Figure 7

Insulin increased the expression of Hnf-1 mRNA and protein in Hepa1–6 cells (a,b). After 12 h from cell splits, Hepa1–6 cells were incubated with serum-free medium for 10 h and then transferred to a medium including 10 nM insulin. The cells were then collected at the indicated time points. (a) Total RNA isolated at each time point was subjected to qPCR to quantify Hnf-1α mRNA levels. Data represent mean ± SEM from triplicate PCR samples and the experiment was repeated once with similar results. Asterisks indicate a significant difference in the mRNA levels in Hepa1–6 cells at 0 min (***p < 0.001). (b) Whole cell lysates were prepared and subjected to immunoblotting using anti-Hnf-1, anti-p-Akt and anti-cyclophilin antibodies. The p-AKT was phosphorylated Akt. The blot of Hnf-1, the blot of p-AKT and the blot of cyclophilin were cropped from different parts of the same membrane. The blot of Angptl8 and cyclophilin was cropped from different membrane using the same whole cell lysates used for anti-Hnf-1, p-AKT and cyclophilin blotting. (c,d) Incubation with 10 nM insulin stimulated the promoter activity of Angptl8, including consensus binding motif for Hnf-1, in Hepa1–6 cells (c) and mouse primary hepatocytes (d). The −2291/+101 reporter construct with the wildtype Hnf-1 binding motif, −2291/+101 construct with the mutated Hnf-1 binding motif, or −59/+101 construct lacking the Hnf-1 binding site were transfected into Hepa1–6 cells or mouse primary hepatocytes. Ten hours after transfection, the cells were incubated with serum-free medium for 4 h. The cells were then incubated 2 h with or without 10 nM insulin. Promoter activities are expressed as luciferase activities relative to those of the pGL 4.10 construct in the absence of insulin stimulation, which was assigned a value of 1. Data represent mean ± SEM from three independent transcription with triplicate determinants. Asterisks indicate the significant difference in the −2291/+101 WT construct with insulin stimulation from the −2291/+101 WT reporter activities in the absence of insulin stimulation (***p < 0.001).