression of GCGR and PCK1 was also up-regulated in the pcDNA3.1-GCGR-EGFP group compared with the control group ( Figure 5D). Compared with the control group, glucose production in the hepatocytes and glucose concentration in the medium increased significantly in the pcDNA3.1-GCGR-EGFP group ( Figure 5E) (p < 0.05), but the lactate and pyruvate concentrations in the medium tended to decrease, though not significantly (p > 0.05).

H89 Inhibits the Glucagon Pathway in Hepatocytes by Targeting PKA
As shown in Figure 6A,B, compared with the control group, glucagon incubation significantly increased the mRNA levels of the measured genes. H89 incubation did not influence the mRNA levels of gcgr, gnas, adcy2, pgc-1α, pck1, and g6pc compared with the control group (p > 0.05). Compared with the glucagon treatment alone, pre-treatment with H89 had no influence on the mRNA levels of gcgr, gnas, and adcy2 (p > 0.05) but significantly decreased the mRNA levels of pgc-1α, pck1, and g6pc in the hepatocytes (p < 0.05). Compared with the control group, the phosphorylation levels of a PKA substrate ( Figure 6C) and CREB at Ser133 ( Figure 6D) were down-regulated significantly by H89 incubation (p < 0.01). Compared with the glucagon group, pre-treatment with H89 decreased the levels of phosphorylated CREB (Ser133) (p > 0.05). As shown in Figure 6E, compared with the control group, H89 significantly decreased glucose production in the hepatocytes but increased the lactate concentration in the medium (p < 0.05). No significant differences were observed in glucose and pyruvate concentrations with the different treatments (p > 0.05).   ( Figure 5B). Compared with the control group, 48 hours after transfecting with pcDNA3.1-GCGR-EGFP, the mRNA levels of adcy2, pka, creb1, pgc-1α, pck1, and g6pc ( Figure 5C) were up-regulated significantly (p < 0.01), while the gnas mRNA levels were up-regulated but not significantly (p > 0.05). The protein expression of GCGR and PCK1 was also up-regulated in the pcDNA3.1-GCGR-EGFP group compared with the control group ( Figure 5D). Compared with the control group, glucose production in the hepatocytes and glucose concentration in the medium increased significantly in the pcDNA3.1-GCGR-EGFP group ( Figure 5E) (p < 0.05), but the lactate and pyruvate concentrations in the medium tended to decrease, though not significantly (p > 0.05).

H89 Inhibits the Glucagon Pathway in Hepatocytes by Targeting PKA
As shown in Figure 6A,B, compared with the control group, glucagon incubation significantly increased the mRNA levels of the measured genes. H89 incubation did not influence the mRNA levels of gcgr, gnas, adcy2, pgc-1α, pck1, and g6pc compared with the control group (p > 0.05). Compared with the glucagon treatment alone, pre-treatment with H89 had no influence on the mRNA levels of gcgr, gnas, and adcy2 (p > 0.05) but significantly decreased the mRNA levels of pgc-1α, pck1, and g6pc in the hepatocytes (p < 0.05). Compared with the control group, the phosphorylation levels of a PKA substrate ( Figure 6C) and CREB at Ser133 ( Figure 6D) were down-regulated significantly by H89 incubation (p < 0.01). Compared with the glucagon group, pre-treatment with H89 decreased the levels of phosphorylated CREB (Ser133) (p > 0.05). As shown in Figure 6E,  , p values were determined by two-way ANOVA with Tukey post-hoc test. Values with different letters mean statistical differences (p < 0.05). G: glucagon treatments; H: H89 treatments; G × H: interaction between glucagon and H89 treatments. For C, p values were determined by two-tailed t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

The Molecule 666-15 Inhibits the Glucagon Pathway in Hepatocytes by Targeting CREB
As shown in Figure 7A,B, compared with the control group, glucagon incubation increased the mRNA levels of the measured genes. Incubation of the hepatocytes with an inhibitor of CREB (666-15) significantly decreased the creb1 mRNA levels compared with the control group (p < 0.05). Following pre-treatment with 666-15, the stimulatory effects of glucagon on creb1 mRNA levels were abolished (p < 0.05). The mRNA levels of g6pc showed a similar trend as the creb1 mRNA levels. Compared with the control group, 666-15 incubation had no effect on the mRNA levels of gcgr, gnas, adcy2, pka, pgc-1α, and

