FIG 7.

SHBs elevates hepatic AC1 expression. (A) Relative mRNA expression levels of ACs in Ad-GFP and Ad-SHBs-infected mouse primary hepatocytes exposed to glucagon (100 nM, top) and in Huh-Control and Huh7-SHBs cells exposed to FSK (10 μM, bottom) for 2 h determined by RT-qPCR. (B) Effect of SHBs on AC1 protein levels in mouse primary hepatocytes exposed to glucagon (100 nM, left) and Huh7 cells exposed to FSK (10 μM, right) for 16 h detected by Western blot analysis. (C, D) The mRNA and protein levels of AC1 in the livers of LSL-SHBs and LSL-SHBs/Alb-Cre mice fasted for 16 h determined by RT-qPCR (C, n = 6 mice/group) and Western blot analysis (D, n = 4 mice/group). (E, F) Effect of AC1 specific inhibitor ST034307 on glucose production (E) and gluconeogenic gene expression (F) in Ad-SHBs-infected mouse primary hepatocytes and in Huh7-SHBs cells. Cells were treated with ST034307 (20 μM) for 1 h and then with glucagon (100 nM) or FSK (10 μM) for 6 h (E) and 2 h (F). (G) Effect of SHBs on promoter activities of AC1 in Huh7 cells. Huh7-Control and Huh7-SHBs cells were transfected with pRL-TK and the reporter genes. After 36 h of 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. (H) Effect of E-box mutant (dE), nuclear receptor binding half site mutant (dH), and BEF mutant on promoter activity of AC1 in Huh7-SHBs cells. 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.