Fig. 4

Effects of IDE deficiency on glucagon signaling and expression of gluconeogenic genes in a cell line of hepatocytes. AML12 cells were serum-starved for 18 h followed by incubation with glucagon (50 ng/mL) at the indicated times and the effects of Ide deficiency on glucagon signaling and expression of gluconeogenic genes were examined. (A) Representative western blots depicting control (white bars) and shRNA-IDE (black bars) hepatocytes treated with glucagon. Densitometric analysis of data in panel A for IDE (B), GCGR (C) and CREB (D). Data are mean ± SEM. n = 3 per group. ^#p value < 0.05 versus untreated cells (time 0) or *p value < 0.05 versus control cells by two-way ANOVA. Gene expression levels of Gcgr (E) or Creb1 (F). Data are mean ± SEM. n = 3 per group. ^#p value < 0.05 versus untreated cells (time 0) or *p value < 0.05 versus control cells by two-way ANOVA. (G) cAMP levels after 30 min of glucagon stimulation in control and IDE-deficient cells. Data are mean ± SEM. n = 3 per group. *p value < 0.05 versus control cells by Students´ T-test. (H) Representative western blots of p-PKA substrates for control and shRNA-IDE cells treated with glucagon (50 ng/mL) at the indicated times. (I) Densitometric analysis of data in panel H for p-PKA substrates. Data are mean ± SEM. n = 3 per group. ^#p value < 0.05 versus untreated cells (time 0) or *p value < 0.05 versus control cells by two-way ANOVA. (J) Densitometric analysis of data in panel A for p-CREB and the ratio p-CREB/CREB (K). Data are mean ± SEM. n = 3 per group. ^#p value < 0.05 versus untreated cells (time 0) or *p value < 0.05 versus control cells by two-way ANOVA. Gene expression levels of G6pc (L) or Pck1 (M). Data are mean ± SEM. n = 3 per group. ^#p value < 0.05 versus untreated cells (time 0) or *p value < 0.05 versus control cells by two-way ANOVA.