The Molecule 666-15 Inhibits the Glucagon Pathway in Hepatocytes by Targeting CREB
As shown in Figure 7A,B, compared with the control group, glucagon incubation increased the mRNA levels of the measured genes. Incubation of the hepatocytes with an inhibitor of CREB (666-15) significantly decreased the creb1 mRNA levels compared with the control group (p < 0.05). Following pre-treatment with 666-15, the stimulatory effects of glucagon on creb1 mRNA levels were abolished (p < 0.05). The mRNA levels of g6pc showed a similar trend as the creb1 mRNA levels. Compared with the control group, 666-15 incubation had no effect on the mRNA levels of gcgr, gnas, adcy2, pka, pgc-1α, and pck1 (p > 0.05). Furthermore, the mRNA levels of gcgr, gnas, adcy2, and pka did not change significantly in the glucagon-plus-666-15 group compared with those in the glucagon group (p > 0.05). Regarding pgc-1α and pck1, 666-15 abolished the stimulatory effects of glucagon on their mRNA levels (p < 0.05). Compared with the control group, phosphorylated CREB and PGC-1α protein expression were down-regulated significantly by 666-15 incubation (p < 0.05) ( Figure 7C). Glucagon-induced phosphorylation of CREB and PGC-1α protein expression could be blocked by a pre-treatment with 666-15 (p < 0.05). Compared with the control group, 666-15 decreased glucose production in the hepatocytes (p > 0.05) but significantly increased the pyruvate concentration in the medium (p < 0.05) ( Figure 7D). Compared with the glucagon treatment alone, pre-treatment with 666-15 significantly decreased glucose production in the hepatocytes but increased the pyruvate concentration in the medium (p < 0.05). No significant difference was observed in glucose concentration in the medium upon different treatments (p > 0.05).

SR-18292 Inhibits the Glucagon Pathway in Hepatocytes by Targeting PGC-1α
As shown in Figure 8A,B, compared with the control group, SR-18292 had no effect on the mRNA levels of the measured genes (p > 0.05). Following pre-treatment with SR-18292, the stimulatory effects of glucagon on the mRNA levels of pck1 and g6pc were abolished (p < 0.05). However, pre-treatment with SR-18292 had no effect on the gcgr, gnas, adcy2, pka, creb1, and pgc-1α mRNA levels compared with glucagon treatment alone (p > 0.05). Compared with glucagon treatment, pre-treatment with SR-18292 significantly decreased glucose production in the hepatocytes and the glucose concentration in the medium (p < 0.05) ( Figure 8C). The lactate and pyruvate concentrations showed upward trends following SR-18292 treatment (p > 0.05).

SR-18292 Inhibits the Glucagon Pathway in Hepatocytes by Targeting PGC-1α
As shown in Figure 8A,B, compared with the control group, SR-18292 had no effect on the mRNA levels of the measured genes (p > 0.05). Following pre-treatment with SR-18292, the stimulatory effects of glucagon on the mRNA levels of pck1 and g6pc were abolished (p < 0.05). However, pre-treatment with SR-18292 had no effect on the gcgr, gnas, adcy2, pka, creb1, and pgc-1α mRNA levels compared with glucagon treatment alone (p > 0.05). Compared with glucagon treatment, pre-treatment with SR-18292 significantly decreased glucose production in the hepatocytes and the glucose concentration in the medium (p < 0.05) ( Figure 8C). The lactate and pyruvate concentrations showed upward trends following SR-18292 treatment (p > 0.05).

