Insulin Controls Clock Gene Expression in the Liver of Goldfish Probably via Pi3k/Akt Pathway

Abstract

The liver circadian clock plays a pivotal role in driving metabolic rhythms, being primarily entrained by the feeding schedule, although the underlying mechanisms remain elusive. This study aimed to investigate the potential role of insulin as an intake signal mediating liver entrainment in fish. To achieve this, the expression of clock genes, which form the molecular basis of endogenous oscillators, was analyzed in goldfish liver explants treated with insulin. The presence of insulin directly increased the abundance of per1a and per2 transcripts in the liver. The dependency of protein translation for such insulin effects was evaluated using cycloheximide, which revealed that intermediate protein translation is seemingly unnecessary for the observed insulin actions. Furthermore, the putative interaction between insulin and glucocorticoid signaling in the liver was examined, with the results suggesting that both hormones exert their effects by independent mechanisms. Finally, to investigate the specific pathways involved in the insulin effects, inhibitors targeting PI3K/AKT and MEK/ERK were employed. Notably, inhibition of PI3K/AKT pathway prevented the induction of per genes by insulin, supporting its involvement in this process. Together, these findings suggest a role of insulin in fish as a key element of the multifactorial system that entrains the liver clock to the feeding schedule.

Keywords: Carassius auratus; circadian system; fish; food-entrainable oscillator (FEO); insulin; liver; period genes; phosphatidylinositol 3-kinase (PI3K).

Figure 1

Dose–response effects of insulin treatment on per gene mRNA abundance. Relative mRNA abundance of the clock genes per1a, per1b, and per2 in cultured goldfish livers treated with different concentrations of insulin for 2 h (A,D,G), 4 h (B,E,H), or 8 h (C,F,I). Data are shown as mean ± SEM; n = 8. Data were analyzed by one-way ANOVA (* p < 0.05; ** p < 0.005; *** p < 0.001). Significant differences among groups according to SNK test are represented by different letters.

Figure 2

Dose–response effects of insulin treatment on clock gene mRNA abundance. Relative mRNA abundance of the clock genes clock1a, bmal1a, and rev-erbα in cultured goldfish liver treated with different concentrations of insulin for 2 h (A,D,G), 4 h (B,E,H), or 8 h (C,F,I). Data are shown as mean ± SEM; n = 8. Data were analyzed by one-way ANOVA.

Figure 3

Effects of cycloheximide on insulin action over per mRNA abundance. Relative mRNA abundance of clock genes per1a and per2 in cultured goldfish liver treated with 1 µM insulin or 10 µM cycloheximide alone or both drugs together for 2 h (A,C) or 8 h (B,D). Data are shown as mean ± SEM; n = 8. Data were analyzed by two-way ANOVA. INS = insulin. CHX = cycloheximide; ** p < 0.005 INS; *** p < 0.001 INS. # p < 0.05 CHX; ## p < 0.005 CHX.

Figure 4

Effects of dexamethasone on insulin action over per mRNA abundance. Relative mRNA abundance of clock genes per1a and per2 in cultured goldfish liver, treated with 0.1 µM insulin (+), 1 µM insulin (++), and 0.1 µM dexamethasone alone or both drugs together for 2 h (A,C) or 8 h (B,D). Data are shown as mean ± SEM; n = 8. Data were analyzed by two-way ANOVA. When an interaction between factors occurred, a one-way ANOVA followed by a SNK test was performed, and differences among groups were represented by different letters (B). INS = insulin. DX = dexamethasone. * p < 0.05 INS; *** p < 0.001 INS. ## p < 0.005 DX; ### p < 0.001 DX. $ interaction between drugs (p < 0.05).

Figure 5

Involvement of PI3K/AKT pathway in insulin action on clock gene mRNA abundance. Western blot analysis of the phosphorylation status of AKT protein in cultured liver of goldfish after 4 h of treatment with control medium (C) or 1 µM insulin (INS). The graph (A) represents the p-AKT/AKT ratio from Western blots performed on 8 individual samples per treatment (mean ± SEM); and (B) shows representative blots. ** indicates significant differences (p < 0.005) between the groups according to Student’s t-test. (C,D) Effect of the PI3K/Akt pathway inhibitor LY294002 on insulin action over per1a and per2 mRNA abundance (mean ± SEM; n = 8). Data were analyzed by two-way ANOVA. When an interaction between factors occurred, a one-way ANOVA followed by a SNK test was performed, and differences among groups were represented by different letters. INS = insulin. LY = LY294002. $ interaction between factors (p < 0.05).

Figure 6

Involvement of the MEK pathway in insulin action on clock gene expression: (A,B) Western blot analysis of the phosphorylation status of MEK protein in cultured liver of goldfish after 4 h of treatment with control medium or 1 µM insulin (INS). The graph (A) represents the p-MEK/MEK ratio of Western blots performed on 7 individual samples per treatment (mean ± SEM); (B) shows representative blots. (C,D) Effect of the MEK pathway inhibitor PD98059 on insulin action over per1a and per2 mRNA abundance (mean ± SEM; n = 8). Data were analyzed by two-way ANOVA (* p < 0.05 for insulin factor). INS = insulin. PD = PD95059.