FIG 4.

SHBs promotes hepatic gluconeogenesis via activation of CREB. (A) Relative mRNA expression levels of CREB in the livers of LSL-SHBs and LSL-SHBs/Alb-Cre mice after 16 h fasting (left, n = 5 mice/group), in the Ad-GFP and Ad-SHBs infected mouse primary hepatocytes after glucagon (100 nM) stimulation for 2 h (middle), and in Huh7-Control and Huh7-SHBs cells exposed to FSK (10 μM) for 2 h (right). Relative mRNA levels were determined by RT-qPCR. (B) Phosphorylated and total CREB levels in the livers of LSL-SHBs and LSL-SHBs/Alb-Cre mice after 16 h fasting, determined by Western blot analysis (n = 4 mice/group). (C) Western blot analysis of phosphorylated and total CREB levels of SHBs in SHBs-expressing mouse primary hepatocytes (left) and Huh7 cells (right) treated with glucagon (100 nM) or FSK (10 μM), respectively, for 0.5 h and 1 h. Arrowhead points to phospho-CREB. (D) Effect of CREB knockdown in Huh7-SHBs cells treated with 10 μM FSK for 2 h on G6pc and PEPCK mRNA expression. shRNA-mediated knockdown of CREB in Huh7-Control and Huh7-SHBs cells confirmed by Western blot analysis. (E, F) Effect of CRE mutation (E) and CREB knockdown (F) on promoter activities of G6pc and PEPCK in Huh7-SHBs cells. Huh7-Control and Huh7-SHBs cells were transfected with pRL-TK and the reporter genes. After 36 h transfection, cells were serum starved overnight and treated with FSK (10 μM) for 6 h. The dual-luciferase reporter gene assays were then carried out and the luciferase activity of the control group was normalized to 1. The glycosylated (gp) and nonglycosylated (p) forms of SHBs were indicated. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significant difference.