Discussion
Mammals such as humans, pigs, and rats were found to have only one GCG gene, which encodes several peptide hormones, including glucagon and glucagon-like peptides-1 and -2 (glp1 and glp2) [32]. In the present study, the amino acid sequence of GCG in the Japanese flounder showed more than 50% homology with the corresponding sequences of mammalian GCG. In some teleost, there are two GCG genes with distinct 3 ends that generate GCG a and b [33]. GCG is a gene that encodes glucagon, glp1, and glp2 and is mainly expressed in the intestine. The GCG b gene encodes glucagon and glp1 and is mainly expressed in the pancreas [34,35]. In the present study, the cloned GCG of the Japanese flounder was shown to share a high sequence similarity with GCG b of other fish species and encodes glucagon and glp1. Glucagon of the Japanese flounder was then chemically synthesized according to the deduced amino acid sequence for incubation with hepatocytes.
In the present study, in vitro incubation with glucagon activated gluconeogenesis and induced glucose production in hepatocytes of the Japanese flounder. In line with the present study, bovine glucagon intraperitoneally injected in blunt snout bream (Megalobrama amblycephala) and infused in rainbow trout increased the hepatic glucose production and the mRNA levels of gluconeogenesis-related genes [36,37]. Earlier studies concerning the glucagon signaling pathway in fish found that bovine glucagon bound GCGR (by measuring the radioactivity of hepatocyte-bound 125 I-labelled glucagon) and increased PKA activity in American eels (Anguilla rostrata) and brown bullheads (Ictalurus nebulosus) hepatocytes [38,39]. However, the glucagon used in these experiments was not from the fish examined, and the components involved in the signaling pathway were not comprehensively explored. In the present study, more genes downstream of GCGR in the Japanese flounder were investigated; it was found that the mRNA expression of creb1, pgc-1α, pck1, and g6pc in the hepatocytes of the Japanese flounder was activated by synthetic glucagon identical to the fish glucagon in a time-and dose-dependent manner.
Adomeglivant is an effective antagonist of GCGR that inhibits the transcription of gcgr [40]. In the present study, adomeglivant significantly inhibited the stimulation of the mRNA expression of gcgr, gnas, adcy2, pka, creb1, pgc-1α, pck1, g6pc and the protein expression of phosphorylated-CREB by glucagon and decreased glucose production in the hepatocytes. Overexpression of GCGR increased the mRNA levels of the above genes, the protein expression of PCK1, and glucose production in the hepatocytes. These results indicated that the CREB1, PGC-1α, PCK1, and G6PC are downstream of GCGR, which is consistent with findings in mammals [3].
In the present study, an inhibitor of PKA (H89) decreased the mRNA expression of pgc-1α, pck1, g6pc, the protein expression of phosphorylated-CREB, and glucose production in the hepatocytes. CREB is a transcription factor residing in the nucleus that is capable of being activated by multiple pathways [41]. In mammals, CREB is activated by glucagonmediated signaling and induces hepatic gluconeogenesis [42]. However, this characteristic has not been addressed in studies carried out in fish. In grass carp hepatocytes, glucagon treatment could elevate the creb mRNA levels and induce CREB phosphorylation [43]. A study in goldfish showed that CREB was activated by glucagon and targeted the leptin gene [44]. To determine the role of CREB in glucagon-mediated gluconeogenesis in the Japanese flounder, 666-15, an antagonist of CREB, was employed to inhibit the transcription of creb. We found that 666-15 significantly decreased the stimulation of glucagon on the mRNA expression of creb1, pgc-1α, and gluconeogenic genes, as well as the protein expression of phosphorylated-CREB and PGC-1α, with glucose production in hepatocytes decreasing accordingly. However, no significant difference in the gcgr, gnas, adcy2, and pka mRNA levels was observed following the incubation of the hepatocytes with 666-15. These results demonstrated that CREB is involved in the glucagon-mediated gluconeogenesis pathway. Furthermore, CREB was confirmed to be downstream of PKA and upstream of PGC-1α, G6PC, and PCK1. In mice carrying a targeted disruption of the CREB gene or expressing a dominant negative CREB protein in the liver, the induction of pgc-1α, g6pc, and pck1 by glucagon was blocked, and this led to severe hypoglycemia [2]. The results showed that CREB induced the expression of gluconeogenic genes through PGC-1α in response to glucagon stimulation.
The transcriptional co-activator PGC-1α plays a vital role in regulating cellular energy metabolism [45]. Accumulating evidence has indicated that PGC-1α is a key regulator of glucose production and is also an attractive target for anti-diabetic therapy [46][47][48]. However, whether PGC-1α participates in the glucagon-regulated gluconeogenic pathway in fish remains unknown. An antagonist of PGC-1α, SR-18292, which suppresses gluconeogenic gene expression and reduces glucose production in mice hepatocytes [49], was used in the present study. The results indicated that treatment of the hepatocytes with SR-18292 significantly reduced the ability of glucagon to stimulate pck1 and g6pc gene expression and glucose production. This was achieved without disrupting the glucagoninduced increase in the mRNA levels of gcgr, gnas, adcy2, pka, and creb1. These results indicated that PGC-1α is downstream of the glucagon-mediated CREB signaling pathway and mediates the activation of gluconeogenesis in the Japanese flounder.

Conclusions
In summary, the present study confirmed that glucagon promoted gluconeogenesis through the GCGR/PKA/CREB/PGC-1α pathway in the Japanese flounder ( Figure 9). These results illustrate the presence of similar pathways regulating gluconeogenesis by glucagon in the Japanese flounder and mammals. They provide a basis for determining whether glucagon-regulated gluconeogenesis is one of the reasons for glucose intolerance in carnivorous fish and further finding targets to improve this disorder. cagon-mediated gluconeogenesis pathway. Furthermore, CREB was confirmed to be downstream of PKA and upstream of PGC-1α, G6PC, and PCK1. In mice carrying a targeted disruption of the CREB gene or expressing a dominant negative CREB protein in the liver, the induction of pgc-1α, g6pc, and pck1 by glucagon was blocked, and this led to severe hypoglycemia [2]. The results showed that CREB induced the expression of gluconeogenic genes through PGC-1α in response to glucagon stimulation.
The transcriptional co-activator PGC-1α plays a vital role in regulating cellular energy metabolism [45]. Accumulating evidence has indicated that PGC-1α is a key regulator of glucose production and is also an attractive target for anti-diabetic therapy [46][47][48]. However, whether PGC-1α participates in the glucagon-regulated gluconeogenic pathway in fish remains unknown. An antagonist of PGC-1α, SR-18292, which suppresses gluconeogenic gene expression and reduces glucose production in mice hepatocytes [49], was used in the present study. The results indicated that treatment of the hepatocytes with SR-18292 significantly reduced the ability of glucagon to stimulate pck1 and g6pc gene expression and glucose production. This was achieved without disrupting the glucagon-induced increase in the mRNA levels of gcgr, gnas, adcy2, pka, and creb1. These results indicated that PGC-1α is downstream of the glucagon-mediated CREB signaling pathway and mediates the activation of gluconeogenesis in the Japanese flounder.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.